A Sponsored Supplement to Science

Your Practical Guide to

Basic Laboratory Techniques

Produced by the
Science/AAAS Custom
Sponsored by Publishing Office
 TAB L E O F C O NT E NT S
B I O LO GY ¡ E A RT H ¡ E N V I R O N M E N T ¡ L I F E ¡ I N T E R D I S C I P L I N A RY ¡ P H YS I CA L ¡ M AT E R I A L ¡ S O C I A L S C I E N C E S

Introductions
2 Ready . . . Set . . . Pipet!
Sean Sanders, Ph.D.

Your
Science/AAAS

O P E N A C C E S S , D I G I TA L , A N D F R E E T O A L L R E A D E R S Practical 3 Taking the First Steps

Ferencz Paldy, Ph.D.

Guide to
Head of Segment Marketing Academia, Sartorius

Fiona Coats, Ph.D.

Basic
Head of Life Science Research Marketing, Sartorius

Laboratory Feature Articles

Techniques

4 Building Skills in Basic Lab Techniques:
Useful Tips from the Experts
Mike May, Ph.D.

7 The Paperless Lab

Chris Tachibana

Technical Notes

10 How to Avoid Contamination in Pipetting

13 Concentration to a Defined Final Volume with

Vivaspin® Turbo 15, Vivaspin® Turbo 4 and
Vivaspin® 500

15 Cell Culture Expansion in Fully Closed Erlenmeyer
Shake Flasks Outside the Biosafety Cabinet with
MYCAP™ CCX

20 How to Achieve Optimal Weighing Performance

Cover Image: © abscent/shutterstock.com

24 Vivaflow® and Vivaspin® Workflow in Protein
Research Laboratories
Original content (pages 4–9) has not

been peer-reviewed or assessed
by Science.
29 Minimizing Syringe Filter Consumption for Monoclonal
This booklet was produced by the Science/ Antibody Harvest from CHO Cell Culture Supernatants
AAAS Custom Publishing Office and

34
sponsored by Sartorius.
Correlation Between Colony Forming Units and Genome
Pushing the Boundaries of Knowledge Editor: Sean Sanders, Ph.D.
Proofreader/Copyeditor: Bob French
Designer: Amy Hardcastle


Copies of 9 Different Mycoplasma Species Using Quantified
CFU and GC Standards for Validation
ROGER GONCALVES,

As AAAS’s first multidisciplinary, open access journal, Science Advances publishes

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Custom Publishing
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38 Scouting Protein Purification Conditions Using
Vivapure Centrifugal Ion Exchange Membrane Absorbers
research that reflects the selectivity of high impact, innovative research you expect rgoncalves@science-int.co.uk
+41-43-243-1358
from the Science family of journals, published in an open access format to serve a © 2018 by The American Association

42
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vast and growing global audience. Check out the latest findings or learn how to All rights reserved. 26 October 2018 Additional Resources
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1
SCIENCE sciencemag.org
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
“
Y I
ou need to learn to walk before you can run” is a
saying many of us probably heard when we were
children. The clear message here is that there are
some basic skills we need to master before we can
move on to the next level. And there are plenty of
good reasons that following this mantra will set one up for success,
not the least of which—to continue the metaphor—is to avoid tripping
and falling on your face.
In a scientific laboratory, there are also fundamental skills that
require mastering before more complex tasks can be undertaken.
Building a solid foundation of core lab skills is critical not only to
producing accurate, reproducible experimental results, but also
to prevent damage to expensive equipment and maintain a safe
environment for ourselves and our fellow labmates.
Gaining competence in accurately weighing dry reagents is a
Ready . . . Set critical skill, particularly when making stock solutions that might be
used across multiple experiments and by multiple researchers in the
. . . Pipet! lab. When an experiment doesn’t work, we often don’t know why—
but we certainly don’t want its failure to be the result of incorrectly
It behooves all prepared solutions due to poor weighing proficiency.
researchers to ensure Filtration is a foundational technique used ubiquitously in the
biological sciences and is an essential step in many protocols. One
that their core lab of its common applications is the generation of clean water needed
skills are solid and in many aspects of lab work, but probably most importantly for
making up and diluting reagents. Impurities in improperly filtered
up to date. water, even at low levels, can negatively impact biological processes
or, even worse, generate spurious results. Filtration is also essential
for purification and/or concentration of solutions as well as the
sterilization of biological reagents for which autoclaving is not an
option due to heat sensitivity.
Most, if not all, life science laboratories have at least one set of
micropipettes. If they’re lucky, some might even have a set for each
researcher. Correct pipetting technique for small volumes of reagents
is an essential skill for researchers performing almost any type of
molecular biology experiment. Knowing how to accurately pipet
a range of fluids—from viscous glycerol to highly volatile phenol—
can make the difference between a successful experiment and yet
another confusing result. And anyone who remembers learning to
pipet will recall that it’s nowhere near as easy as it looks.
With increasing focus in the scientific community on reproducibility
of results, it behooves all researchers to ensure that their core lab
skills are solid and up to date. The latest advances in lab techniques
need to be studied and absorbed, and basic skills revisited and
refreshed. In other words, keep practicing your walking skills so that
you’re able to sprint when it’s really needed!
Sean Sanders, Ph.D.
Senior Editor, Custom Publishing
Science/AAAS
2 sciencemag.org SCIENCE
I NT R O D UC T I O NS
n a scientific world that is more competitive than ever before,
it is imperative to gain a deep understanding of biological no-
velties and phenomena at both a macro and micro scale, and to
do this as quickly and accurately as possible. This knowledge
will potentially enable scientists to formulate novel hypotheses,
Taking the make new discoveries, and share their findings with the world. The
creation and dissemination of scientific information is the corner-
First Steps stone of scientific and societal advancement.
“A Journey of a With such a strong focus on exciting discoveries—like the next
generation of cancer therapies—it is easy to forget that it all starts
Thousand Miles with the basics. As the Chinese philosopher Laozi once said, “The
Begins with a Single journey of a thousand miles begins with a single step.“ Every cell
culture medium, and every sample of DNA, RNA, or purified protein,
Step.”—Laozi needs at some point during the experimentation process to undergo
a variety of different treatments. These materials may need to be
dissolved or diluted in purified water, weighed, filtered, pipetted,
or generally experience aseptic handling or transfer. All these small,
seemingly insignificant steps and minor details tend to be forgotten
as a user gains experience and confidence in the daily routines of their
laboratory, or even disregarded when it comes to complete beginners.
Since nothing that stands the test of time can have a weak foun-
dation, it is extremely important for today’s young scientists entering
the lab world for the first time to be able to build a robust foundation
in basic lab techniques, starting on day one. This underpinning is
crucial to their future success. It is equally important that experienced
scientists revisit these basic topics in order to remedy potential
misconceptions, and to fill in the gaps in their knowledge that have
developed over time.
Sartorius, a global laboratory products and services supplier for the
academic and (bio)pharma markets, has been dedicated to providing
solutions that strengthen scientific experimentation for more than
140 years. Sartorius engages with its customers over the full spectrum
of their work, catering not only to their basic laboratory needs (such
as weighing, pipetting, and filtering), but also by offering high-end
and high-throughput (live) cell-analysis instrumentation. By offering
this booklet in partnership with Science/AAAS, we hope that we can
contribute to building a secure and prosperous scientific future for
the benefit of all stakeholders involved.
Ferencz Paldy, Ph.D.
Head of Segment Marketing Academia, Sartorius
Fiona Coats, Ph.D.
Head of Life Science Research Marketing, Sartorius
SCIENCE sciencemag.org 3
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
Building Skills in Basic Lab Techniques:
Useful Tips from the Experts
Safety and competency in a science laboratory depend on a set of basic skills. As science advances, so do some of the
capabilities required for it. Nonetheless, some skills are almost as old as science itself, and these remain vital—even
though the way of doing these tasks has evolved. With both old and new techniques, beginner and experienced scientists
alike need to maintain their competency in the use of numerous standard methods. Even after learning and mastering a
technique, a refresher never hurts, and keeping current on changing methods maintains the foundation of a lab and the
integrity of its findings. By Mike May, Ph.D.
H
ere, we’ll explore familiar, everyday methods along
with some newer ones—all aimed at helping scientists
build and maintain a skillset. Many of these skills will
apply to various applications. For example, Donald
Spratt, assistant professor of chemistry and biochemistry at
Clark University (Worcester, Massachusetts), says, “Protein
scientists need, for example, to have excellent planning and
organizational skills so they can design and successfully execute
their experiments.” He adds, “These skills are translatable to many
different scientific disciplines.” In fact, most lab skills build on
others and help scientists learn new ones.
Weigh it right
Weighing samples is one of the oldest procedures in all of
science. It’s one of the first things that scientists learn how to do,
and a skill that most of them need throughout their careers. The
ubiquity of weighing makes it a top-priority skill for scientists at
all levels. The first step to weighing involves picking the right
balance. “People do not need a four-place analytical balance for
routine powder dispensing, and conversely they cannot achieve
4 5
Others agree on the value of the right attitude with this
process. “The most important thing to keep in mind while
pipetting is slowing down and taking my time,” says VJ Tocco,
lecturer in the department of chemical engineering at the
University of Florida, Gainesville. “Sometimes, I get tempted to
rush, which can lead to mistakes.”
Tocco suggests other things to remember as well, including
picking the right pipette. “You should use the pipette that dis-
penses the smallest volume,” he says. “For example, to pipet
18 microliters of fluid, use the 20-microliter pipette, not the
100-microliter pipette.” And the pipette tip should be wet before
using it. As Tocco says, “It’s best to aspirate liquid and dispense it
at least once before actually pipetting your liquid.” Lastly, Tocco
reminds scientists to take their time and not to “aspirate so quick-
precise weighing on a top-loading balance,” says Kevin Olsen, ly that bubbles form in the solution.” Those bubbles cause errors
instrumentation specialist in the chemistry and biochemistry in volume measurement.
department at New Jersey’s Montclair State University. “It is
important to understand the limitations of whatever kind of Purifying the processes
balance you are using.” Many protocols in a lab require a variety of solutions,
For instance, any balance produces a more accurate weight including culture media, buffers, and more. And these solutions
for larger over smaller samples. “This is why we typically weigh usually require water. In most cases, not just any water will do.
out an analytical standard in the grams range and dilute it rather Instead, water for lab processes must be filtered and purified,
than weighing the same material in the milligram range,” Olsen and the application determines the level of purity required.
IMAGES: (FROM TOP) © M.LORAINE/SHUTTERSTOCK.COM; MAKYZZ/SHUTTERSTOCK.COM
explains. “Different balance models have different features and if According to ASTM International, water can be categorized
they are used incorrectly, the weighing may not be accurate.” as Type I–IV, with Type I being the purest. One metric that distin-
For every balance, keeping it clean and calibrated impacts all guishes these categories is resistivity (Ω-cm); water with fewer
weight measurements. So, a little care goes a long way. impurities shows higher resistivity. For example, the resistivity
of Type I and IV water is 18 and 0.2 megaΩ-cm, respectively.
Proper pipetting The less-pure Type IV water can be used as a source for a lab
After weighing samples, the next most common technique, distiller, for example, and ultrapure Type I water is used for cell
at least in the biological sciences, might be pipetting. For culture, gas chromatography, high-performance liquid chroma-
some scientists, pipetting could even be the most important tography (HPLC), and other applications that are very sensitive
skill to master. To get it right, scientists need to pay attention, to impurities.
and not just to the proper technique. In fact, becoming The secret is matching the right water to an application, and
distracted is a common mistake in pipetting, according to not overspending to make water that is more purified than
Tamara Mandell, associate director of education and training necessary. The volume of water necessary will also determine
at the University of Florida’s Biotility, a center that prepares how to make it. In some situations, a water system for a lab
people for the biotech industry. is enough, while other applications require building-wide
purification systems. In the latter case, a building-wide system
might make reasonably pure water, for example, Type III; and
then lab systems can further treat that water as needed.
FE AT UR E ART I C L E S
One critical role
for filtration in
these industries
is sterilization,
since the use of
heat to sterilize
would cause
undesirable
product
degradation.
Filtering fluids
To remove unwanted solids from a sample and increase
purity, scientists often use various forms of filtration, which
extend from a simple piece of filter paper in a funnel to
advanced membrane-based devices. Many molecular methods
include filtration to concentrate a sample. Filtration is used
extensively to concentrate and purify proteins or DNA,
for example, for crystallography studies or for use in the
polymerase chain reaction (PCR).
Filtration processes can also be distinguished by general
application. One of the most common applications for
analytical filtration is sample preparation for HPLC. Filtering out
particles is essential to prevent blocking of the column, which
can lead to failure of the analysis; it also reduces background in
the chromatogram and improves sensitivity and accuracy.
The biotechnology and pharmaceutical industries also re-
quire filtration in many processes, using a variety of membranes
and devices that often have the added requirement of meeting
specific criteria, such as ASTM International standards or good
manufacturing practices (GMP) regulations. One critical role for
filtration in these industries is sterilization, since the use of heat
to sterilize would cause undesirable product degradation.
Keeping cultures healthy
From basic science to biotechnology and pharmaceutical
sciences, many labs include cell or tissue culture as a standard
method. The basic idea of keeping cells alive in culture is over
130 years old—starting in 1885 with work by German zoologist
Wilhelm Roux, who cultured chicken embryonic cells in a
saline solution. Today, scientists culture cells in two and three
dimensions, even mimicking complete organs in some cases.
Despite its increasing complexity, some of the steps for cell
culture are easier than ever.
 LIFE SCIENCE TECHNOLOGIES
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
DIGITAL LAB
FEMANAGEMENT
AT UR E ART I C L E S
LIFE SCIENCE TECHNOLOGIES
Processing proteins maintain neutrality throughout,” says Shawn Douglas, LIMSwiki
DIGITAL LAB MANAGEMENT
Many protocols in life science and clinical labs involve pro- curator, “avoiding marketing and self-promotion. The wiki is an
teins. When asked about the top skill required for working with evolving tool, and we’re always looking for quality contributors.”
these molecules, Daniel J. Kosman, SUNY Distinguished Profes- LIMSwiki
maintain provides
neutrality definitions
throughout,” for Shawn
says terms such as ELN
Douglas, (elec-
LIMSwiki
sor in biochemistry at the University of Buffalo’s Jacobs School of curator, “avoiding marketing and self-promotion. The wiki experi-
tronic laboratory notebook, generally used to document is an
Medicine and Biomedical Sciences, picks the ability to use fast ments) and
evolving tool,LIMS
and we’re (laboratory
alwaysinformation
looking formanagement
quality contributors.” systems,
protein liquid chromatography (FPLC), which can isolate proteins traditionally used for definitions
LIMSwiki provides tracking standardized
for terms such processes
as ELN such (elec-as
in a mixture. He also notes that protein scientists must be able to production). But the distinction between informatics products
tronic laboratory notebook, generally used to document experi-
perform heterologous expression, in which DNA or RNA from one is blurring, says Markus Dathe, good manufacturing
ments) and LIMS (laboratory information managementpractice systems,
species is expressed in another to create a specific protein. With and computer at Roche,
traditionally usedsystem validation
for tracking coordinator
standardized processes such because
as
this technique, though, Kosman notes that the key challenges are “convergence is happening.” ELNs, LIMS, and equipment soft-
production). But the distinction between informatics products
ensuring the “correct folding and posttranslational modification of ware are expanding functions, interconnecting, and overlapping.
is blurring, says Markus Dathe, good manufacturing practice
heterologously expressed proteins.” Informatics
and computerpackages increasingly
system validation aim to cover
coordinator the entire
at Roche, life-
because
Spratt also points out the need for protein-expression capabili- cycle of an R&D project including reagent inventories, regulatory
“convergence is happening.” ELNs, LIMS, and equipment soft-
ties. When asked about the most common technique for obtain- forms,
ware areand work requests
expanding functions, in addition to experimental
interconnecting, details.
and overlapping.
ing proteins for further research, he selects bacterial expression Most researchers start small, though, with a homegrown ELN
Informatics packages increasingly aim to cover the entire life-
in Escherichia coli using recombinant DNA technology, calling it with ofprotocols
cycle an R&D in text documents
project including reagent and electronic
inventories, data regulatory
files.
Scientists who have been thinking about “the most common and cheapest way to make a protein.” With this “Everyone
forms, and worksees the value
requests of ELNs,
in addition to from scientistsdetails.
experimental to principal
writing all along can get a head start by using technique, the overexpressed protein “can then be purified using investigators to lab managers,” says Erik Alsmyr, senior director
Most researchers start small, though, with a homegrown ELN
an electronic lab notebook to keep track of
protocols and results.
chromatography, based on its unique physicochemical properties,
such as size, charge, affinity, solubility, and/or oligomeric state,”
Spratt explains. “Once the protein is pure, it needs to be quanti-
The Paperless Lab of software
Contur’s
“Everyone
development
iLabber)
sees for
for the
the small-to-medium-sized
Accelrys
with protocols in text documents and electronic data files.
value of ELNs, from scientists
Notebook
research
(previously
to groups.
principal
Alsmyr says most labs start with all-purpose
investigators to lab managers,” says Erik Alsmyr, senior director organizing and
fied prior to further biochemical examination.”
In the early 1980s, for example, I worked in a cell-culture lab,
and we made most of what we needed, including materials like
In fact, getting adequately pure protein for downstream tech-
niques can be challenging. “Many protein biochemists have
The Paperless Lab
Some scientists keep experimental records on sticky notes.
Some groups maintain ordering information in the head of a
single technician. But for researchers looking for more sta-
ofsharing
software
Contur’s
(IP)
software
ize theyiLabber)
development
need more
protection.
such asfor
for storage
Electronic
Evernote
the Accelrys or SharePoint,
Notebookthen
capacity or intellectual
small-to-medium-sized
systems provide
research
24/7
real-
(previously
property
global
groups.
access
rat-tail collagen to coat the coverslips on which the cells grew. to contend with frustrating obstacles, including protein yield, Alsmyr says most labs start with all-purpose organizing and
ble, searchable, and sharable records,
Some scientists keep experimental records on sticky notes. digital options such as sharing software such as Evernote or SharePoint, then real-are
to your records, says Alsmyr, and most commercial ELNs
Today, scientists can purchase a wide variety of media and solubility, and degradation issues,” Spratt says. “Speaking from
electronic laboratory notebooks (ELNs)
Some groups maintain ordering information in the head of a and laboratory infor- compliant
ize they need with
more regulatory
storagerequirements for electronic
capacity or intellectual records,
property
reagents as well as labware designed for specific culture tech- personal experience, it can take many attempts to overcome
mation management systems (LIMS) are
single technician. But for researchers looking for more sta- readily available. for example Part 11 of the Code of
(IP) protection. Electronic systems provide 24/7 global accessFederal Regulations Title 21,
niques, such as 3D culture. these challenges.”
Scientists can start with a simple online notebook
ble, searchable, and sharable records, digital options such as or choose which covers the U.S. Food and
to your records, says Alsmyr, and most commercial ELNs areDrug Administration, and Euro-
Still, some of the key skills remain the same. “The most impor- That brings up perhaps the most crucial lab skills of all: patience
a complete lab management package to
electronic laboratory notebooks (ELNs) and laboratory infor- track the entire life- pean Union Annex 11 for the European
compliant with regulatory requirements for electronic records, market.
tant aspect of tissue culture is good sterile technique,” says Katy and persistence.
cycle of
mation their projects.
management By Chris
systems (LIMS)Tachibana
are readily available. Researchers
for example Part 11 areofstilltheslowCode adopters,
of Federal though, particularly
Regulations Title at
21,
Phelan, director of the cytogenetics laboratory at Florida Can-

A
universities. That’s why LabArchives offers a free ELN in Euro-
ad-
cer Specialists & Research Institute (Fort Myers, Florida). “This Writing up the results Scientists can start with a simple online notebook or choose which covers the U.S. Food and Drug Administration, and
a complete paperlab notebook
management seems like it should
package to track last
theforever.
entire After
life- dition
pean to a subscription-based
Union Annex 11 for the European version market.
with more storage and
applies to initial setup of cultures as well as feeding, subcultur- Once those skills pay off, it’s time to write. Scientists who have
all, Gutenberg Bibles
cycle of their projects. By Chris Tachibana have survived since the 1400s. features. “Our research says that in
Researchers are still slow adopters, though, particularly academia, about 95%atof
ing, and cryopreservation.” This means that everything—culture been thinking about writing all along can get a head start by using
Still, paper is not perfect. Consider these true stories: universities. That’s why LabArchives offers a freeBeutler,
scientists still use a paper notebook,” says Earl ELN in LabAr-

A
media and additives, pipettes, culture vessels, and other equip- an electronic lab notebook to keep track of protocols and results. ad-
At an Australian university, 30 years of notebooks
paper notebook seems like it should last forever. After became a chives’ chief executive officer. Beutler,
dition to a subscription-based version with more storage whose entire familyandare
ment—must be kept sterile and tested to confirm sterility. “Prac- At the very least, they can cut and paste methods and results to
pile of loose pages after the have
bindings crumbled
sinceduring relo- scientists (including a Nobel Prize in winner), thinks it’s95%
time of for
ticing good sterile technique will reduce the chance that cul- get started on an article. all, Gutenberg Bibles survived the 1400s. features. “Our research says that academia, about
cation. Still,
In the United States, a postdoc spent days combing labs to go digital.
use a“I’ve worked around smart,Earltechnologically
tures will become contaminated,” Phelan explains. “Valuable cell Beyond collecting all the information, more challenges arise in paper is not perfect. Consider these true stories: scientists still paper notebook,” says Beutler, LabAr-
lines can be lost or compromised due to failure to practice good knowing how to describe the work. For even seasoned writers, Atthrough three-ring
an Australian binders30
university, foryears
experimental
of notebooks details requested
became a proficient
chives’ chief scientists
executive my entireBeutler,
officer. life,” hewhose
says, “and entireI’m amazed
family are
by reviewers. In a positive example of going paperless, a Swiss scientists (including a Nobel Prize winner), thinks it’s timeprinting
that their state-of-the-art is still taking a photo of a gel,
sterile technique.” In fact, keeping cultures contamination-free it’s worth reading “The Science of Scientific Writing” by writing pile of loose pages after the bindings crumbled during relo- for
consultant George Gopen and Judith Swan, associate director for contract manufacturing organization
a postdocwowed clients with real- it out, anddigital.
gluing“I’ve it into a paper notebook.”
is one of the biggest challenges of this general method. cation. In the United States, spent days combing labs to go worked around smart, technologically
writing in science and engineering at Princeton University (Ameri- time, online chromatography runs of their samples. Electronic Realizing that adhesives disintegrate and notesI’m on laptops
Plus, it’s crucial to ensure that a culture includes only what is through three-ring binders for experimental details requested proficient scientists my entire life,” he says, “and amazed
can Scientist, November–December 1990). As they concluded, “In laboratory tools have definite advantages, but scientists have don’t have the strongest IP protection, universities are buying
intended. “A common mistake in cell culture is sample mix-up by reviewers. In a positive example of going paperless, a Swiss that their state-of-the-art is still taking a photo of a gel, printing
or cross-contamination of samples,” Phelan explains. “Various real and important ways, the structure of the prose becomes the been reluctant adopters. The major barriers
contract manufacturing organization wowed clients with real- for going digital are informatics site licenses that
it out, and gluing it into a paper notebook.” cover entire departments, says
techniques can be employed in an attempt to prevent this error, structure of the scientific argument.” cost,online
time, the activation energy required
chromatography to change
runs of their samples. work habits, and
Electronic Beutler.
RealizingThisthatremoves
adhesives the cost barrier for
disintegrate and scientists
notes onand ensures
laptops
such as working with only one sample at a time in the tissue cul- To build the best structure, make an outline or develop the daunting
laboratory tools number of options.
have definite advantages, but scientists have proper
don’t have archiving
the strongestof potentially patentable
IP protection, results. LabArchives
universities are buying

PHOTO: PAVEL IGNATOV/SHUTTERSTOCK.COM

ture hood, avoiding the use of prelabeled flasks or petri dishes, some organization before writing begins. It doesn’t need to be been reluctant adopters. The major barriers for going digital are also targets
informatics anlicenses
site audience thatthat doesn’t
cover entirehave paper nostalgia:
departments, says
and double-checking two unique identifiers on all paperwork a formal system of Roman numerals or capital letters, but just Where to Start
cost, the activation energy required to change work habits, and students. “Many of our users
Beutler. This removes the cost barrier for scientistsare academic researchers who
and ensures
and culture vessels.” something that works for the writer. A research article comes LIMSwiki is an excellent
the daunting number of options. starting point for laboratory infor- teach, so we created our classroom
proper archiving of potentially patentable results. LabArchives ELN at their request,” says
with an overall organization, including introduction, methods, matics newbies. The online resource is a community service also targets an audience that doesn’t have paper nostalgia: and
Beutler. “It lets instructors provide background information
PHOTO: © ABSCENT/SHUTTERSTOCK.COM

Increasingly, scientists must ensure the integrity of cultures.

PHOTO: PAVEL IGNATOV/SHUTTERSTOCK.COM

“In a research lab, a sample mix-up can lead to false and discussion, and conclusion. So it’s worth making time to organize from the
Where to StartLaboratory Informatics Institute, a trade organization give and “Many
students. grade assignments
of our users are electronically. The largest who
academic researchers class it’s
unreliable results,” Phelan notes. In a diagnostics lab, however, topics within each section. In short, know what you want to write founded
LIMSwiki in 2006
is an by LabLynx,
excellent a vendor
starting pointofforbrowser-based
laboratory infor- re- been so
teach, used weincreated
was more our than 2,000 ELN
classroom students.”
at their request,” says
such an error could be deadly for a patient. Many journals before you write it. searchnewbies.
matics management software.
The online LabLynx
resource emphasizesservice
is a community transpar- Tammy
Beutler. Morrish
“It lets is an academic
instructors researcher information
provide background who went digital and
require that researchers authenticate cell lines used, and this Writing and the other techniques described here take time and ency, for example in pricing, and LIMSwiki
from the Laboratory Informatics Institute, a trade organizationprovides prices when from day one, setting up her laboratory
give and grade assignments electronically. The largest class with Labguru, a web-it’s
can be done using DNA fingerprinting. The American Type practice. Also, these scientific skills should be refreshed as need- possibleinin2006
founded its up-to-date
by LabLynx, vendor descriptions.
a vendor “We’ve tried
of browser-based re- to based
been usedresearch
in wasmanagement
more than 2,000 system. As a postdoc, Morrish
students.”
Culture Collection (ATCC), says Phelan, “actually provides a ed. Only then can scientists produce their best work. search management software. LabLynx emphasizes transpar- Tammy Morrish is an academic researcher who went digital
service for human cell authentication and has an online course ency, for example in pricing, and LIMSwiki provides prices when from day one, setting up her laboratory with Labguru, a web-
called Cell Line Authentication Training.” Mike May is a publishing consultant for science and technology. possible in its up-to-date vendor descriptions. “We’ve Originallytried to
published based
25 research
July 2014 management system. As a postdoc, Morrish
in SCIENCE

6 468 sciencemag.org/products SCIENCE

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LIFELIFE
SCIENCE TECHNOLOGIES
SCIENCE TECHNOLOGIES
Y OLAB
DIGITAL U R PR
DIGITAL A C TMANAGEMENT
I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
MANAGEMENT
LAB FE AT UR E ART I C L E S

kept a kepthomemadea homemade database of

database ministration
of encourage
ministration encourageelectronic documentation,
electronic documentation, Dathe says,Dathe says, of scientific informatics Featured Participants Santa Cruz has traveled
projectproject
resources but wanted
resources but wanted“The pharmaceutical
“The pharmaceutical industry is generally
industry conservative,
is generally and it’sand it’s
conservative, and better data integra- Accelrys University of Toledo the entire DIY lab notebook
an advanced,
an advanced, sharable system
sharable system often easier and cheaper
often easier and cheaperto stayto with
stay a with
papera system
paper system that is that is tion, says Elliott, “is www.accelrys.com www.utoledo.edu journey. Boettiger started
when she when started as an as
she started assis-
an assis-knownknown to be accepted
to be accepted by regulatory agencies.”
by regulatory agencies.” the move toward more Atrium Research & Consulting keeping publicly accessible
tant professor at the at
tant professor Universi-
the UniversityAt LEOAtPharma LEO Pharma in Denmark, in Denmark,head ofhead discovery informatics
of discovery informatics collaborative work.” www.atriumresearch.com
Additional Resources lab records in the Open-
ty of Toledo
of Toledo Biochemistry
Biochemistry andandand data andmanagement
data management Ulrik Nicolai de Lichtenberg
Ulrik Nicolai de Lichtenberg developed developed To the wish list of LabArchives WetWare platform. “It’s a
Core Informatics
CancerCancer BiologyBiology
Department.Department.a model for committing
a model for committing to a commercial
to a commercial informatics system.
informatics system. informatics improvements, www.labarchives.com bit radical,” says Boettiger.
www.corelims.com
That’s That’s
a greatatime greattotimeset up
to set upStart with Start in-depth stakeholder
with in-depth analyses,
stakeholder he says.
analyses, heDefine your your
says. Define Dathe adds features that Labguru “Anyone can go in and
Evernote
a new a system,
new system,she says, shebe-
says, be-needs needs and goals and andgoals “how
andmuch “how pain muchyou pain canyou putcanupput with,”
up with,” give data context: when www.labguru.com edit other peoples’ notes,
www.evernote.com
cause causeyou know you all
know the all
mice,
the mice, meaning the money,
meaning time, and
the money, time, effort
andavailable for implementing
effort available for implementing and where they were LabKey Software although that rarely hap-
GitHub
cell lines,celland
lines, plasmids
and plasmidsyou youa new a system.
new system. RealizeRealize
that your thatELN yourorELN LIMS orisLIMS
just ais part
just of an of an
a part collected and for what www.labkey.com pens.” After OpenWetWare,
www.github.com
have available for projects.
have available for projects. information ecosystem.
information ecosystem.LEO Pharma LEO Pharma chose chose the Accelrys
the AccelrysELN ELN project. Data should be Boettiger moved to plat-
In this
In[nearly
this [nearly sci-fi]sci-fi] visionvision MorrishMorrish praisespraises
Labguru’s Labguru’s for its Medicinal
for its MedicinalChemistry R&D Department,
Chemistry R&D Department, but thebut ELN theisELN is linked to relevant molecular
LabLynx
www.lablynx.com
IDBS
forms that give him increas-
www.idbs.com
of theoffuture
the future laboratory, laboratory,customer serviceservice
customer and says and says just one element
just in a comprehensive
one element in a comprehensive infrastructure designed
infrastructure by
designed by and clinical information and LIMSwiki
Jekyll
ing control over his research
the system is a huge
the system is atime-
huge time- de Lichtenberg’s
de Lichtenberg’s team. Theirteam.systemTheir systemwill capture, validate,
will capture, and and
validate, the entire data-generating www.limswiki.org records, starting with Word-
scientists simply
www.jekyllrb.com
scientists simply saver. Itsaver. streamlines ordering
It streamlines orderingpermanentlypermanentlystore records
store recordsso theysoare theyaccessible,
are accessible,searchable,searchable, process, including the type LEO Pharma Press, which is usually used
OpenWetWare
do their workwork
do their whilewhile an an by putting
by product
putting numbers,
product numbers, and legally
and defensible
legally in
defensible case inof IP
case disputes.
of IP It’s
disputes. a complex
It’s a complex and status of equipment www.leo-pharma.com
www.openwetware.org for blogging. Boettiger now
vendors, and current
vendors, orders orders
and current in project
in and deand
project Lichtenberg
de Lichtenberg recommends recommends seeking adviceadvice
seeking from from used. “Without context,” Roche uses the online software
automated
automated tracking tracking one place, she says.
one place, sheLabguru
says. Labguru independent
independentconsultants who understand
consultants who understand the ever-changing
the ever-changing says Dathe, “the mountain www.roche.com
PerkinElmer
www.perkinelmer.com development site GitHub as
system simultaneously
system simultaneouslyholds her holdslaboratory’s mousemouse
her laboratory’s informatics market.market.
informatics of data we can collect is University of California,
Santa Cruz
Thermo Fisher Scientific
his note-book and Jekyll
recordsrecordswith fullwithgenotypes,
full genotypes, meaningless.” www.thermofisher.com website-generating soft-
keepskeeps records. records. and plasmid information
and plasmid in-
information in- Looking Lookingto theto Cloud
the CloudAnd Beyond And Beyond
www.ucsc.edu
ware to publish his note-
cludingcluding
maps. maps.MorrishMorrish
says says Michael Elliott, Elliott,
Michael chief executive
chief executive of Atrium
officer officer Research
of Atrium Research Being Open-Minded book online.
the system is particularly
the system helpfulhelpful
is particularly for locating items. items.
for locating “Think “Think
how how & Consulting,
& Consulting, advised de Lichtenberg
advised de Lichtenberg and endorsesand endorses his ap-his ap- Scaling the data mountain is Britt Piehler’s job. Piehler is A blog-type ELN creates a robust, cached history of your
much time
muchwe waste
time we looking
waste lookingfor things,” she says.
for things,” she“Now
says. when“Now when proach.proach.
“Don’t “Don’t
get enamored
get enamored with a with demo,” he says.
a demo,” he“Look
says. “Look president of LabKey Software, which develops tools for data research, says Boettiger. It discourages fraud because any
I need Isomething,
need something, even ifevenotherifpeople
other peoplearen’t around to ask,to
aren’t around I ask, I under the under hoodtheandhood check out theout
and check capabilities
the capabilitiesof an informatics
of an informatics management and integration. The trend toward globalization changes leave records. You choose what is public, private,
can typecanit type
into theit intodatabase and find
the database andit. find
Of course,” she adds,
it. Of course,” she adds,system.” ClientsClients
system.” dream dream of a single of a system
single system that streamlines
that streamlines pro- pro- and multisite collaboration, he says, means project managers and password protected. And think of the advantages when
“people have tohave
“people put things back where
to put things they found
back where they them.”
found them.”Her Hercess managementcess management and securely
and securelyand permanently
and permanently stores stores
data data must coordinate data collected at far-flung sites under diverse talking to people at conferences or answering reviewer
lab haslaba technician
has a technician who checkswho checks inventories againstagainst
inventories the data- the data-while rapidly retrieving
while rapidly neededneeded
retrieving information. An ideal
information. Ansystem
ideal system conditions with a variety of instruments. “That’s where requests, he says. You can just pull up records on a handheld
base weekly.
base weekly. would would
even find even “dark
finddata”—previous
“dark data”—previous work that work could
that answer
could answer LabKey comes in,” says Piehler. “We build tools for specific device to see what you tried and when, and how it worked out.
At a higher level, the
At a higher system
level, facilitates
the system group interactions,
facilitates group interactions, currentcurrent research questions
research but is buried
questions in disorganized
but is buried in disorganizedfiles. files. tasks, usually data integration for multisite collaborative
for example by making
for example data sharing
by making data sharingeasy. Iteasy.
also It teaches best bestClientsClients
also teaches want scalability, a user-friendly
want scalability, interface,
a user-friendly and outstand-
interface, and outstand- projects that need to standardize heterogeneous data.” An What’s Next
practices. “It helps
practices. “Itstudents
helps studentslearn that learn with
thatanywith database,”
any database,”says says ing global support.
ing global However,
support. products
However, vary invary
products these in capabilities,
these capabilities, unusual feature of LabKey Software is that its product is open “The trends in laboratory records,” says Boettiger, “are
Morrish, “you have
Morrish, “youtohave entertoinformation
enter information correctly and consis-
correctly and consis- says Elliott. “Don’t “Don’t
says Elliott. choosechoose based based on a presentation
on a presentation or brand or brand source. toward more open and collaborative, more secure, and
tently or you or
tently won’t
you be ablebetoable
won’t find to it.”find it.” name. name.
Think carefully about your
Think carefully aboutneeds now and
your needs now in and
the future.”
in the future.” “We grew out of the academic community,” says LabKey’s more automated.” Although Boettiger and Dathe should
If expandability
If expandabilityand ease andofease use of areuse priorities, a cloud-
are priorities, a cloud- Science Outreach Director Elizabeth Nelson, “so we believe have different perspectives as an ecology researcher in
GoingGoingDigitalDigital But Maintaining
But Maintaining Control Control based based
system, for example
system, from Core
for example fromInformatics,
Core Informatics, might might it’s an advantage for the software platform to be freely Santa Cruz and a pharma development and information
Science-based
Science-based businessesbusinessesalso appreciate
also appreciatethe efficiency of
the efficiency be the be
of answer. In principle,
the answer. the cloud
In principle, the can
cloud house unlimited
can house unlimited available.” Open source code allows researchers to tailor their technology specialist in Basel, respectively, they share a nearly
digital digital
research management,
research management, but long-term
but long-termstability is a high
stability is a highamounts of dataofand
amounts data hasand a familiar interface
has a familiar since accessed
interface since accessed is is systems, says Piehler, and building and sharing LabKey tools sci-fi vision of the future laboratory. In this vision, scientists
priority,priority,
too. “The too.challenge
“The challengeis assuring the accessibility
is assuring and and through
the accessibility a web abrowser.
through web browser. Brower-based
Brower-based systems don’t require
systems don’t require creates a community. simply do their work while an automated tracking system
usability of dataof20data
usability years 20 from
yearsnow,”from says now,”Dathe. Choosing
says Dathe. Choosing specialized software,
specialized so they’re
software, easy toeasy
so they’re upgrade. Informatics
to upgrade. Informatics If the code is free, what does LabKey offer? simultaneously keeps records. Barcoding will note reagents,
a majora informatics
major informatics supplier such as
supplier IDBS,
such as PerkinElmer,
IDBS, PerkinElmer, or or vendors are also
vendors are creating user-friendly
also creating modular
user-friendly packages.
modular packages. “Customization,” says Piehler. LabKey Software experts samples, and instruments used, providing context to the data
Accelrys might give
Accelrys might some
give assurance
some assurance of permanence,
of permanence, but but SimilarSimilar
to choosing mobilemobile
to choosing phone phone apps, users apps, select only the
users select only the can create tools that directly address Dathe’s call for for subsequent analysis. The entire process will be recorded,
the market is so dynamic
the market that any
is so dynamic that vendor will likely
any vendor willundergo
likely undergo components components they need.they need. giving context to data, for example by adding demographic showing the provenance of every byte and definitively
changes. In the past
changes. In thedecades,
past decades,Thermo Fisher Fisher
Thermo ScientificScientific Also onAlso the on horizon is greater
the horizon mobility
is greater and compatibility.
mobility and compatibility. information. And in August 2013, open source and open establishing IP claims. “It will give a much more extensive
acquired InnaPhase;
acquired PerkinElmer
InnaPhase; PerkinElmer purchased Labtronics,
purchased Labtronics, Researchers
Researchersare taking smartphones
are taking smartphones and tablets into theinto the
and tablets access came together via LabKey to promote scientific record that can be transparent or shared if you want,” says
CambridgeSoft
CambridgeSoft and ArtusLabs;
and ArtusLabs;Accelrys, which has
Accelrys, which itshas its laboratory so informatics
laboratory so informaticsdevelopers are making
developers products
are making products transparency and reproducibility. For a clinical trial of a Boettiger. A fully automated system would simplify research
PHOTO: BRUCE ROLFF/SHUTTERSTOCK.COM

PHOTO: BRUCE ROLFF/SHUTTERSTOCK.COM

own lengthy
own lengthymergermerger and acquisition
and acquisition history,history,
was recently
was recently compatible with handheld
compatible with handheld devices. Increasingly,
devices. data needs
Increasingly, data needs vasculitis therapy published in the New England Journal of by capturing experimental details with no manual data entry.
acquired by the by
acquired French software
the French company
software Dassault.
company Still, after
Dassault. Still, afterto be compiled
to be compiled acrossacross different instruments
different instrumentsand informatics
and informatics Medicine, the LabKey open source platform was used to Then, all we’d need is a robot to return reagents to the right
consolidating,
consolidating, companies strive to
companies retain
strive to users. “We still
retain users. “Wecarry
still carryplatforms, so Pedersen
platforms, so Pedersen says he is personally
says he is personally pushing pushing create a web portal with free public access to participant-level shelves.
software developed
software developedin the 1990s
in the 1990sand we’ve and always
we’ve alwaysshownshown for increased for increased standardization
standardization to facilitate information
to facilitate information data, stripped of identifying information.
customers a path aforward,”
customers says Leif
path forward,” says Pedersen,
Leif Pedersen, senior senior
vice vice sharing. Ever the
sharing. realist,
Ever though,
the realist, Elliott says
though, Elliottprogress
says progress Researchers who are committed to transparency and are
president at Accelrys.
president at Accelrys. in standardization
in standardization is slowisbecauseslow because even within a single
even within a single also do-it-yourselfers have a choice of open source workflow Chris Tachibana is a science writer based in Seattle, USA, and
Nonetheless,
Nonetheless, industries are notare
industries uniformly adopting
not uniformly laboratory
adopting laboratorydepartment,
department, users might
users employ
might employ different terminology
different terminology and and management tools. Carl Boettiger, an ecology and evolu- Copenhagen, Denmark.
informatics. Although
informatics. agencies
Although such as
agencies the as
such FoodtheandFood Drug
andAd-Drug Ad- definitions. The force
definitions. The that forcecould drive both
that could drivestandardization
both standardization tion postdoctoral researcher at the University of California, DOI: 10.1126/science.opms.p1400087

Originally published 25 July 2014 in SCIENCE Originally published 25 July 2014 in SCIENCE

8 sciencemag.org/products
SCIENCE
SCIENCE sciencemag.org/products 469 469 470 9
sciencemag.org/products SCIENCE
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
Introduction
Preventing contamination in pipetting is paramount to
achieving reliable results. It requires identification of the
potential contamination mechanisms in order that they
can all be addressed.
Aerosols, suspensions of solid or liquid particles in a
How to
HowAvoid
to Avoid Contamination in Pipetting
gas, are formed in many laboratory activities such as
pipetting with air-displacement pipettes, and aerosols
Contamination in Pipetting are the major contamination source in pipetting. They
may transfer into the pipette body when non-filter
#05 pipette tips are used and consequently contaminate
subsequent samples. A slow and careful pipetting
rhythm helps minimize aerosol formation.
Application #01
This paper addresses the three contamination types
Note that originate from pipetting: pipette-to-sample
contamination, sample-to-pipette contamination, and
Practical methods #02 sample-to-sample contamination.
for avoiding
contamination in
Pipette-to-Sample Contamination
pipetting. #03 This type of contamination occurs when a contaminated
pipette or pipette tip contaminates the sample.
#04 Pipette tips are available in multiple purity grades from
most manufacturers. Purity grades can be divided into
three categories:
– no purity certification
– certified free of contaminants like DNase, RNase,
and endotoxins
– sterilized to be free of microbial life
Contaminants such as DNase, RNase, and endotoxins
are difficult to remove by any sterilization method, so
it is very important to prevent contamination during
manufacturing. The absence of these contaminants is
separately tested, usually by a third-party laboratory.
Sterilization after manufacturing ensures that the tips
do not contain any microbial life (bacteria, viruses, etc.)
when delivered to customers.
Pipette tips can also be a potential source of leachables
– trace amounts of chemicals originating from materials
or process equipment that can contaminate the
samples. Examples of potential leachables are – Always change the pipette tip after each sample.
heavy metals, UV stabilizers, antioxidants, pigments,
release agents, biocides, and surfactants. High-quality
10
AP P L I C AT I O N NO T E S
tips manufactured from 100% virgin polypropylene in
a high-quality manufacturing facility do not contain
leachables. It is recommended that you confirm this with
the tip manufacturer.
In daily laboratory work, pipette-to-sample contam-
ination can be avoided by following these simple
guidelines:
– Select a tip with the relevant purity class for your
application.
– Use (sterilized) filter tips or positive displacement
tips. Alternatively, you may be able to use tip-cone
filters with some manufacturers’ pipettes. The filters
prevent aerosols from reaching the pipette body and
potentially contaminating subsequent samples.
– Always change the pipette tip after each sample.
– Regularly autoclave, or disinfect, the pipette or
the components that may come into contact with
the sample.
Sample-to-Pipette Contamination
This type of contamination takes place when the
pipetted liquid or aerosol particles from it enter the
pipette body. To minimize the risk of sample-to-
pipette contamination, the following precautions are
recommended:
– A lways release the pipette’s push button slowly to
prevent aerosol formation and uncontrolled liquid
splashing within the pipette tip.
– Hold the pipette in a vertical position during pipetting
and store the pipette in an upright position. This
prevents liquids from running into the pipette body.
Sample-to-Sample Contamination
Sample-to-sample contamination (or carryover
contamination) occurs when aerosol or liquid residue
from one sample is carried over to the next sample.
This may take place, for example, when the same
pipette tips are used multiple times. To avoid carryover
contamination:
– Use filter tips or positive displacement tips to prevent
aerosol transfer from the sample into the pipette
body, and again to the next sample. Alternatively,
filters can be used on pipette tip cones.
– If you suspect pipette contamination, autoclave or
disinfect the pipette according to the manufacturer’s
instructions.
11
 Definitions:
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Decontamination Any activity that reduces microbial load to Antisepsis The application of an antimicrobial
Definitions: prevent contamination. Includes methods chemical to living tissue to destroy
for sterilization, disinfection, and antisepsis. microorganisms.
Decontamination that reduces microbial The
Any activity Sterilization loaddestruction
to of all microbialThe
Antisepsis life,application ofDNase
an antimicrobial Powerful enzymes (nucleases) that degrade
including bacterial endospores.chemical
prevent contamination. Includes methods Can be to living tissue to destroy DNA by hydrolyzing it into short fragments.
for sterilization, disinfection, and antisepsis.
accomplished, e.g., using steam,microorganisms.
heating, Even trace amounts of DNases can lead to
Sterilization The destruction of all microbial life,chemicals, or radiation.
DNase Powerful enzymes (nucleases) that degrade low or no yields in DNA techniques such as
including bacterial endospores. Can Autoclaving
be DNA by hydrolyzing it into short fragments. PCR, or to degradation during DNA
Autoclaving (moist heat) is an efficient
purification. Contamination sources: human
Concentration toinaPipetting
Defined Final Volume
accomplished, e.g., using steam, heating, Even trace
sterilization method for laboratories. A hot, amounts of DNases can lead to
chemicals, or radiation. low or no
pressurized, and saturated steam is applied yields in DNA techniques such contact,
as saliva, bacteria. How to Avoid Contamination
PCR, or to degradation during DNA
Autoclaving to destroy microorganisms and
Autoclaving (moist heat) is an efficient RNase Powerful enzymes (nucleases) that catalyze
sterilization method for laboratories.decontaminate,
A hot,
pressurized, and saturated steam is applied
purification. Contamination sources: human
e.g., laboratorycontact,
plastic and
glassware. Exposure time and temperature
saliva, bacteria.
the degradation of RNA into short
fragments. Very stable enzymes that are
with Vivaspin® Turbo 15, Vivaspin® Turbo 4
#05
to destroy microorganisms and are critical.RNase
decontaminate, e.g., laboratory plastic
glassware. Exposure time and temperature
and
penetrate
Moreover, the steam Powerful
needs to
the to
through the entire load
enzymes (nucleases) that catalyze
degradation
be of RNA into short
difficult to remove. Contamination sources:
oils from skin, as well as hair, tears,
fragments. Very stable enzymes that are bacteria.
and Vivaspin® 500
efficient.
are critical. Moreover, the steam needs to difficult to remove. Contamination sources: Application #01
Disinfection
penetrate through the entire load toThe
be elimination of virtually all pathogenic Endotoxins
oils from skin, as well as hair, tears, Lipopolysaccharides, large molecules that
efficient. microorganisms (excluding bacterial
bacteria. are part of the outer membrane of Rik McRae1, Hannes Landmann2,* Note
Disinfection endospores)Endotoxins
The elimination of virtually all pathogenic and reduction of the microbial
Lipopolysaccharides, large molecules thatGram-negative bacteria such as E. coli,
microorganisms (excluding bacterial contamination to an acceptable level.
are part of the outer membrane of Salmonella, Shigella, Pseudomonas, and 1. Sartorius Stedim Lab Ltd, Sperryway, Stonehouse, Gloucestershire, GL10 methods
Practical 3UT, UK #02
A practical
endospores) and reduction of the microbial method for surface Gram-negative bacteria such as E. coli, Haemophilus . Cause fever in humans and
2. Sartorius Lab Instruments GmbH & Co. KG, for
Otto-Brenner-Straße avoiding
20, 37079 Göttingen, Germany
contamination to an acceptable level.decontamination. The disinfectant (e.g., , Shigella, Pseudomonas, and impair the growth of cell cultures. Are * Correspondence
Salmonella contamination in
A practical method for surface halogens), . Cause fever in humans andreleased into the environment when E-Mail: hannes.landmann@sartorius.com
alcohols, phenolic compounds, Haemophilus
concentration, and exposure time
decontamination. The disinfectant (e.g., should
impair be
the growth of cell cultures. Are bacteria die and the cell wall is destroyed.
pipetting. #03
alcohols, phenolic compounds, halogens), released into the environment when
selected according to the assumed Contamination sources: Endotoxins are
concentration, and exposure time should be
contamination type. bacteria die and the cell wall is destroyed.present wherever bacteria are able to grow,
selected according to the assumed Contamination sources: Endotoxins are i.e., air, water, soil, skin, raw materials, any #04
contamination type. present wherever bacteria are able to grow, non-sterile environment.
i.e., air, water, soil, skin, raw materials, any
non-sterile environment.
Abstract
This short Application Note describes how you can use Vivaspin® Turbo 15, Vivaspin®

Specifications subject to change without notice. Printed and copyrighted by Sartorius Biohit Liquid Handling Oy.
Turbo 4 and Vivaspin® 500 concentrators to concentrate to defined final volumes. By

Specifications subject to change without notice. Printed and copyrighted by Sartorius Biohit Liquid Handling Oy.
adding a particular volume to the filtrate vessel prior to the concentration, the final
volume of the concentrate can be adjusted accurately.

Publication No.: SUL1002-e 150601 ∙ Order No.: 85037-550-68∙ Ver. 06| 2015
Publication No.: SUL1002-e 150601 ∙ Order No.: 85037-550-68∙ Ver. 06| 2015

Sartorius Lab Instruments GmbH & Co. KG

Sartorius Lab Instruments GmbH & Co. KG
Weender Landstrasse 94-108 Weender Landstrasse 94-108
37075 Goettingen, Germany 37075 Goettingen, Germany
Phone +49.551.3080 Phone +49.551.3080
Fax +49.551.308.3289 Fax +49.551.308.3289

Sartorius Biohit Liquid Handling Oy Sartorius Biohit Liquid Handling Oy

Laippatie 1 Laippatie 1
00880 Helsinki, Finland 00880 Helsinki, Finland
Phone +358.9.755.951 Phone +358.9.755.951
Fax +358.9.755.95.220 Fax +358.9.755.95.220
www.sartorius.com www.sartorius.com
12
13
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Introduction
It is sometimes desirable to be able to preselect a defined final volume for a concentration step, especially when
parallel concentrations are being performed. Vivaspin ® centrifugal concentrators have a built-in deadstop feature,
which prevents overconcentration to dryness. Due to the fast concentration rates possible with the
patented vertical membrane design in the Vivaspin ®, the drying out of the sample would otherwise be a possibility.
Introduction
This note describes a method for achieving reproducible
– Sartorius Precision Lab Balance
defined® D-16C
– Centrisart final volumes using
Centrifuge with Vivaspin
swing out rotor Turbo 15, Vivaspin ®
® for 50 ml Cell Culture Expansion in Fully Closed
How Erlenmeyer Shake Flasks Outside the
It is sometimes desirable to ®be able to preselect a defined final and 15 ml falcon tubes
Turbo 4 and Vivaspin 500 centrifugal concentrators. The method does not rely on the deadstop pocket but is
volume for a concentration step, especially when parallel concen- – Centrisart A-14C Centrifuge with fixed angle rotor
increasing the performed.
trations are being retained Vivaspin
volume ® by adding liquid to the filtrate vessel prior to centrifugation.
centrifugal concentrators for 24 1.5 | 2.2 ml tubes to Avoid Contamination in Pipetting
Biosafety Cabinet with MYCAP™ CCX
have a built-in deadstop feature, which prevents overconcentration
Equipment
to dryness. Due to the fast concentration rates possible with the Reagents
Reagents
–patented
Vivaspinvertical
® membrane
Turbo 15 10 design the Vivaspin®, the drying
kDainMWCO 1 mg/ml Bovine Serum Albumin labelled with Bromophenol blue
1 mg/mL Bovine Serum Albumin labeled with
out of the sample would otherwise be a possibility.
– Vivaspin Turbo 4 10 kDa MWCO
®
Bromophenol blue
#05
–This
Vivaspin 500a 10
note describes
®
kDafor
method MWCO
achieving reproducible defined Methods Charles Meadows1* (Senior Product Manager), Gregory Bremer1 (Upstream Engineer,
final volumes using Vivaspin® Turbo 15, Vivaspin® Turbo 4 and
– Tacta® 5 mL mechanical pipette and Optifit pipette tips Methods Research & Development), Dr. Michael Zumbrum1 Application
(Director of Research & Development,
Vivaspin 500 centrifugal concentrators. The method does not
–rely
Tacta
on the1000 μL mechanical
deadstop pipette the
pocket but is increasing andretained
Optifitvolume
1. Add defined amount of water to the filtrate tube (see table
pipette tips 1. Add defined amount of water to the filtrate tube (see New Oxford)
#01
by adding liquid to the filtrate vessel prior to centrifugation.
– Tacta 200 μL mechanical pipette and Optifit pipette tips
below).
table
2. Put the below).
concentrator insert into the filtrate tube
Note
and 2. Put thesolution.
add sample concentrator insert into the filtrate tube and 1. Sartorius Stedim Biotech GmbH, August-Spindler-Strasse 11, 37079 Goettingen
-Equipment
arium pro ultrapure water system
®
3. Close the concentrator cap (for Vivaspin® Turbo 15 or Practical methods #02
® add sample screw
solution. *
Correspondence
– Vivaspin
– Sartorius Turbo 15 10kDa MWCO
Precision Lab Balance Vivaspin Turbo 4) or close the cap (Vivaspin® 500) and place
®
for avoiding
– Vivaspin® Turbo 4 10kDa MWCO in the3.centrifuge.
Close the concentrator screw cap (for Vivaspin ® Turbo E-Mail: charles.meadows@sartorius-stedim.com
–– Vivaspin
Centrisart ®
D-16C Centrifuge with swing-out rotor for 15 or contamination in
theVivaspin
sample. ® Turbo 4) or close the cap (Vivaspin ®
®
500 10kDa MWCO 4. Concentrate
– Tacta
50 mL5 mland 15 mL pipette
mechanical falconandtubes
Optifit pipette tips
– Tacta 1000 µl mechanical pipette and Optifit pipette tips
5. Remove the concentrator
500) and placeinsert andcentrifuge.
in the recover the concentrate pipetting. #03
–– Tacta
Centrisart A-14C Centrifuge with fixed-angle rotor with a pipette.
200 µl mechanical pipette and Optifit pipette tips 4. Concentrate the sample.
- arium
for 24
®
1.5ultrapure
pro | 2.2 mL tubes
water system 5. Remove the concentrator insert and recover the
concentrate with a pipette. Abstract #04
Results
Results
Expansion of suspension cell culture from cell banks to seed bioreactor is performed
Results for Vivaspin® Turbo 15
Volume of water added Volume of sample solution added Spin conditions Final concentrate volume
through passages of successively larger Erlenmeyer shake flasks. The traditional cap of an
to the filtrate tube to the concentrator insert (average of 8 devices) Erlenmeyer flask is unscrewed for each fluid transfer. Risk of contamination is mitigated by
11.5 mL 15 mL 20 min @ 4,000 x g 1.50 ± 0.02 mL performing these fluid transfers in a biosafety cabinet (BSC) or laminar flow hood.
9.5 mL 15 mL 20 min @ 4,000 x g 0.96 ± 0.01 mL
7.5 mL 15 mL 20 min @ 4,000 x g 0.53 ± 0.02 mL
Work in a BSC is not preferred because of high maintenance and operating costs, intensive
®
Results for Vivaspin Turbo 4 cleaning and decontamination procedures, and the risk and inconvenience of performing
Volume of water added Volume of sample solution added Spin conditions Final concentrate volume operations in the BSC.
to the filtrate tube to the concentrator insert (average of 8 devices)
2.0 mL 4 mL 20 min @ 4,000 x g 0.34 ± 0.03 mL
1.5 mL 4 mL 20 min @ 4,000 x g 0.15 ± 0.02 mL Despite working in a BSC, expansion processes include passages with backup flasks to be
1.2 mL 4 mL 20 min @ 4,000 x g 80 ± 10 µL used in the case of contamination. Backup flasks are a material waste and multiply labor-
Results for Vivaspin® 500 in 40° fixed-angle rotor
intensive BSC work.
Volume of water added Volume of sample solution added Spin conditions Final concentrate volume
to the filtrate tube to the concentrator insert (average of 8 devices) Sartorius’ MYCAP™ CCX includes integral tubing and a specially designed gas exchange
500 µL 500 µL 15 min @ 15,000 x g 103 µL ± 13 µL
Sartorius Lab Instruments cartridge. Integral tubing supports good aseptic technique to prevent contamination.
380 µL 500 µL 15 min @ 15,000 x g 51 µL
GmbH± 11 µLKG
& Co.
250 µL 500 µL 15 min @ 15,000 x g Otto-Brenner-Strasse 20
30 µL ± 5 µL
All fluid transfers are done outside the BSC. The gas exchange cartridge has a high filter
37079 Goettingen, Germany
200 µL 500 µL 15 min @ 15,000 x g 23 µL ± +49.551.308.0
Phone 7 µL surface area to support passive gas exchange and vibrant cell growth in the incubator.
email: LF-info@sartorius.com
www.sartorius.com
Conclusion
Conclusion Sartorius Lab Instruments
Reproducible defined final concentrate volumes can beGmbH
quickly
USA Toll-free +1.800.635.2906
and easily achieved
& Co. KG with Vivaspin ® Turbo 15,
UK +44.1372.737159
Otto-Brenner-Strasse 20 France +33.1.70.62.50.00
Vivaspin ®
ReproducibleTurbo
defined4, and
final Vivaspinvolumes
concentrate ®
500.can be quickly 37079 Goettingen, Germany Italy +39.0362.5557.11
® ®
and easily achieved with Vivaspin Turbo 15, Vivaspin Turbo 4, Phone +49.551.308.0 Spain +34.913.586.095
14 Vivaspin 500.
and ®
email: LF-info@sartorius.com Russian Federation +7.812.327.53.27 15
www.sartorius.com Japan +81.3.3740.5408

USA Toll-free +1.800.635.2906 Specifications subject to change without notice.

 hose barb filter membrane.
culture wasonmoved
the filter
to ahousing.
flask withCell
thegrowth resumed
traditional flaskonce
cap. the
culture was moved to a flask with the traditional flask cap. The pH change of the solution on flasks with the MYCAP® CCX cap
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S
The pH
and change
flasks with of
thethe solution vented
traditional on flasks
capwith
arethe MYCAP
virtually CCX cap
identical.
®
Traditional flasks have a filter membrane embedded in the cap.
Traditional flasks allows
have a for
filter membraneair embedded in the and flasks with the traditional vented cap are virtually identical.
The arrangement unrestricted flow across thecap.
entire
Introduction The arrangement
filter allowsthe
surface. However, forfilter
unrestricted
membrane air flow across
occupies thethe entire
entire 1L Flask
Gas Exchange Study
Bottle closures with integral tubing are widely filtersurface
surface. However, the filter membrane occupies the entire Sartorius performed an evaluation to
cap leaving no room for integral tubing for aseptic fluid
14,00
7,50 1L Flask
used in bioprocessing because they reduce or cap surface leaving no room for integral tubing for aseptic fluid
12,00
14,00 compare gas exchange across the MYCAP™
transfers. 7,40
7,50

eliminate the risk of contamination from poor transfers. 7,30

10,00
12,00
CCX cap closure and the traditional vented

CO2 Concentration
7,40

pH of Solution
aseptic technique. Good aseptic technique cap closure.
8,00
10,00
7,20
7,30 pH MYCAP™® CCX 1L

CO2 Concentration
pH of Solution
6,00
8,00 pH Traditional 1L
is especially important upstream where
7,10
7,20

Sartorius’ Solution
pH MYCAP
CO
®
CCX 1L
Concentration
2
7,00 4,00
6,00 pH Traditional 1L
preserving axenic, or monoculture conditions is 1L and 3L flasks were modified to accept a
7,10

Sartorius’
The Solutionprocess of the patented MYCAP® bottle closure
manufacturing 6,90
7,00
2,00
4,00
CO2 Concentration

compulsory. pH probe in the side wall so that the probe

The
is anmanufacturing process Components,
enabling technology. of the patented MYCAP
usually tube
®
bottle closure
assemblies, 6,80
6,90
2 3 4 5 6 7 8 9 10
0,00
2,00

Time (hours) would be in direct contact with solution to

is an enabling technology. Components, usually tube
are inserted into pre-formed holes. Silicone elastomer is dispensedassemblies, 6,80
2 3 4 5 6 7 8 9 10
0,00

Tubing materials of the cap closure are Time (hours)

read pH changes. Flasks were filled with
are inserted
into the cap into pre-formed seal
to hermetically holes. Silicone
the elastomer
installed is dispensed
components in
commonly thermoplastic elastomer (e.g., phosphate buffered saline (PBS) solution
into the
place andcap
to to hermetically
create the highlyseal the installed
compliant, componentsbottle
plasticizer-free in 3L Flask
C-Flex®), which can be aseptically welded to containing sodium bicarbonate buffer. Test
place and to create the highly compliant, plasticizer-free bottle
closure.
14,00
7,50 3L Flask
another tube of the same size. Alternatively, 12,00 articles were placed in an incubator and CO2
closure.
14,00
7,40
7,50

aseptic connecting devices (Sartorius Opta®, 10,00

12,00
concentrations changed every two hours.
Inserted components are not restricted to tube assemblies. 7,30

CO2 Concentration
7,40

pH of Solution
Colder Aseptiquik®, Pall Kleenpak®, etc.) may
8,00
10,00
7,20
Inserted components
Sartorius developed the areMYCAP
not restricted
®
CCX gastoexchange
tube assemblies.
cartridge
7,30 pH MYCAP® CCX 3L

CO2 Concentration
pH of Solution
be installed at the tube ends. In either case, Change in pH of the solution indicates gas
6,00
8,00 pH Traditional 3L
7,10
7,20

Sartorius
with the MYCAP® CCX gas exchange cartridge
developedobjectives:
the following
™
pH MYCAP
CO
®
CCX 3L
Concentration
2
4,00
6,00
the bottle can be aseptically connected to receive or Traditional flasks have a filter membrane embedded in exchange across the filter membrane.
7,00
7,10 pH Traditional 3L

–with the following

Provide adequately objectives:
large filter surface area 6,90
7,00
2,00
4,00
CO2 Concentration

dispatch fluids in non-classified spaces without the risk the cap. The arrangement allows for unrestricted air flow
Provide
– Allow adequatelyair
unrestricted large
flowfilter surface
across filterarea
membrane 6,80
6,90 0,00
2,00

of introducing a contaminant. across the entire filter surface. However, the filter mem- The pH change of the solution on flasks with
2 3 4 5 6 7 8 9 10

– Reduce
Allow unrestricted air flow across filter
the filter footprint allowing space membrane
for integral tubing
Time (hours)
Cell Growth Study Conclusion
6,80 0,00
2 3 4 5 6 7 8 9 10
brane occupies the entire cap surface, leaving no room for the MYCAP™ CCX cap and flasks with the
– Reduce the filter footprint allowing space for integral tubing
Time (hours)

Key customers approached Sartorius to improve aseptic integral tubing for aseptic fluid transfers.
Sartorius performed a study comparing cell growth in flasks with traditional vented cap are virtually identical.
2 the MYCAP ®
CCX cap to flasks with the traditional vented cap. Expansion of suspension cell cultures using Erlenmeyer
technique in cell expansion with the following objectives: 2 Cell Growth Study Conclusion
Sartorius performed a study comparing cell growth in flasks with a BSC is a labor-intensive process. The flask’s cap is remo
– Eliminate contamination risk Sartorius’ Solution
the MYCAP CCX cap to flasks with the traditional vented cap.
® at eachofpassage
Expansion suspension andcell
fluid transfers
cultures including
using Erlenmeyermedia addi
flasks
– Enable fluid transfers in non-classified spaces The manufacturing process of the patented MYCAP™ Thaw Cell Growth Study
500 mL inoculation
a BSC and sampling
is a labor-intensive areThe
process. done, typically
flask’s cap is by hand-pi
removed
– Reduce waste from requisite backup passages bottle closure is an enabling technology. Components, Sartorius performed a study comparing cell
These
at each operations
passage are performed
and fluid under
transfers including laminar
media flow in th
addition,
– Achieve comparable culture growth rates & usually tube assemblies, are inserted into pre-formed Thaw growth in flasks with the MYCAP™ CCX cap to
500 mL inoculation and sampling are done, typically by
BSC to prevent contamination. Yet, contamination risk p hand-pipetting
doubling times to incumbent expansion methods holes. Silicone elastomer is dispensed into the cap to flasks with the traditional vented cap.
Passage 1 These
sooperations
back-up flasks are performed under laminar
are maintained for use in flow
theinevent
the of
hermetically seal the installed components in 500 mL 500 mL
BSCcontamination.
to prevent contamination.
In a GMP seed Yet, contamination
expansion process, risk persists
a typi
Cellular respiration consumes O2 and produces CO2 as a place and to create the highly compliant, plasticizer-free Passage 1 CHO DG44
so back-up flasks cells
are were directly
maintained for thawed
use in into
the a of a
event
passage requires three to four operators; the hood techn
byproduct. Cell cultures starved of O2 will not propagate. bottle closure. 500 mL 500 mL traditional In
contamination. flask and seed
aand
GMP thenexpansion
split into two trains:
process, a typical
Passage 2 hood assistant data/batch record recorder(s).
Cultures with an overabundance of CO2 become acidic 1000 mL 500 mL 500 mL 1000 mL
Train
passage 1 utilized
requires MYCAP
three to
™
four CCX flasks;
operators; and
the hood technician,
and impair cell viability. The exchange of O2 and CO2 Inserted components are not restricted to tube Passage 2 hood Train 2
assistant utilized
andhas traditional
data/batch flasks. Cells
record allowing were
recorder(s).
MYCAP ®
CCX integral tubing for aseptic fluid
across the filter membrane is critical to cell growth. assemblies. Sartorius developed the MYCAP™ CCX 1000 mL 500 mL 500 mL 1000 mL sub-cultured consecutively for three additional
in the open space of a workbench. The number of opera
gas exchange cartridge with the following objectives: Passage 3 MYCAP passages
®
CCX hasinintegral
varioustubing
size flasks
allowingup to
for3000 mL.
aseptic fluid trans
3000 mL 500 mL 500 mL 3000 mL in half, contamination risk is eliminated and wasteful ba
It is customary to attach a disc filter to integrated tubing – Provide adequately large filter surface area in the open space of a workbench. The number of operators is
Passage 3 flasks are not necessary.
in a cap for air venting during fluid transfer. Early testing – Allow unrestricted air flow across filter membrane 3000 mL 500 mL 500 mL 3000 mL Two-tailed
in half, T-Tests
contamination were
risk performed
is eliminated and wasteful back-up
of closures on Erlenmeyer flasks with a 50 mm disc – Reduce the filter footprint allowing space for Passage 4 flaskscomparing
are not necessary.
the doubling times between
Carefully™controlled conditions for cell growth in a shak
filter showed slowed or fully halted cell growth. Despite integral tubing 3000 mL 500 mL 500 mL 3000 mL MYCAP CCX and traditional flasks of
Passage 4 in an controlled
Carefully incubator are required. In particular,
growth inthe unrestric
the large filter surface area (3 in2 | 20 cm2) of the 50 mm 3000 mL 500 mL 500 mL 3000 mL the same size. conditions
There wasfor nocell
statistically a shake flask
Figure
Fig. 1: Cell
1: Cell Growth
Growth Study Study
Process Process
Diagram Diagram exchange
in ansignificant of
incubator are CO and
required. O between
In the
particular, cell
the culture and th
unrestricted
disc filter, the gas exchange across the membrane The MYCAP™ CCX gas exchange cartridge is a three- difference in growth rates between
2 2

Fig. 1: Cell Growth Study Process Diagram incubator

exchange of COenvironment
and O2with is critical
between to culture
achieving
theconfidence
cell andtargeted
the c
was inadequate for cell growth. Air flow through the dimensional, stadium-shaped part. Two generous 0.2 μm, the two systems,
2 a 95%
density
incubator and viability.
membrane is restricted at the 8 in. (3.2 mm) orifice of hydrophobic filter membranes extend into the neck of the Table 1: Process Parameters level.environment is critical to achieving targeted cell
Incubator Parameter Description | Set Point density and viability.
the hose barb on the filter housing. Cell growth resumed flask. The orientation of the filter membranes protects and
Incubator
TemperatureParameter Description
36.8°C | Set Point Gas exchange, as measured by a change in pH of solutio
once the culture was moved to a flask with the traditional places them in position for unrestricted gas exchange Average culture doubling times for each flask
Temperature 36.8°C Gas response
exchange,toasa measured
change inby concentration,
COa2 change in pH ofbetween
solution in MYC
flask cap. between the culture and the incubator environment. The Carbon Dioxide % 7.5% size were graphed. The graph (next page)
Carbon Dioxide % 7.5%
and traditional
response to a changeflasksin COwas compared
concentration, and found
between to be
MYCAP sub
®
CC
stadium shape conserves space on the cap for integral Agitation 500 mL, 1000 mL 120 rpm illustrates the comparability2
of doubling times
and equivalent.
traditional flasks was compared and found to be substantia
tubing for media addition, inoculum addition, sampling Agitation 500 mL,mL
3000 1000 mL 12080rpm
rpm for MYCAP™ CCX flasks and traditional flasks.
equivalent.
and transfer. 3000 mL 80 rpm
Table 1: Process Parameters Successful passages in an expansion process are benchm
Table 1: Process Parameters Successful passages
cell growth ratesinandan expansion
cell culture process are benchmarked
doublings. A comparis
cell culture
growth rates and cell culture doublings.
doublings between MYCAP CCX and ® A comparison of c
traditional
16 CHO DG44 cells were directly thawed into a traditional flask and 17
CHO culture doublings betweenwereMYCAP
®
CCXbeand traditional flasks
thenDG44 cells two
split into weretrains.
directly thawed
Train into a MYCAP
1 utilized traditional
® flask
CCX and
flasks; ®
across 4 passages found to
across 4 passages were found to be equivalent.
equivalent.
 The graph illustrates the comparability of doubling times for
YMYCAP
®
CCX
O U R PR A C T I C A flasks
L G U IDE and
TO BA SIC traditional
LA BORATORY flasks.
TECHNIQUES
streamline experimental design, data collection and data analysis.
AP P L I C AT I O N NO T E S

Recommendations
The Tool generates charts to visualize growth rates and performs
MYCAP CCX Validation Template Tool: ™

Culture Doubling Times between Traditional Flasks and MYCAP® CCX A validation study comparing rates of growth in – Supports up to 6 Passages

30
the Student’s
MYCAP CCX T-Test totraditional
flasks with compare
™
the datasets.
flasks should – Complete MYCAP CCX materials list including “Where ™

performed before implementing. Sartorius offers the Used Guide”

MYCAP™ CCX Validation Template Tool to streamline – Record and maintain experimental conditions; flask
25
MYCAP CCX Validation Template Tool makes it quick and easy
experimental design,Template
® ® CCX Validation
MYCAP
– Supports up to 6 Passages
data collection
Tool: and data analysis. size, culture volume, shaker speed, incubator
temperature, CO2 concentration

20 to make a scientifically sound and informed decision if

– Complete
The MYCAP® CCX
Tool generates
Guide’
materials
charts list including
to visualize ‘Whererates
growth Used and – Compare against required performance criteria; growth
performs the Student’s T-Test to compare the datasets. rate, cell count targets, cell viability
MYCAP CCX is an acceptable replacement of incumbent
–®
Doubling Time

Record and maintain experimental conditions; flask size, culture

– Visual and Statistical Analysis including:
Culture

volume,™shaker speed, incubator temperature, CO2 concentration

MYCAP
– Compare CCX Validation Template Tool makes it quick – Doubling Time and Growth Rate Graphs at
(hr)

15
technology for use in a production process.
and easy
against
to make
required performance
a scientifically
cell count targets, cell viability
criteria;
sound and
growth rate,
informed each Passage
decision if Statistical
– Visual and MYCAP™Analysis
CCX isincluding:
an acceptable replacement – Overall Doubling Time and Growth Rate Graphs
10
of–incumbent
Doubling Time and Growthfor
technology Rate
useGraphs at each Passage
in a production – Student’s T-Test
– Overall Doubling Time and Growth Rate Graphs
process.
5 – Student’s T-Test

Growth Rate at each Passage Overall Growth Rate

0 0,0350 0,0350
500 mL Flask 1000 mL Flask 3000 mL Flask
0,0300 0,0300

Traditional Flask MYCAP CCX Flask

®
0,0250
0,0250

Growth Rate
0,0200

Growth Rate
MYCAP™ CCX 0,0200
MYCAP® CCX
0,0150 Traditional Flask
Traditional Flask
Acceptable Growth 0,0150
0,0100

Conclusion Carefully controlled conditions for cell growth in a 0,0050

0,0100

Expansion of suspension cell cultures using Erlenmeyer shake flask in an incubator are required. In particular, 0,0000 0,0050

flasks in a BSC is a labor-intensive process. The flask’s the unrestricted exchange of CO2 and O2 between the
1000 mL 1000 mL 3000 mL 3000 mL 3000 mL Seed Reactor
Vessel Size 0,0000

cap is removed at each passage and fluid transfers

including media addition, inoculation and sampling are
cell culture and the incubator environment is critical to
achieving targeted cell density and viability.
3
done, typically by hand-pipetting. These operations Doubling Time at each Passage
Overall Doubling Time
35,00
are performed under laminar flow in the BSC to prevent Gas exchange, as measured by a change in pH of 30,00

ithout notice. Copyright Sartorius Lab Instruments GmbH & Co. KG. Printed in the EU on paper bleached without chlorine.
contamination. Yet, contamination risk persists, so solution in response to a change in CO2 concentration, 25,00
30,00

backup flasks are maintained for use in the event of a was compared between MYCAP™ CCX and traditional 25,00

Doubling Time (hrs)

20,00

contamination. In a GMP seed expansion process, a flasks and found to be substantially equivalent.

Growth Rate
15,00 20,00

typical passage requires three to four operators; the

MYCAP™ CCX MYCAP® CCX
Traditional Flask Traditional Flask
15,00
hood technician, hood assistant and data/batch record Successful passages in an expansion process are
10,00
Acceptable Doubling Time

recorder(s). benchmarked by cell growth rates and cell culture 5,00 10,00

doublings. A comparison of cell culture doublings was 0,00 5,00

1000 mL 1000 mL 3000 mL 3000 mL 3000 mL Seed Reactor
MYCAP CCX has integral tubing allowing for aseptic
™
compared between MYCAP™ CCX and traditional Vessel Size
0,00

fluid transfers in the open space of a workbench.
The number of operators is cut in half, contamination
risk is eliminated and wasteful backup flasks are
not necessary.
flasks across 4 passages were found to be equivalent.
MYCAP™ CCX should be considered a suitable
replacement for traditional Erlenmeyer flasks to reduce
MYCAP™ CCX and Opta ® are registered trademarks of Sartorius-Stedim Biotech
waste, eliminate contaminations and streamline cell
AseptiQuik ® is a registered trademark of Colder Products Company
expansion operations.

· Order No.: 85037-561-00 · Ver. 01 | 2018

Kleenpak ® is a registered trademark of Pall Corporation
C-Flex ® is a registered trademark of St. Gobain Performance Plastics

MYCAP® CCX and Opta® are registered trademarks of Sartorius-Stedim Biotech Sartorius Lab Instruments
AseptiQuik® is a registered trademark of Colder Products Company GmbH & Co. KG
Kleenpak® is a registered trademark of Pall Corporation Otto-Brenner-Strasse 20
18 37079 Goettingen, Germany 19
C-Flex® is a registered trademark of St. Gobain Performance Plastics
Phone +49.551.308.0
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Choose a
1
Direct weighing of even the smallest quantities of a
substance in large glass flasks enables straightforward, Quiet Pla
How to Achieve accurate and efficient preparation of stock solutions
and reference standards, e.g., for HPLC analysis. This 1. The table should be
whenever possible,
Optimal Weighing Performance
eliminates the need for transferring a microsample from a
synthetic stone.
weighing boat into a volumetric flask, which can result in
1. 2. 3. 2. Avoid causing the
errors. Weighing directly in a large container reduces both deflect even slight
sample loss and contamination. use it to prop up y
How to Avoid Contamination in Pipetting 3. Set up the balance
This application requirement that a balance needs to 4. 5. 6. location. Ensure th
meet poses an even greater challenge to its weighing or engines that gen
electromagnetic fi
#05 technology. The reason is that the smaller the sample
Magnetism must b
quantities used, the greater the relative measuring errors may not be made o
7.
become; and the larger the tare container size employed, 4. Do not position th
Application #01 the higher the influence of environmental conditions will of the room, but n
be on weighing accuracy. To ensure high accuracy during better, in the corne
Note weight measurements and excellent repeatability of the
is where the vibrat
generally at their l
results, you need to observe certain basic rules and
Practical methods #02 requirements.
for avoiding
contamination in
External environmental influences or improper handling
pipetting. #03 can lead to inaccurate results or poor weighing
performance, which are not caused by the balance.

#04

1
Choose a Stable Weighing
Table in a Quiet Place to Set
Up Your Balance
1. The table should be solid-built and, whenever
possible, be made of stone or synthetic stone.
2. Avoid causing the tabletop to sag or deflect
Introduction even slightly; for example, never use it to prop
With full-resolution 1 μg readability up to 61 g, the new Sartorius high-capacity microbalances are pushing up your arm.
back the limits of what is possible in weighing technology: They set a new record in accuracy with 60 million
divisions. Their exceptional weighing performance and the impressive quality of their weighing results are 3. Set up the balance in a vibration-free location.
clearly revealed when they are checked with certified weights. Ensure that there are no machines or engines
that generate vibrations or electromagnetic
But perfect measurement of weights is not the application this balance was designed for. Sartorius high- fields near the balance. Magnetism must be
capacity microbalances enable optimal minimum weights within the USP 41 operating range to be measured ruled out (e.g., tables may not be made of
in heavy glass vessels, such as long-necked, volumetric flasks. stainless steel).
4. Do not position the table in the middle of the
room, but near a wall or, even better, in the
corner of a room, as this is where the vibration
amplitudes are generally at their lowest.
5. Avoid exposing your balance to sunlight and
infrared radiation emitted by lamps or heaters.
6. The location may only be slightly ventilated.
Exposure to drafts needs to be avoided, and the
air flow rate should be below 0.2 m/s.
7. Cold air currents from air conditioners may not
pass directly across or over the draft shield,
as this can result in an inversion layer of air
inside the draft shield. This, in turn, can cause
unstable weight readouts.

20 21
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Work in the Lab under Consistently During the Measuring Sequence,

2 4
2 4
Constant Climate Conditions. Ensure That …
Work in the Lab under During the next Measuring
Consistently
1. Avoid significant Constant
temperature changes 4. Use the Sartorius ionizer option to elimi- 1. … the vessels used are acclimatized
to your balance; Sequence,
i.e., have adapted toEnsure
the
4. Avoid touching a vessel with your bare
That
fingers ...times, as a single fingerprint
at all
or spikes. nate electrostatic influences. Electrostatic
2. Keep theClimate Conditions
relative humidity as constant charges on glass vessels dissipate only very temperature conditions in the same room. can weigh up to 50 μg and therefore have
1. >40% 2. 3. as possible. Prevent the relative humidity slowly, particularly when these vessels have 1. 2. 3. 2. … you do not 1. ... the
touch vessels used
the container with are acclimatized
a major impact nexton to
theyour
accuracy of your
1. Avoid significant temperature changes
from dropping below 40%, as this will or surfaces,
very clean spikes. especially when they your hands whenbalance,
positioningi.e.,
it on the weight measurement
have adapted to the temperature result.
significantly increase interference by static are used freshly from a laboratory glass- weighing pan or in a sample holder. 5. When weighing, ensure that no powder
conditions in the same room.
electricity. ware washer. Electrostatic influences are Touching the sample vessel with your falls onto the weighing pan next to the vess
2. Keep the relative humidity as constant as ••••••••• •
hand usually
4. 3. Use the Sartorius climate sensor option easy to detect by the continuous drift of 4.
•••••
•
5. 6. as this will mean that the displayed sample
possible.
(temperature, Prevent
barometric the and
pressure relative humidity from
weight readouts. Increase the air humidity increases the 2. ... you doofnot
temperature the touch
vessel. the container
weight is with youris actually in the vessel.
not what
dropping below 40%,
relative humidity) to monitor climate as this will significantly
to levels up to 60%, and use an ionizer to Buoyancy and airhands
currentwhen
effectspositioning
influence 6.it Avoid
on thetheweighing
complete pan
interchange of air whe
conditions.increase interference by static electricity.
reduce these effects on the resulting weighing results. or in a sample
Remember that itholder.
takes Touching
openingthethesample
draft shield by opening only o
weight readings. 7. 8. ten minutes for these
vessel effects
withtoyour
subside. door,increases
hand usually where possible.
the Opt for using the dra
Use a pair of tweezers or forceps to posi- shield learning capability to open the door
3. Use the Sartorius climate sensor option temperature of the vessel. Buoyancy and air
tion the vessel. only as far as actually necessary.
(temperature, barometric pressure and relative 3. Avoid placing yourcurrent effects
hand inside the influence
draft 7.weighing results.
Carefully place the tare container on
humidity) to monitor climate conditions. Remember
shield to ensure that that it takes ten
no unnecessary theminutes
weighingfor these
pan or in the sample holder.
interchange of aireffects
outsideto subside.
and inside theUse a pair
Avoidofapplying
tweezers anyorexcessive force.
4. Use the Sartorius ionizer option to eliminate draft shield takesforceps
place andtothat no heat 8.
position the vessel.Do not lean on or against the weighing
electrostatic influences. Electrostatic charges is transferred into the draft shield. table or rest your arm arm on it during
the weighing procedure.
on glass vessels dissipate only very slowly, 3. Avoid placing your hand inside the draft shield
particularly when these vessels have very to ensure that no unnecessary interchange of air
clean surfaces, especially when they are used outside and inside the draft shield takes place
freshly from a laboratory glassware washer. and that no heat is transferred into the draft
Ensure That the Balance Is
3
Electrostatic influences are easy to detect by shield.
the continuous drift of weight readouts. Increase
Leveled and Calibrated.
the air humidity to levels up to 60%, and use an 4. Avoid touching a vessel with your bare fingers at
ionizer to reduce these effects on the resulting all times, as a single fingerprint can weigh up to
1. Sartorius high-capacity micro
weight balances will
readings. 50 μg and therefore have a major impact on the
isoCAL support you in using the calibration | ad- accuracy of your weight measurement result.
justment function isoCAL, and the Q-Level

3
1. 2. function implemented in the balance
for leveling continuously maintains the 5. When weighing, ensure that no powder falls
accuracy of the weighing results within onto the weighing pan next to the vessel, as this
a narrow toleranceEnsure
range. That the Balance is will mean that the displayed sample weight is
2. Moreover, routinelyLeveled and Calibrated
check the balance not what is actually in the vessel.
using an external, certified weight.
1. Sartorius high-capacity microbalances will 6. Avoid the complete interchange of air when
support you in using the calibration | adjustment opening the draft shield by opening only one
function isoCAL; and the Q-Level function door, where possible. Opt for using the draft
implemented in the balance for leveling shield learning capability to open the door only
continuously maintains the accuracy of the as far as actually necessary.
weighing results within a narrow tolerance range.
7. Carefully place the tare container on the
2. Moreover, routinely check the balance using an weighing pan or in the sample holder. Avoid
external, certified weight. applying any excessive force.

8. Do not lean on or against the weighing table

or rest your arm on it during the weighing
procedure.

22 23
 to final concentration | desalting of the to 2 l using dd-H2O. After every prepara-
purified protein. This protocol shows in tion, concentration and purification step,
detail the recoveries after each step along 1 ml sample was set aside for SDS gel
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S
with the time needed for every purification analysis at the end of the preparation.
and concentration step.
Ion Exchange chromatography was cho-
Concentration and Purification Efficiency
of and efficacyPart 1 – cycle
of a multiple Creating sen as and Concentrating
the method of choice for purifying
Proteins in Cell Culture Supernatant
experimental procedurethe Culture Medium the cell culture supernatant,
was performed lysozyme from
Using Sartorius Vivaflow ®, Vivaspin
®
using Vivaflow
® tangential flow cassettes especially from the “contaminant” BSA. For
for initial concentration and diafiltration this, the 2 l cell culture supernatant needed
and Vivapure Products
®
2 bottles
of a cell culture supernatant, (4 g)byof RPMI-1640
followed were dissolved
to be concentrated into 1.8
and then diafiltered to L
Vivapure® Ion Exchangedd-H O, andfor4 g ofadjust
spin columns
2
sodium acetate
the sample was
to the added.
starting conditions
® ®
Vivaflow and Vivaspin Workflow This protocol demonstrates how the Vivaflow cassettes,
the®protein purification step and finally needed for the ion exchange chromatogra-

Vivaflow and Vivaspin Workflow

Vivaspin® 20 ultrafiltration devices for the
® ® Vivapure ® Ion Exchange spin columns and Vivaspin ®
phy binding step.
The pH was adjusted to 7.2 using 1M HCl. 2 g of BSA
final sample concentration and desalting.

in Protein Research Laboratories

devices can be used in order to perform a An complete
artificial mixture ofand 1 g inofa lysozyme
proteins RPMI- Forwere added as
concentration andprotein samples,
diafiltration, the
How to Avoid Contamination in Pipetting
in Protein Research Laboratories
protein purification workflow, from concentration andmedium was
1640 culture meant to to
created bemimic
separated by ®chromatography.
Vivaflow 200 was used with a 5The volume
kDa PES
®
the type of
diafiltration of the original protein source, a cell cultureproduct that many researchers membrane. Vivaflow 200
of the cell culture supernatant sample was brought up to is a ready-to-
culture using e.g. the UniVessel device. use laboratory crossflow cassette in an
supernatant, to final concentration | desalting of the 2 L using dd-H O. After every preparation, concentration,
This procedure further reflects a method 2 acrylic housing, which allows caustic clean-
#05 purified protein. This protocol shows in detail
that the
can be adapted toand purification
a large number of step, ing 1
andmL4-5sample wascassettes
re-uses. Two set asidecan for
be SDS
recoveries after each step along with the time needed
protein gel analysis
purification protocols, adapting at the end
run inofparallel
the preparation.
for the concentration of up
for every purification and concentration step.MWCOs and device sizes where necessary. to 5 l sample volumes. For the 2 l sample
Application to be concentrated in this experiment, one

Note
#01 #03 cassetteIon
with anwas
wasexchange
sufficient. Achromatography
Easychosen
Load, sizeas
Masterflex pump
16 the
pumpmethod
head was
used toofrunchoice for purifying
the Vivaflow ®
200 cassette.

Application
®
Figure lysozyme
1a. and 1 b. show
fromthe theVivaflow 200
cell culture
Practical methods #02 set up before and during the concentration
for avoiding process.supernatant, especially from the
“contaminant” BSA. For this,
contamination in
pipetting. #03 Note #04 ®
the 2 L200
The Vivaflow system
cell was set
culture up and
supernatant
run at 3 bar. Once 1.8 l of filtrate had been
needed to be concentrated and
collected, the pump was stopped, the tubes
removedthen
fromdiafiltered to adjust
the cell culture mediumthe
sample
concentrate and to the and
filtrate starting conditions
the Vivaflow ®

#04 systemneeded
was purged with dd-H O. This
for the ion2 exchange solu-

#05 tion now contained a 10 fold concentration

chromatography binding step.
of the constituent proteins from the origi-
nal culture-medium.
For concentration and
diafiltration, the Vivaflow® 200
was used with a 5 kDa PES

#06 membrane. Vivaflow® 200

is a ready-to-use laboratory
crossflow cassette in an acrylic
Efficiency and efficacy of a multiple cycle experimental housing, which allows caustic cleaning and 4-5 reuses.
procedure was performed using Vivaflow® tangential Two cassettes can be run in parallel for the concentration

#07
flow cassettes for initial concentration and diafiltration
of a cell culture supernatant, followed by Vivapure ® Ion
Exchange spin columns for the protein purification
step and finally Vivaspin ® 20 ultrafiltration devices
for the final sample concentration and desalting.
An artificial mixture of proteins in a RPMI-1640
culture medium was created to mimic the type of
product that many researchers culture using, e.g., the
UniVessel device. This procedure further reflects a
method that can be adapted to a large number of
protein purification protocols, adapting MWCOs
and device sizes where necessary.
of up to 5 L sample volumes. For the 2 L sample to
be concentrated in this experiment, one cassette was
sufficient. A Masterflex pump with an Easy Load, size 16
pump head was used to run the Vivaflow® 200 cassette.
Figure 1a and 1b show the Vivaflow® 200 setup before and
during the concentration process.
The Vivaflow® 200 system was set up and run at 3 bar.
Once 1.8 L of filtrate had been collected, the pump was
stopped, the tubes removed from the cell culture medium
concentrate and filtrate, and the Vivaflow® system was
purged with dd-H2O. This solution now contained a
10-fold concentration of the constituent proteins from
the original culture medium.

24 25
 with further 10 ml of 25 mM Sodium Acetate, discarding the
flow through, followed by an elution step with 5 ml of 1 M NaCl in
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S
25 mM Sodium. A BCA test revealed a 95 % lysozyme recovery.

A BCA protein detection test conveyed a 100% recovery Part 2 – Buffer Exchange of Culture Part 3 – Purification of Lysozyme,
AofBCA protein
protein detection
after test concentration
this first conveyed a 100%step.recovery of 1
Table Medium
Part 2 – BufferConcentrate exchange of culture medium concentrate PartProtein
the 3 – Purification
of Interestof Lysozyme, the protein of interest The eluate was then con
protein after this first concentration step. Table 1 indicates the The Vivaflow® 200 system was used for fast and easy diafiltration.
indicates the time needed for the sample concentration.
time needed for the sample concentration. To this end, the diafiltration cup, a Vivaflow® accessory, was filled The purification of lysozyme was performed using a Vivapure® MWCO), Figure 5., and c
A BCA protein detection test conveyed a 100% recovery of Thethe
Part 2Vivaflow
– 200
Buffer
®
200 System
exchange was Figure
of sample.
culture used 2for
medium fastthe
concentrate and The purification of lysozyme was performed using ®
protein after this first concentration step. aTable
with
easy ®
ml concentrated
diafiltration. To this end, the
shows
diafiltration
diafiltra-
cup,
cation exchange membrane adsorber devices (Vivapure Maxi H S). approximately 2 ml of c
a Vivapure cation exchange membrane adsorber
®
100%1 recovery
indicatesofthe The Vivaflow 200 system was used for fast and easy diafiltration.
®
A BCA protein detection test conveyed Part 2 – Buffer exchange of culture medium concentratehowev-
tion set up. The Vivaflow 200 system was set up as before,
time protein
neededafter
for the
thissample concentration.
first concentration step. Table 1 indicates the To
er this
aThe
Vivaflowend, the
attaching
Vivaflow ®
an diafiltration
® accessory,
additional
200 system was cup,
was
tubeusedtoathe
Vivaflow
filled fastwith
®
andaccessory,
for diafiltration the lid
easy 200 was
and mL filled
placing
diafiltration. The membrane
device (Vivapure Maxi
® adsorber
H S). Thematrix
membrane holds the active ligands and per-
adsorber was then re-filled with 1
with
this the
new 200
inlet
concentrated sample. ml
tube concentrated
into a 25 mMsample.
Sodium Figure
Acetate 2 shows
Figure 2 shows the diafiltration (pH the
5.5) diafiltra-
buffer matrix
formsholdslikethe active ligands and performs like a
®
time needed for the sample concentration. To
tion
(needed
this
withset the
end,
up.
to 200
the diafiltration
ThemlVivaflow
re-adjust the ®®sample
concentrated
cup,
200 sample.
a
system
Vivaflow
was
concentrate
Figure
accessory,
set2forup as before,
the
shows
was
ionic
theas
filled
howev-
starting
diafiltra-
a traditional cation exchanger. Membrane adsorbers pH 7.2 to 20 ml for a fin
setup. The Vivaflow 200 system was set up before, traditional cation exchanger. Membrane adsorbers
er attaching
conditions
tion set up.ofThe
however
an
attaching
additional
theVivaflow
ion exchange ® tube to the diafiltration lid and placing
an25
chromatography
200 system
additional
was set up asstep
tube to the
which
before, was to
howev-
diafiltration represent
represent a special
a special form
form of of chromatography
chromatography matrix. matrix. Unlike tradi- purified sample. The sam
this newThis
er attaching
follow). inlet antube
leads tointo
theaconcentration
additional mMtoSodium
tube ofAcetate
the diafiltration
the sample (pHand
lid 5.5)
in buffer
placing
the reser-
lid
(neededand
newtoplacing
thisand
voir re-adjust
inlet
the tube extent this
the
into inanew
sample
25 mM
which inlet tubeAcetate
concentrate
theSodium
original into forathe
buffer (pH25 mM
is ionic
5.5)
removed Sodium
starting
buffer and tionaltraditional
Unlike chromatography
chromatography resins,
resins,they make use of convective trans-
they make sample volume of 2 ml h
Acetate
(needed to
conditions
collected as(pH the5.5)
ofre-adjust
waste ion buffer
the
(filtrate), sample
exchange (needed
new concentrate to readjust
chromatography
buffer (25 mMfor thestep
Sodium the
ionic sample
starting
which was to
Acetate) use of convective transport to bring proteins to the ion
port to bring proteins to the ion exchange surface; hence, binding, 97 % lysozyme recovery
is conditions
follow).
suckedThis
concentrate into of the
leads
the for ion
tothe
closed exchange
the ionic
system, chromatography
concentration
starting
gradually ofconditions
the
leading steptowhich
sample ainofthe was
the
buffer to
reser-
ion exchange surface; hence, binding, washing and elution
follow).
voir
exchangeand This
to the
while leads
extentto the
keeping in concentration
which
the
exchange chromatography step which was to follow).samplethe of thebuffer
original
volume sample
constant is in the
removed
at 200 reser-
and
ml. washing
is performedand elution
quickly, is performed
and high quickly
binding capacities are and high binding capaci-
voirsystem
collected
The and as towasthe
waste extent 3inbar.
(filtrate), whichnew the 1original
buffer (25 buffer
mMhad is been
removed
Sodium and
Acetate)
This
collectedleads as torun
waste the atconcentration
(filtrate),
Once
new buffer
l ofof buffer
(25 the
mM sample
Sodium in the
Acetate) ties achieved
even are even achieved
at high at This
flow rates. highallows
flowtherates.
use This allows the use of the
Figure
Fig. 1a 1and
1a. and 1b: Vivaflow
b: Vivaflow ® ®
200 set up 200
beforesetup before
(1a) and (1a)theand
during (1b) sample is sucked into the closed
exchanged, the filtration was stopped. system, gradually leading to a buffer
reservoir and to the system,
extent in volume
which the original
to a bufferbuffer of the chromatography matrix in fast and convenient
during (1b) the sample concentration process.
concentration process. The
is sucked while
exchange into
200 ml solution
exchange while
the closed
keeping
keeping
now the sample
contained
the sample
gradually leading
the correct
volume
constant
bufferatto200
constant at new
ml.
maintain
200 ml.
chromatography matrix in fast and convenient centrifugal spin
Figure 4: Vivapure Maxi spin columns can be used in a
®
is
The removed
system was and
run collected
at 3 bar. Once as 1 waste
l
the stability of the proteins of interest for the next part of the of (filtrate),
buffer had been buffer centrifugal spin columns (Figure 3).
columns (Fig. 3). Fig. 4: Vivapure Maxi spin columns can be used in a centrifuge for fast and
®
The system was run at 3 bar. Once 1 l of buffer had been centrifuge for fast and easy protein purification.
Vivaflow ® 1 b: Vivaflow® 200 set up before (1a) and during (1b) the sample
200 (5 kDa MWCO)
Fig. 1a. and (25 mMand
exchanged,
protocol sodium
the theacetate)
hadfiltration correct waspH isandsucked
stopped. into the closed
salt concentration for the ion easy protein purification.
Table 1:1a.
Fig. Vivaflow
concentration and
process. 200, ®PES,
1 b: ®Vivaflow 200 set5upkDa MWCO
before (1a) and during (1b) the sample exchanged, the filtration was stopped.
The 200
system, ml solution now contained the correct buffer to maintain
Filtrate Volume (m
concentration
concentration L)
speed.
process. Time taken (h:min:s) The 200 mlgradually
exchange binding
solution nowstep. leading
BCA protein
contained to the
a buffer
correctexchange
quantification again
buffer to maintain while
showed
athe stability
100%
keeping protein
the stability theof the proteins
recovery.
of sample
the proteins
of
volume interest
of interest
for
constant the
for the next
next
at 200 part
part mL.
of the
of theThe The eluate was then concentrated in a Vivaspin® 20
0Vivaflow® 200® (5 kDa MWCO) 0:00:00 protocol and had the correct pH and salt concentration for theionion
Vivaflow 200 (5 kDa MWCO) protocol and
system washad run theatcorrect
3 bar.pHOnce and salt 1 concentration
L of buffer had for the been (PES, 5 kDa MWCO), shown in Figure 5, and centrifuged
100
Filtrate Volume (mL(m) L) 0:03:16
Time taken (h:min:s) exchange
Table 2 showsbinding the step.
time BCA
needed protein
for quantification
diafiltration
exchange binding step. BCA protein quantification again showed of again
200 ml showed
sample
Filtrate Volume
Part
200 2 – Buffer exchange of culture Time taken
medium
0:06:50
(h:min:s)
concentrate aexchanged,
a100%
against
100%1000protein
protein mlthe filtration
recovery.
exchange
recovery. buffer,was stopped.
again using Vivaflow® 200 with at 5000 x g for 10 min or until approximately 2 mL
0 0 0:00:00
0:00:00 of concentrate had been collected. The device was
he The Vivaflow® 200 system was used for fast and easy diafiltration. a 5 kDa PES membrane.
300
100 0:10:45 ®
To end, the diafiltration cup, a0:03:16
this100 0:03:16 accessory, was filled
Vivaflow The
Table2200
Table 2shows
showsmLthe solution
the time
time needed nowfor
needed contained
for diafiltration
diafiltration the correct
ofof200200mlmlsample buffer
sample Part 3 – Purification of Lysozyme, the protein of interest then refilled
The eluate waswith
then18 mL 50 mM
concentrated in potassium
a Vivaspin® 20phosphate
(PES, 5 kDa
400
with
200 the 0:14:38 against 1000 ml exchange buffer, again using Vivaflow ® ® 200 with
200200 ml concentrated sample. Figure 2 shows the diafiltra- ®
0:06:50
0:06:50 to maintain
against 1000 ml the stability
exchange of the
buffer, again proteins
using Vivaflowof interest 200 with for The purification of lysozyme was performed using a Vivapure MWCO), Figure 5., and centrifuged at 5000 xg for
buffer, pH 7.2 to 20 mL for a final buffer exchange 10 min. or until
® a a55kDa PES membrane.
tion
500 set
300 300 up. The Vivaflow 200 system was
0:18:36
0:10:45 set up as before, howev-
the kDanext PESpart membrane.
of the protocol and had the correct pH cation exchange membrane adsorber devices (Vivapure® Maxi H S). and approximately 2 ml of concentrate had been
desalting of the purified sample. The samplecollected. The device
0:10:45
er
600attaching an additional tube to the diafiltration
0:22:43 lid and placing The membrane adsorber matrix holds the active ligands and per- was then re-filled with 18ml 50mM Potassium Phosphate buffer,
400new400inlet tube into a 25 mM Sodium 0:14:38
0:14:38 and salt concentration for the ion exchange binding was again centrifuged until a final sample volume of
this Acetate (pH 5.5) buffer forms like a traditional cation exchanger. Membrane adsorbers pH 7.2 to 20 ml for a final buffer exchange and desalting of the
700 0:26:57 step. BCA protein quantification again showed a 100% 2purified
mL had beenTheattained. A BCA
500 500to re-adjust the sample concentrate
(needed 0:18:36 for the ionic starting
0:18:36 represent a special form of chromatography matrix. Unlike tradi- sample. sample was againtest revealed
centrifuged a 97%
until a final
800
conditions
600 600 of the ion exchange chromatography0:31:14
0:22:43
0:22:43 step which was to protein recovery. tional chromatography resins, they make use of convective trans- lysozyme
sample recovery.
volume of 2 ml had been attained. A BCA test revealed a
Figure 3: The electron microscopic image of
follow).
900
700 700This leads to the concentration 0:36:01of the sample in the reser-
0:26:57
0:26:57 Fig.
port 3:
to The
bring electron
proteins to microscopic
the ion exchange
chromatography gel beads (upper right) in image
surface; of chromatography
hence, binding, 97 % gel beads
lysozyme (upper
recovery.
voir and to the extent in which the0:40:50 original buffer is removed and washinginand elution is performed
1000
800 800 0:31:14
0:31:14
Table 2 shows the time needed for diafiltration of 200 right)
comparison comparison
to a Q ion to a Qquickly
exchange and high binding
ion exchange
membrane capaci- adsorber (background)
membrane
collected as waste (filtrate), new buffer (25 mM Sodium Acetate) Fig. 2: Diafiltration system set up for buffer exchange. Culture medium ties are even
adsorber achieved at reveals
(background) high flow rates. This
100-fold allows the use of the
larger
mL sample against 1000 mL exchange buffer, again reveals 100 fold larger
in fastpore sizes of the membranespin adsorber.
1100
is900 900
sucked into the closed system, gradually 0:45:46
0:36:01
0:36:01 leading to a buffer concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate
using Vivaflow pore sizes of thematrix
chromatography membrane adsorber.
and convenient centrifugal
buffer) can be200seen with a 5tokDa PES onmembrane.
®
1200
exchange1000 while keeping the sample 0:50:36
0:40:50 (exchange connected the system the left of the
1000 0:40:50 constant at 200 ml.
volume
Fig.
image. 2: Diafiltration system set up for buffer exchange. Culture medium columns (Fig. 3).
The
1300 system
1100 was run at 3 bar. Once 0:55:32 1 l 0:45:46
of buffer had been Fig. 2: Diafiltration system set up for buffer exchange. Culture medium
1100 0:45:46 concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate
® H S type devices (Figure 4) were
mple exchanged,
1400
1200
1200 the filtration was stopped. 0:50:36
1:00:24
concentrate
Table
(exchange
(exchange
can be seen in the
2: buffer)
Diafiltration
buffer)
can be seen
can be seen
center of the
of connected
200 mL
connected
to
to
image. 1 L 25 mM Sodium
concentrated
the system on thecell
the system on
culture
left of
the left
the
of
Acetate
the
TwoVivapure
Two Vivapure ®
Maxi Maxi H S type devices (Fig. 4) were equilibrated
The 200 ml solution now contained0:50:36 the correct buffer to maintain supernatant
image. containing the proteins lysozyme and BSA equilibrated with 10 mL of 25 mM sodium acetate,
1500 1300 0:55:32
1:05:26 image. Volume (mL)
Filtrate Time taken (h:min:s)
with 10 ml of 25 mM Sodium Acetate, pH 5.5 each, by filling with
the
1300stability of the proteins of interest for the next part of the
0:55:32 against 1000 mL 25 mM sodium acetate. pH 5.5 each, by filling with 10 mL of this buffer and
1400 1:00:24 0 0:00:00
1600
protocol
1400 and had the correct pH and
1500binding step. BCA protein
1:10:28
salt
1:00:24 concentration for the ion
1:05:26 Filtrate Volume (mL)
100 Time taken (h:min:s)
0:06:58
10 ml of this
centrifuging for 5 buffer
min in a swingand centrifuging
bucket centrifugefor at 5 min. in a swing bucket
exchange
1700 quantification
1:15:52 again showed Filtrate Volume (mL) Time taken (h:min:s)
1500
a 100%
1800
protein
1600 recovery.
1:05:26
1:10:28
1:21:50
0
200
0
0:00:00
0:14:16
0:00:00
centrifuge
500 at 500 the
x g and discarding xgflowandthrough.
discardingUsing the the flow through. Fig. 5: Vivaspin® 20 ultrafiltr
1600 1:10:28 100 0:06:58 concentrated and buffer exchanged sample from Part
Table
1700
170021:shows the time needed for1:15:52
1:15:52
diafiltration of 200 ml sample
300
100 0:22:39
0:06:58 Using the concentrated and buffer exchanged sample from Part
2, 10 mL sample were pipetted into each of these two
which allows pressurization o
Table 1800 1:21:50 200 0:14:16 Fig. 3: The electron microscopic image of chromatography gel beads (upper
against
Vivaflow® 200, PES, 5 kDa MWCO concentration
1800 1000 ml exchange buffer, 1:21:50
speed.
again using Vivaflow® 200 with 400
200
300
0:29:40
0:14:16
0:22:39 2, 10in comparison
right) ml sample
equilibrated Vivapure ®were pipetted into each of these two equilibrated
to a Q ion devices and centrifuged
exchange membrane again
adsorber (background)
in a centrifuge.
a 5 kDa PES membrane. 500 0:37:02 for 5 min in ®alarger
swingporebucket
Table 1: Vivaflow® 200, PES, 5 kDa MWCO concentration speed. 300
400
600
0:22:39
0:29:40
0:44:15
Vivapure
reveals 100 fold
devices andcentrifuge
sizes of the at 500 xagain
centrifuged
membrane adsorber. g. The for 5 min. in a swing
Table 1: Vivaflow® 200, PES, 5 kDa MWCO concentration speed. 400 0:29:40 Vivapure devices were washed with further 10 mL of ®
®
Figure 2: Diafiltration system 500
700
500
0:37:02
0:51:34
0:37:02
bucket
TwomM
25 Vivapurecentrifuge
sodium
®
Maxi atdevices
500(Fig.
H S typediscarding
acetate,
xg.4) The
the were Vivapure devices were washed
flow equilibrated
through,
set up for buffer exchange. 600 0:44:15
Culture medium concentrate 800
600
700
0:58:54
0:44:15
0:51:34 with
with 10further
followed mlbyofan 10Sodium
25 elution
mM mlstepof 255mM
Acetate,
with pH
mL5.5ofSodium
each,
1 M by
NaCl
10 ml of this buffer and centrifuging for 5 min. in a swing bucket
Acetate,
filling with discarding
Figure 5: Vivaspinthe®
20 ultrafiltration device, on the right with
can be seen in the center of in 25 mM sodium. A BCA test revealed a 95%
the image. 1 L 25 mM sodium
900
700
800 1:06:03
0:51:34
0:58:54 flow through,
centrifuge at 500 xg andfollowed by flow
discarding the an through.
elution step with 5 ml
a pressure of ® 1
cap M
that NaClpressurization
allows
Fig. 5: Vivaspin 20 ultrafiltration in on the rightofwith
device,
the device as
a pressure cap
1000 1:13:02 lysozyme recovery. and buffer exchanged sample from Part well, and the regular utilization in a centrifuge.
Using the concentrated
acetate (exchange buffer) can 800
900 0:58:54
1:06:03 25 mM Sodium. A BCA test revealed a 95 % lysozyme
2, 10 ml sample were pipetted into each of these two equilibrated
recovery.
which allows pressurization of the device as well and the regular utilization
in a centrifuge.
be seen connected to the 1000
900 1:13:02
1:06:03
system on the left of the image. Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain- Vivapure® devices and centrifuged again for 5 min. in a swing
1000
ing 1:13:02
the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate. bucket centrifuge at 500 xg. The Vivapure® devices were washed
Fig. 2: Diafiltration system set up for buffer exchange. Culture medium Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain-
26
concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate ing the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate. with further 10 ml of 25 mM Sodium Acetate, discarding the 27
Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain- flow through, followed by an elution step with 5 ml of 1 M NaCl in
(exchange buffer) can be seen connected to the system on the left of the
ing the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate.
 1 ml sample taken during the experiment were diluted with 95µl 1 working day, including SDS gel analysis, utilizing this time saving
reducing sample buffer, of which 20 µl were loaded onto a 12% strategy, when adapted to individual needs.
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S
tris-HCl SDS gel (Fig. 6)

Figure 6: Coomassie stained 12% Task Time Recovery

Tris-HCl SDS gel loaded with
20 μL sample preparations. Lane 1: Vivaflow® 200 set up and run through 1 h 25 min 100%
Marker (SDS Broad Range marker);
Lane 2: Original sample; Lane 3: Vivaflow® 200 Diafiltration set up 1 h 20 min 100%
Original sample filtrate (Part 1); Lane and run through
4: Marker; Lane 5: Buffer exchange
concentrate (Part 2); Lane 6: Filtrate
after binding (Part 3); Lane 7: Marker;
Vivapure® purification Minimizing45 min Syringe
95% Filter Consumption
tofor Monoclonal Antibody
Pipetting Harvest from
®
Lane 8: Filtrate after eluting (Part 3); Vivaspin Lysozyme How
desalting |Avoid Contamination
30 min 97%
in
Lane 9: Filtrate after concentrating concentration
and desalting (Part 3); Lane 10:
Concentrate after concentrating
and desalting.
Total CHO Cell 3 h 45Culture
min 92%Supernatants
#05

Michael Grauf1*, Klaus Schöne2 Application #01

1. Sartorius Stedim Cellca, Erwin-Rentschler-Straße 21, 88471 Note
Laupheim
2. Sartorius Lab Instruments GmbH & Co.KG, Otto-Brenner-Str 20, 37079 Göttingen
Products used in this Correspondence
experiment
*
Order No. Practical methods #02
Fig. 6: Coomassie stained 12% tris-HCl SDS gel loaded with 20 µl sample E-Mail: michael.grauf@sartorius.com for avoiding
preparations. Lane 1: Marker (SDS Broad range marker); Lane 2: Original sam- Vivaflow® 200, PES, 5kDa VF20P1 contamination in
Part
Part 44– – Analyzing
Analyzing the Samples
the samples The complete
The complete setsetup
up andand completion of protein
ple; Lane 3: Original sample filtrate (Part 1);completion of protein
Lane 4: Marker; Lanepurification takes
5: Buffer pipetting. #03
The
The samples
samples ofofthethe individual
individual stepssteps were analyzed
were analyzed byusing purification
by SDS gel, approx. 3.45 htakes approx.
using this 3.45
method, h using
starting form this method,
a culture super- 500 mL Diafiltration cup VFA006
reducing sample buffer (prepared by adding exchange
50 µl concentratenatant,
2-mercaptoe- (Part 2);
with Lane
high 6:protein
Filtrate after binding
recoveries in each (Part
step 3); Table
(see Lane 3)7:
SDS gel, using reducing sample buffer (prepared by starting from a culture supernatant, with high protein
thanol
Part 4 to 950µl Laemmli sample buffer). Marker; Lane 8: Filtrate after eluting (Part 3); Lane 9: Filtrate after concen-
adding –50Analyzing the samples
μL 2-mercaptoethanol to For
950allμL
steps, 5µl of the
Laemmli The
The total protein
complete
recoveries inset purification
up and
each step procedure
completion
(see can be purification
of protein
Table 3). The completed within
takes
total protein Vivapure® S H Maxi VS-IX20SH08
1 mlsamples
The sample of
taken
the during the steps
individual experiment
were weretrating
diluted
analyzed
sample buffer). For all steps, 5 μL of the 1 mL sample by and
with
SDS desalting
95µl
gel, using 1(Part
approx.3);3.45
working Lane
day,
h 10: Concentrate
including
using this after concentrating
SDS gel analysis,
method, starting
purification procedure can be completed within 1
utilizing
form a andsaving
this time
culture super- #04
reducing
reducing sample
taken
sample buffer,
duringgel
tris-HCl
thanol toSDS
the
of which 20
buffer (prepared
experiment
(Fig.
950µl Laemmli
by µl
adding desalting.
were 50
loaded onto a 12%
µl 2-mercaptoe-
were diluted with 95 μL
6) sample buffer). For all steps, 5µl of the
strategy, when
natant, with adapted
high proteintorecoveries
individualinneeds.
each step (see Table 3)
working day, including SDS gel analysis, utilizing this
The total protein purification procedure can be completed within
Vivaspin® 20, 5 kDa Abstract VS2011
reducing sample buffer, of which 20 were
1 ml sample taken during the experiment μL were loaded
diluted with 95µl time-saving
1 working day,strategy, when
including SDS geladapted to individual
analysis, utilizing needs.
this time saving
onto a 12% Tris-HCl SDS gel (Figure 6). Conclusion The clarification of cell culture supernatants with volumes < 25 mL for harvesting
reducing sample buffer, of which 20 µl were loaded onto a 12% strategy, when adapted to individual needs.
Task Time Recovery
tris-HCl SDS gel (Fig. 6) The overall result
shows that
® a standard and straightforward 100% monoclonal antibodies by using syringe filters is often a laborious and sometimes an
Vivaflow
Table 3 200 set up and run through 1 h 25 min
procedure can be followed
Vivaflow® 200 toDiafiltration
concentrate,set up purify,
1 h isolate
20 min and100%
exhausting work step. Therefore, a proper selection of the suitable filter model could be
Conclusion Taskrun through
analyze a protein of interest
and Time
from a cell culturing device, using Recovery paramount. In this work, we compared two syringe filter models with a similar effective
The overall result shows that a standard and ®
Vivaflow ® 200 set up and run through 451 hmin
25 min 100% filtration area from two suppliers regarding their clarification characteristics of CHO cell
Vivaflow® 200 tangential
Vivapure flow units for cell culture
purification supernatant
95%
straightforward procedure can be followed to ®
Vivaspin
concentration andand
200 Diafiltration
Vivaflow® Lysozyme
diafiltration,
set| ®up
desalting 1
30hmin
20 min 100%
97% culture supernatant samples. To obtain robust results we examined 10 combinations
concentrate, purify, isolate and analyze a protein of run through Vivapure for ion exchange
concentration
chromatography
interest from a cell culturing device, using Vivaflow® 200 followed ®
® by Vivaspin 20 for final sample
of cultivation methods and monoclonal antibody products like IgG1, IgG2, fc fusion
Vivapure
Total purification 45
3 h min
45 min 95%
92%
concentration andVivaspin
tangential flow units for cell culture supernatant desalting.
®
Lysozyme desalting | 30 min 97%
proteins, and bispecific antibodies with regard to turbidity, mAb recovery, relative yield,
concentration and diafiltration, Vivapure ® for ion concentration and throughput. As a result, we found that syringe filter model Minisart® High Flow shows
exchange chromatography, followed by In Vivaspin 20 for
many cases dialysis,
®
Total which is an overnight procedure
3 h 45 min would be
92% an average throughput of 18.0 mL compared to 9.3 mL of another premium brand at cell
final sample concentration and desalting.performed instead of the much quicker alternative ultrafiltration. densities between 38.3 and 163.6 x 105 cells/mL. For the other parameters, we could
Here, we show how time saving and efficient ultrafiltration is not find any significant differences. This finding emphasizes the importance of carefully
In many cases dialysis, which is an overnight procedure, Products used in this experiment Order No.
Fig. 6: Coomassie stained 12% tris-HCl SDS gel loaded for diafiltration
and desalting applications, as well as for protein selecting the syringe filter model that reduces the number of both devices needed and
would be performed instead of the muchwith 20
quickerµl sample
preparations. Lane 1: Marker (SDS Broad range marker); Lane 2: Original sam-
alternative, ultrafiltration. concentration. Vivaflow® 200, PES, 5kDa VF20P1 thus the workload.
ple; Lane 3: Original sample filtrateHere,
(Part 1);we
Laneshow howLane
4: Marker; time-
5: Buffer
saving
exchangeand efficient
concentrate (Partultrafiltration
2); Lane 6: Filtrateisafter
for binding
diafiltration
(Part 3); Lane 7: 500 mL Diafiltration
Products used in thiscup
experiment VFA006
Order No.
Fig. 6: Coomassie
Marker; stained
Lane 8: Filtrate 12%
after tris-HCl
eluting SDS3);gel
(Part loaded with 20 µl sample
and desalting
preparations. Laneapplications,
1: Marker wellLane
as range as9:for
Filtrate after
protein concen-
Vivapure ®
S H Maxi
Vivaflow® 200, PES, 5kDa VS-IX20SH08
VF20P1
trating and desalting (Part 3);(SDS
LaneBroad marker);
10: Concentrate afterLane 2: Original and
concentrating sam-
concentration.
ple; Lane
desalting.3: Original sample filtrate (Part 1); Lane 4: Marker; Lane 5: Buffer
Vivaspin ®
20, 5 kDa cup VS2011
exchange concentrate (Part 2); Lane 6: Filtrate after binding (Part 3); Lane 7: 500 mL Diafiltration VFA006
Marker; Lane 8: Filtrate after eluting (Part 3); Lane 9: Filtrate after concen-
Conclusion Vivapure® S H Maxi VS-IX20SH08
trating and desalting (Part 3); Lane 10: Concentrate after concentrating and
The overall result shows that a standard and straightforward
desalting. ®
Vivaspin 20, 5 kDa VS2011
procedure can be followed to concentrate, purify, isolate and
analyze a protein of interest from a cell culturing device, using
Conclusion
28 29
®
The overall200
Vivaflow tangential
result flowa units
shows that for cell
standard andculture supernatant
straightforward
concentration and diafiltration, Vivapure® for ion exchange
means of the parameters: turbidity, mAb recovery, relative yield, throughput, and filter consumption.
Y O13
In U Rcultivation
PR A C T I C A batches
L G U IDE 10
TOcombinations
BA SIC LA BORATORY
of targetTECHNIQUES
proteins and cultivation methods were used AP P L I C AT I O N NO T E S

(Table 1). In addition to 125 and 1000 mL shaking flasks, 5 L stirred tank reactor (UniVessel, Sartorius)
Introduction
were also used. The cell density and viability was examined with the Vi-CELL XR from Beckman In addition to 125 and 1000 mL shaking flasks, 5 L stirred tank reactor (UniVessel, Sartorius) were also used. The
Coulter. As of
Clarification target proteins,
mammalian cell CHO cell
culture lines were
samples selected with
for preparative mAb from
or analytical different
purposes IgG1 types,
is a necessary IgG2,
step fc
to enable cell density and viability was examined with the Vi-CELL XR from BeckmanCoulter. As target proteins, CHO cell
both a subsequent purification step and a smooth operation of analytical instruments.
fusion protein and a bispecific antibody. The specific designation has been anonymized, due to The overall aim of the clarification lines were selected with mAb from different IgG1 types, IgG2, fc fusion protein and a bispecific antibody. The
step is to removeagreements.
confidentiality cells, cellular debris and other particles from the cell culture while simultaneously allowing the target specific designation has been anonymized, due to confidentiality agreements.
product to be recovered with a sufficient yield. The conventional procedure for clarification of small volumes (approx.
<All
25cell
mL)culture
is a combination of centrifugation
batches were harvestedof the cell
after cultureFrom
14 days. sample followed
every bytwo
batch, a microfiltration
samples were of the supernatant
taken All cell culture batches were harvested after 14 days. From every batch, two samples were taken (max. 31 mL per
obtained.
(max. 31 While centrifugation
ml per sample), one removes
samplecoarse and high-density
destined particles,
for clarification withmicrofiltration
Minisart® Highis frequently
Flow andnecessary
one for to sample), one sample destined for clarification with Minisart ® High Flow and one for another premium brand. The
pull out small or low-density particles from the centrifugation supernatant. Microfiltration can serve as a simultaneous samples were clarified by centrifugation for 60 min at 4000 g, and the supernatants were filtered with the respective
another premium brand. The samples were clarified by centrifugation for 60 min at 4,000 g and the
sterilization step by using 0.2 or 0.22 μm rated sterile filters. syringe filters (Figure 1).
supernatants were filtered with the respective syringe filters (Figure 1).
The mAb titer was determined in the unharvested and harvested cell culture fluid using the Octet QKe system
equipped with a protein A Biosensor (ProA) from FortéBio without any interfering sample preparation.
TheAs sample
recovery waswith different
calculated volumes
by the values between 25 and 31 ml were compared, the
determined. relative mAb
calculated (Equation 1).
𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣 [mL
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 ] × 𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣 �mL �
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣
𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚 × 100% = yield [%]
[
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑠𝑠𝑠𝑠𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑠𝑠𝑠𝑠𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 mL ]
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 × 𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 �mL �
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣
Equation 1: Calculation formula of the relative mAb yield [%].This was necessary to compare the results from d
Equation 1: Calculation formula of the relative mAb yield [%].This was necessary to compare the results from different
sample
sample volumes
volumes ranging
ranging from 24from
mL–3124 - 31
mL. ml.
CCF CCFculture
= cell = cellfluid
culture
(= cellfluid (= cell
culture culture broth).
broth).

As Turbidity
samples withvalues
differentwere measured
volumes between 25 before
and and after
titers (cell clarification
culture) betweenby
0.4 using
and 8.8the TurbiCheck
mg/mL. This WL
31 mL were compared, the relative mAb yield was diversity was the prerequisite for a robust statement in
turbidimeter from Lovibond (white light source). Afterwards the reduction of turbidity was
calculated (Equation 1). terms of the syringe filter suitability.
determined by calculation of the ratio of values from harvested and unharvested samples.
Turbidity values were measured before and after To determine the particle reduction, we examined the
clarification by using the TurbiCheck WL turbidimeter turbidity of the cell culture and the filtrate. We found that
Figure 1: Clarification and sterile filtration of cell culture supernatants under aseptic conditions by using the
syringe filter Minisart® High Flow with a pore size of 0.22 μm.
Results and Discussion
from Lovibond (white light source). Afterwards the both filter models removed particles efficiently from the
reduction of turbidity was determined by calculation supernatant. The filtrate of the Minisart® High Flow showed
Goal of the study was to compare
of the ratio of values from harvested and unharvested the suitability
an average of oftwo
17.6 different
NTU, and other syringe
premium filter models for c
brands
Even though centrifugation removes the vast majority Methods and Materials of mAb supernatants in regard to particle reduction,
samples. showed an average mAbturbidity
recovery, yield,
of 17.7 NTU. and consumptio
Considering the
of particles, clogging of filters is often a problem and In an attempt to facilitate the filtration of cell culture entire clarification process, including centrifugation and
Figure
can 1: Clarification
lead and sterile
to an increased filtration of cell
consumption culture
of filter supernatantssupernatants
devices under aseptic conditions by using theof
for the development syringe filter a
cell lines,
units. and Discussion
Results filtration, this leads to a relative reduction in turbidity
Minisart® High Flow with a pore size of 0.22 µm.
or to ergonomic handling issues. However, both the comparative study was performed. The study was The goal of the study was to compare the suitability between 93.8 and 98.8%. Remarkably, the turbidity in
reduction of filter consumption and the associated executed by using CHO cell culture samples from real Fordifferent
of two the experiments, we used
syringe filter models both various
for clarification of thecultivation
filtrate does notsystems
depend on and theexpression
initial turbidity vectors.
of the Wi
The mAb titer was determined in the unharvested and harvested cell culture fluid using the Octet
operation time can be achieved by a well-considered
QKe system equipped with a protein A Biosensor (ProA) from
projects spread over a period of 3 months. The syringe
FortéBio without any interfering sample
mAbapproach,
supernatants we generated
in regard a heterogeneous
to particle reduction, mAb range
cell of(Figure
culture characteristics
3). with respect to viable ce
choice of the right filtration device. filter models Minisart® High Flow (Sartorius, order no. recovery, yield, and consumption of filter units. The various cell culture samples had titers of monoclonal
preparation. The recovery was calculated by the values determined.
16532-K, 0.22 μm PES membrane, EFA = 6.2 cm2) and viability, turbidity, mAb product, and titer (Table antibodies1). in aIn particular,
range of 0.2 to 8.8theg/L. turbidity
The mAb titersof the cell c
In the present work, we show that a proper choice another premium brand (0.2 μm PES membrane, Forharvest ranged
the experiments, wefrom 457various
used both to 1431 NTU, theofviable
cultivation cell
the filtrate count
ranged fromranged
0.2 to 8.2from
g/L for 4 x 106 to 16 x 10
both
of the syringe filter device for the clarification of EFA = 5.8 cm2) and were examined regarding their systems and expression vectors. With this approach, manufacturers, resulting in recovery rates between 89.9
CHO cell cultures can improve sample throughput and filtration performance by means of the parameters: weagenerated
viabilitya heterogeneous
from 48 to 89 range%,ofand mAb titers and
characteristics (cell culture)
103.9% between
(average: 97.7%) for0.4 and
Minisart ® 8.8 mg/ml. This d
High Flow and
filter consumption without having a negative impact on turbidity, mAb recovery, relative yield, throughput, and was
with thetoprerequisite
respect viable cell count,for a robust
viability, statement
turbidity, mAb 86.9inandterms
107.3% of(average:
the syringe
98.2%) forfilter suitability.
the other premium
turbidity, recovery of mAb product, and total yield. Two filter consumption. product, and titer (Table 1, next page). In particular, the brand. It should be emphasized that the recovery was
common sterile syringe filters available in the market were turbidity of the cell culture at harvest ranged from 457 independent of the cell culture titer (Figure 3). This is
To determine the particle reduction, we examined the turbidity of the cell culture and the
chosen with a pore size of 0.22 μm, slight difference in In 13 cultivation batches, 10 combinations of target to 1431 NTU, the viable cell count ranged from 4 x 10 6 to important when regularly monitoring mAb titers during
effective filtration areas, and a polyethersulfon membrane. proteins and cultivation methods were used (Table 1). 16 We
x 10 6 found
cells/mL,that bothfrom
a viability filter
48 tomodels
89%, andremoved
mAb particles
cultivation withefficiently
different levelsfrom the concentrations.
of product supernatant. The f
30
the Minisart® High Flow showed an average of 17.6 NTU and other premium brand31showe
average turbidity of 17.7 NTU. Considering the entire clarification process, including centri
 Figure 3: The recovery [%] of mAb products in relation to the mAb titer
These results demonstrate that the Minisart® High Flow allows the filtration of larger volumes before
influenced by the syringe filter modelAPand was on average at 97.7 % for
P L I C AT I O N NO T E S
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES clogging while other parameters like turbidity, mAb recovery, and relative yield showed
premium brand. No impact of thethe same
syringe filter used on the mAb recove
high performance. culture titer.
The relative yield Table 1: Overview of various sample types (expression vectors| mAb products) and their parameters
such as cultivation system (STR = stirred tank reactor and SF = shake flask), viable cell count (VCC) 110
per sample was
and viability after 14 days, and turbidity of the cell culture at harvest. Clarification tests were run Turbidity 100
Rel. Yield
the same for both 90
with both syringe filters, so that the respective volume was clarified with both variants to obtain an
syringe filter models, objective comparison. 80
100

Relative yield mAb [%]

despite differences

Turbidity Reduction [%]

[105 cells/mL] cell culture cell culture volume
[NTU] [mg/mL] [mL] 70
in housing design
V1 | IgG1 STR (5 L) 86.9 58% 1431 7.8 31 60
and number of filters 90
V1 | IgG1 STR (5 L) 155.2 78% 1355 6.0 31
used per sample 50
V1 | IgG1 STR (5 L) 163.6 89% 828 8.8 31
(Figure 4). 40 82% 83%
V2 | fc fusion protein SF (25 mL in 125 mL) 121.0 71% 1031 0.2 25 80
V3 | IgG1 SF (25 mL in 125 mL) 73.0 64% 508 0.9 25 30
In terms of
throughput and filter
V4 | fc fusion protein SF (25 mL in 125 mL) 47.7 67% 457 0.4 24 20
70 Minisart High Flow
V5 | IgG2 SF (25 mL in 125 mL) 112.6 67% 873 0.7 23
consumption, we 10
V6 | fc fusion protein SF (300 mL in 1 L) 42.2 69% 701 1.8 25 100% Reduction
determined for each 0
V6 | fc fusion protein SF (300 mL in 1 L) 43.5 62% 834 1.2 25 60
sample the volume Other premium brand Minisart High Flow
V7 | IgG2 SF (300 mL in 1 L) 38.3 48% 821 0.4 25 400 900 1400
of the supernatant, Figure 4: Average values of the relative yield [%] of
V8 | IgG1 SF (300 mL in 1 L) 69.9 73% 558 1.6 25 Turbidity unharvested cell culture [NTU] Figure
the volume of the various4:mAbAverage values The
products. of the relative
relative yieldof[%]
yield of various
a sample is mAb produc
total filtrate, and the V9 | IgG1 SF (300 mL in 1 L) 52.3 59% 669 0.3 25 Figure 2: Turbidity reduction [%] in relation to the total mAb amount
the relation of the in themAb
total filtrate and inin
amount the
theunharvested
filtrate andcell culture. As
Figure 2: Turbidity
turbidity reduction cell
of the unclarified [%] in relationThe
culture. to the turbidity of the unclarified
clarification cell culture.
models The clarification
irrespective of procedure
theculture.
syringe filter designwe andfound
consumption.
required number V10 | bispecific an body SF (300 mL in 1 L) 46.1 69% 671 0.6 25 in the unharvested cell As a result, no
comprises
procedureacomprises
centrifugation and a microfiltration
a centrifugation and a step.
micro-The reduction of the turbidity does not depend on the turbidity of the
difference between both filter models irrespective
of filter units. The cell culture.
filtration ThisThe
step. is valid for the Minisart®
reduction High Flow
of the turbidity does asnot
well as for the other premium brand (figure not shown because the
of the syringe filter design and consumption.
average throughput for Minisart® High Flow was 18.0 mL and for the other premium brand was 9.3 mL and (Figure 5). data points are virtually the same).
depend on the turbidity of the cell culture. This is valid
This 100% discrepancy cannot be explained by the small difference in the effective filtration area (Minisart® High Flow: for the Minisart® High Flow as well as for the other
premium brand (figure not shown because the data
6.2 cm2, other premium brand: 5.8 cm2). More likely, differences in the structural design of the polyethersulfon membrane 24
points are virtually the same).
utilized in the devices could be the reason for this observation. In consequence, we found an average filter consumption 22
Throughput
per sample of 2.5 pcs for Minisart® High Flow and 1.4 pcs for the other premium brand.

Throughput per filter unit [mL]

20
18.0 mL
These results demonstrate that the Minisart High Flow allows the filtration of larger volumes before clogging, while other
® 120 Recovery 18
+ 94%
16
parameters like turbidity, mAb recovery, and relative yield showed the same high performance.
14
100
12
9.3 mL
Conclusion supernatant clarification can be improved substantially 10

Recovery mAb [%]

80
Microfiltration is most commonly an indispensable step after without impairment of other relevant parameters. 8
centrifugation of a cell culture sample. When processing a 6
60
small number of samples with volumes < 25 mL, syringe Acknowledgment 4
filters are often the perfect choice. Using the right filter Special thanks go to Dr. Noushin Delmdahl and 2
can significantly facilitate the task and reduce the number Dr. Andrea Friße for reviewing the manuscript and 40 Other premium brand
0
of devices needed. In this study, we compared two for their constructive discussion on this subject. Minisart High Flow Other premium brand Minisart High Flow
different filter models with slight differences in filtration 20 100% Recovery
Figure 5: CHO cell culture supernatants were filtered
areas (Sartorius Minisart® High Flow: EFA = 6.2 cm2 Abbreviations Figure 5: CHO cell culturesyringe
supernatants were filtered through two different sy
through two different filter models: Sartorius
and other premium brand: EFA = 5.8 cm2). However, 0 Minisart High Flow and other premium brand. Theper filter unit was d
Flow and ®other premium brand. The average throughput
CHO Chinese hamster ovary models in throughput
throughput were probably
no significant difference in terms of turbidity, recovery, 0 1 2 3 4 5 6 7 8 9 10 average per filter unitnot
wascaused by the deviation in effective
determined.
other premium brand: 5.8 cm 2) but most likely by differences in structural me
and yield per sample could be found. What could be mAb monoclonal antibody mAb titer unharvested cell culture [g/L] Differences between both filter models in throughput
observed is a clear impact of the filter model on the were probably not caused by the deviation in effective
EFA effective filtration area
filtration performance. With its 7% larger filtration area, the
Figure 3: The recovery [%] of mAb products in relation to
Figure 3: The recovery [%] of mAb products in relation to the mAb titer of Conclusion
filtration area (Minisart® High Flow: 6.2 cm2, other
the unclarified
premium brand: cell
5.8 culture.
cm2) butThemost recovery
likely was not
by differences in
PES polyethersulfon the mAb titer of the unclarified cell culture. The recovery
Minisart® High Flow achieved a 94% higher filtrate volume influenced
was by the syringe
not influenced filter
by the modelfilter
syringe and model
was onand average Microfiltration
was at 97.7 % forstructural
the Minisart® High
membraneis most
Flow commonly
and
design. at 98.2 %an for indispensable
the other step after c
per device and thus halved the number of filter units per NTU nephelometric turbidity units premium
on averagebrand. No impact
at 97.7% of Minisart
for the the syringe
® filterFlow
High usedand
on the
at mAb recoveryWhenwas observed
processing in aarange
smallofnumber
0.3 to 8.8ofg/lsamples
of the cell
with volumes < 2
sample. This finding emphasized that the efficiency of culture for
98.2% titer.
the other premium brand. No impact of the perfect choice. Using the right filter can significantly facilitate t
syringe filter used on the mAb recovery was observed in
a range of 0.3 to 8.8 g/L of the cell culture titer. devices needed. In this study, we compared two different filter
100
Rel. Yield filtration areas (Sartorius Minisart® High Flow: EFA = 6.2 cm2 a
90 cm2). However, no significant difference in terms of33turbidity, r
32
80 be found. What could be observed is a clear impact of the filte
[%]
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Abstract using Sartorius’ quantified CFU and GC standards to

In this study, the correlation between genome copies (GC) facilitate implementation and approval of qPCR-based

Correlation Between Colony Forming Units and

and colony forming units (CFU) of 9 different Mycoplasma Mycoplasma detection methods.
species has been investigated using Sartorius’ quantified
CFU and GC standards. As PCR technology only detects Materials and Methods
Genome Copies of 9 Different Mycoplasma genome copies (GC), a correlation between colony
forming units (CFU) and GC is required by different
DNA Extraction of Microsart ® Validation Standard
Each package of Microsart® Validation Standard contains
Species
How Using
to Avoid Quantified CFU
Contamination in and GC Standards
Pipetting
authorities, i.e., the Korean Food and Drug Administration
(KFDA) or the Pharmaceuticals and Medical Devices
3 vials, each containing 10 CFU of the chosen Mycoplasma
species. 250 μL Dulbecco’s Modified Eagle Medium

for Validation Agency (PMDA), Japan, before an assay is accepted to be

used in quality control of cell culture products. The results
(DMEM) + 10% fetal bovine serum (FBS) were added to
two vials to prepare a suspension with a concentration of
#05 of this study show that the number of genome copies vary 40 CFU/mL. 500 μL DMEM +10% FBS were added to one

Correlation between colony forming units and

Caroline Paeschke1, Dr. Alexandra Mueller-Scholz1*, Dr. Susanne Roederstein2, Kai Nesemann2
between Mycoplasma species, but have successfully been
correlated to 20 CFU and 40 CFU respectively.
vial to prepare a suspension with a concentration of 20
CFU/mL. The DNA of the cell suspensions was extracted
Application
genome copies of 9 different Mycoplasma#01 species Introduction
1.Research and Development Department, Sartorius Stedim Biotech, Göttingen, Germany
Note
with Microsart® AMP Extraction Kit in duplicates according
to the protocol. The eluate was used directly for Microsart®

using quantified CFU and GC standards #02 for validation

2. Department Microbiology, Sartorius Lab Instruments, Göttingen, Germany Mycoplasma are the smallest free-living organisms. ATMP Mycoplasma qPCR.
*
Correspondence: They belong to the bacterial class Mollicutes, which are
Practical methods
E-mail: PCR@Sartorius.com distinguished by their lack of cell wall. For that reason they Microsart ® ATMP Mycoplasma qPCR
for avoiding
contamination in are unaffected by many commonly antimicrobial All lyophilized components were rehydrated. For one
agents such as beta-lactam antibiotics [1]. Mycoplasma reaction, 15 μL of Mycoplasma Mix were mixed with
pipetting. #03 are widespread contaminants in cell culture. In fact, 1 μL Internal Control DNA. 15 μL of this mix were added
depending on the laboratory, 10% to 85% of cell lines to each PCR tube. Each test was carried out with at

#04 #01
may be contaminated [2]. Due to their extremely basic
genomes, mycoplasma live as parasites. They compete
least two Non-Template Controls (NTC) and samples in
duplicate. Therefore 10 μL of sample or NTC were added
with host cells for biosynthetic precursors and nutrients to the PCR tubes with Master Mix respectively.
Application
and can alter DNA, RNA, and protein synthesis and induce
chromosomal alterations [2]. Given their tiny size (~0.3 μm– Standard Curve with Microsart® Calibration Reagent
Note #02
0.8 μm) mycoplasma contamination cannot be visualized
with a light microscope [1]. Moreover, altered growth rates
To quantify the DNA extracts of Microsart ® Validation
Standards, it is necessary to generate a standard
and morphological changes in infected cell cultures can curve with known concentrations of genome copies
be minimal or unapparent. Furthermore, mycoplasma- (GC). Therefore Microsart ® Calibration Reagents were
contaminated products represent a human health risk [2]. used. The calibration reagents contain 10 6 GC/μL of

#03
All these facts show clearly the high demand of routine
mycoplasma detection.
the specific organism after rehydration. Dilution series
havebeen prepared in Tris-buffer to achieve final
concentrations of 5 GC/10 μL to 500 GC/10 μL.
Microsart® ATMP Mycoplasma enables a reliable and
sensitive detection of mycoplasma DNA in cell cultures and Microsart ® ATMP Mycoplasma qPCR was performed

#04
cell culture derived biologicals, like autologous transplants,
according to European Pharmacopeia 2.6.7. Regulations
require comparability studies with compendial growth based
methods. As PCR technology only detects genome copies
(GC), a correlation between colony forming units (CFU) and
on the CFX96 Touch Cycler (Bio-Rad; 45 cycles,
3 min 95°C, 30 s 95°C, 30 s 55°C, 45 s 60°C). The
mycoplasma DNA is indicated by an increasing
fluorescence signal in the FAM ® channel. Internal
Control DNA is detected in the ROX® channel in the

#05
GC is required by different authorities, i.e., the Korean Food
and Drug Administration (KFDA) or the Pharmaceuticals and
same tube to indicate a successful reaction in every
individual PCR tube. The analysis of the reaction was
Medical Devices Agency (PMDA), Japan. done with the CFX Manager Software (Bio-Rad). The
limit of detection of all Mycoplasma species listed in
In this study, the correlation between CFU and GC the EP/USP is ≤ 10 CFU/mL.
of 9 different Mycoplasma species was investigated

34 35
the protocol. The eluate was used directly for Microsart® ATMP
Mycoplasma qPCR.
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

Results and Discussion

Table 1: Product overview of Microsart® Validation Standards and Microsart® Calibration Reagents for different mycoplasma species.
Table 1: Product overview of Microsart® Validation Standards and Microsart® Calibration Figureafter 2 and Reagents for different
3 show exemplary Mycoplasma
amplification plots of The study indicated that the GC/CFU ratio varied from approval. These results demonstrate a good data
The Validation Standard contains 10 CFU per vial, the Calibration Reagent contains 106 GC/µl rehydration.
species.The Validation Standard contains 10 CFU per vial, theResults Calibration andReagent
Discussioncontains
A. laidlawii . On10
6
basis GC/μL
of theafter rehydration.
ct values (FAM™ channel) and species to species and lies within a range of 9 GC/CFU basis. Nevertheless it should be kept in mind that
concentrations of the standards the CFX Manager software
Figure 2 andCatalog 3 show No. exemplary amplification Catalog plots No. of to 68 GC/CFU after DNA extraction. significant variations within the GC:CFU ratio might be
created a standard curve (Fig. 1) with a linear equation.
A. laidlawii. On basis of® the ct values (FAM™ channel) and observed if other media | matrices are used for the CFU
Mycoplasma
Mycoplasma species NCTC code ATCC code
concentrations
Results
MicrosartA regression
of standard
and
Validation
the standards
Discussion
coefficient
the
of 0.983
Microsart
CFX Manager
®
is an indication for a good
Calibration
software
Standard curve. The Reagent
efficiency of an optimal qPCR spikes, which affects the DNA isolation efficiency, or if
created a standard Figurecurve 2 and
(Fig. 31)show
with exemplary
a linear amplification
equation. plots isof100 %.
Mycoplasma arginini 10129 23838 Results and Discussion
In
SMB95-2011 that
A. laidlawii case the
. On basis ampliconSMB95-2021DNA will be doubled in eachandcycle. other conditions are used for correlation. The results
A regression coefficient
According of 0.983to the anofindication
isanalysis the ct values
of the for a(FAM™
CFX good channel)
Software the exemplary
concentrations ofanthe standards theisofCFX of this study indicate a higher GC than CFU number, as
Mycoplasma orale 10112 23714 Figure 2 and
standard SMB95-2012
curve. 3 show
The qPCR
exemplaryofamplification
efficiency
run of A. laidlawii
SMB95-2022
optimal ran
plots
qPCR
highly 100Manager
efficient%. with an software
efficiency
A. that
laidlawii created a standard curve (Fig. 1) with a linear equation. expected. The theoretical GC:CFU ratio should be
Mycoplasma gallisepticum 10115 19610 In case. On
the basis
SMB95-2013
ampliconof the
of 101 %DNA ct values
(see will be(FAM™
efficiency SMB95-2023
doubled channel)
in Fig 1). eachand
in cycle.
concentrations theofAanalysis
According toSMB95-2014 regression
the standards
of the coefficient
the CFX
CFX of
Software 0.983
Manager theissoftware
an indication for a good
exemplary 1:1, as 1 GC per cell should ideally be detected.
Mycoplasma pneumoniae 10119 15531 SMB95-2024
standard curve. The efficiency of an
Mycoplasma synoviae 10124 25204
created
qPCR runa standard curveran
A. laidlawii
of SMB95-2015 (Fig. 1) with
highly a linearwith
efficient
SMB95-2025 an optimal
equation. efficiency qPCR is 100 %.
Figure 3: Exemplary amplification plot of Acholeplasma laidlawii, generated
Practically, this ratio is not realizable even if
A regression In that case the amplicon DNA will be doubled in each cycle. mycoplasma cultures are harvested during early
of 101 % (seecoefficient
efficiency of in 0.983
Fig 1). is an indication for a good with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™
Mycoplasma fermentans 10117 19989 standard curve. SMB95-2016
According
The efficiency to theof an SMB95-2026
analysis
optimal of the
qPCR CFX is Software
100 %. the exemplary channel. Black Lines: Non template Control (NTC). Red: 5 GC/PCR. logarithmic growth to prevent detection of DNA
Mycoplasma hyorhinis 10130 17981 qPCR
SMB95-2017 run of A. laidlawii
In that case the amplicon DNA will be doubled in each cycle.with an efficiency
ran
SMB95-2027 highly efficient Blue: 10 GC/PCR. Green: 50 GC/PCR. Yellow: 100 GC/PCR. Brown: 500 GC/PCR.
of 101 % (see efficiency in Fig 1). Figure 3: Exemplary amplification plot of Acholeplasma laidlawii, generated from dead cells in the preparation. This non-equal ratio
Acholeplasma laidlawii 10116 23206 According toSMB95-2018
the analysis of the CFX Software SMB95-2028 the exemplary with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™
20 CFU/ml and 40 CFU/ml of Red:
each5mycoplasma species have been arises because a significant number of the mycoplasma
Spiroplasma citri 10164 27556 A. laidlawii ran highly efficient
qPCR run of SMB95-2019 SMB95-2029 with an efficiency channel. Black Lines: Non template Control (NTC). GC/PCR.
detected
Figure
Blue: 10 GC/PCR. Figure 3: successfully
Exemplary in all
amplification samples
plot of (Assay LOD is ≤ 10 CFU/ml;
500Acholeplasma
Acholeplasma laidlawii, generated cells would not grow to a colony in culture and remain
of 101 % (see efficiency in Fig 1). Green: 50 3:GC/PCR.
Exemplary Yellow:amplification
100 GC/PCR. Brown: plot of GC/PCR.
with Microsart
laidlawii,
determined ®
ATMP Mycoplasma
during
generated kit with
validation). qPCR. Fluorescence
Microsart ® signals in FAM™
ATMP Mycoplasma undetected (i.e., stressed or viable but non-culturable
Results and Discussion 20 CFU/ml
Figure 3: Exemplary
channel. Black Lines: Non template Control (NTC).
andqPCR.
Blue:
Fluorescence
40amplification
CFU/ml
10 GC/PCR. of plot signals in laidlawii
eachofmycoplasma
Green: Acholeplasma
50 GC/PCR. Yellow:
FAM
species ® Red: 5 GC/PCR.
channel.
have
100 ,GC/PCR. been
generated
Black Lines:
Brown: 500 GC/PCR.
cells). Furthermore, mycoplasma cells tend to form
Figures 2 and 3 show exemplary amplification plots detected
with Non-Template
Based
successfully
Microsart ® on the
in all samples
ATMP Mycoplasma Control
linear equation(NTC). of
(Assay LODsignals
qPCR. Fluorescence Red:
the 5 GC/PCR.
standard
FAM™ Blue: 10 GC/
is ≤ 10in CFU/ml;curve the software agglomerates, which would be detected as 1 CFU,
determined
channel. Blackduring PCR. Green:
calculated the
kittemplate 50
validation).GC GC/PCR.
concentrations Yellow: of 100
the GC/PCR.
mycoplasma Brown:
samples
of A. laidlawii. On basis of the ct values (FAM ® Lines: Non
20 CFU/ml and Control (NTC). Red:
each5 mycoplasma
GC/PCR. but in fact combine several cells and therefore
Blue: 10 GC/PCR. 500
(20 GC/PCR.
CFU/ml
Green: 50 and 40
GC/PCR. 40 CFU/ml
CFU/ml
Yellow: 100
of
extracts).
GC/PCR. In table
Brown: 500
species
2 GC/PCR.
the averagehave
GCbeen
to
Microsart
channel) and
®
ATMP Mycoplasma qPCR
concentrations of the standards, the detected successfully in all samples (Assay LOD is ≤ 10 CFU/ml; several GC. Both scenarios lead to a significant
All lyophilized components were rehydrated. For one reaction Microsart® ATMP Mycoplasma qPCR was performed on the CFX96
CFU ratios of 9 different mycoplasma species are shown.
CFX Manager software created a standard curve Based on the linear equation
determined duringof thekit standard
validation). curve the software underestimation of the realistic mycoplasma cell
Figure45 1: cycles,
Exemplary standard 30 sof95Acholeplasma
95 °C,curve °, 30 s 55 °C,laidlawii (Microsart
®
15 µl of Mycoplasma Mix were mixed with 1 µl Internal Control Touch Cycler (Bio-Rad; 3 min 20 CFU/ml the
calculated andGC 40 concentrations
CFU/ml of each of mycoplasma
the mycoplasma speciessamples
have been
(Figure
DNA. 15 1) with
µl of thisa mix
linear
wereequation. A regression
added to each PCR tube. coefficient
Each test Calibration Reagent), using final Genomic Copy (GC) concentrations of
45 s 60 °C). The mycoplasma DNA is indicated by an increasing ® detected successfully in all samples (Assay LOD is 10 CFU/ml; number in the sampled cell culture, as only a portion
(20 CFU/ml andTable 2: Average
40 CFU/ml
Based theextracts). GC to
In CFU
table ofratio
theofaverage
2the ≤ 9 different
GC tothe software
of 0.983
carriedisout
an with
indication
at leastfor
twoaNon
good standard curve. The 5 GC/10 µl to 500 GC/10 µl. generated with Microsart ATMP Mycoplasma. Table 2:onAverage linear equation
GC to CFU ratio of 9 standard curve
different mycoplasma species of the cells would grow to form a CFU. Non-culturable
was Template Controls (NTC) fluorescence signal in the FAM™ channel. Internal Control DNA determined
CFU ratios ofduring 9Mycoplasma thespecies.
kit validation).
different
calculated mycoplasma species areofshown.
GC concentrations the mycoplasma samples
efficiency
and samplesofinan optimalTherefore
duplicate. qPCR is10100%. In thatorcase
µl of sample the
NTC were is detected in the ROX™ channel in the same tube to indicate a
(20 CFU/ml and 40 CFU/ml extracts). In table 2 the average GC to species or viable but non-culturable cells could lead to
Figure 1: Exemplary standard curve of Acholeplasma laidlawii (Microsart®
added
ampliconto the PCR will
DNA tubesbewith MasterinMix
doubled respectively.
each cycle. According successful reaction in every individual PCR tube. The Analysis of Based Mycoplasma
on the linear equation Species Average GC to CFU ratio false-negative results using a growth-based method.
Calibration Reagent), Each using sample
final Genomic and Copy (GC) concentrations
Non-Template Control of showed an
(NTC) amplifi- CFU ratios of 9 of the standard
different mycoplasmacurve the software
species are shown.
the reaction was done with the CFX Manager Software (Bio-Rad). the Mycoplasma
to the analysis of the CFX Software, the exemplary 5 GC/10 µl to 500 GC/10 µl.1:generated
cation
Figure of the
Exemplary with
internal Microsart
standard control
curve
®
ATMP
DNA
of Mycoplasma.
and
Acholeplasmaconsequently
laidlawii calculated
Table®2: Average
fluorescence
(Microsart to CFU arginini
GCGCconcentrations ratio ofof9 the mycoplasma
different 1.1 × samples
mycoplasma 10 GC/CFU
species Undetected mycoplasma contamination because of
The limit of detection of all mycoplasma species listed in the (20
qPCR run of A. laidlawii ran highly efficient with an signal
Calibration in the ROX™
Reagent), channel
using (Ct<40;
final Genomic data
Copy not
(GC) shown).
concentrationsA ofCFU/ml andMycoplasma
success- 40 CFU/ml extracts).
orale In table 2 the 3.5average GC to
× 10 GC/CFU false-negative results in growth-based methods can
EP/USP is ≤ 10 CFU/ml.
efficiency of 101% (see efficiency in Figure 1). 5 fully
GC/10PCR µl towithout
500 GC/10 µl. generated with Microsart ®
ATMP
inhibition was indicated. The NTC didMycoplasma CFU
Mycoplasma. ratios
not show of 9 different
Table 2: Average
Mycoplasma mycoplasma species
GC toAverage
gallisepticum CFU ratioGC are shown.
of 1.7
9 different
× 10 ratiomycoplasma species
GC/CFU result in unsafe products with potential infection risks,
Species to CFU
Each
Figure sample
1: and
Exemplary Non-Template
a fluorescence
standard curve of
Figure 1: Exemplary standard curve of Acholeplasma
Control
signal
Acholeplasmain (NTC)
the FAM™showed
laidlawii an
channel, amplifi-
(Microsart as
®
expected (see Fig. 2
Mycoplasma arginini Mycoplasma pneumoniae 1.1 × 10 GC/CFU 4.3 × 10 GC/CFU especially for patients with immunodeficiency. This
cation of(Microsart
Calibration
laidlawii the internal
Reagent), and® control
using 3).final
Calibration DNAReagent),
Consequently
Genomic and
Copy aconsequently
mycoplasma
(GC)using
concentrations
final fluorescence
free preparation
of
genome of the PCR Mycoplasma Species Average GC to CFU ratio
Each sample and Non-Template Control (NTC) showed Mycoplasma study shows that the correlation between GC and
5 GC/10in
signal
copy
µl the
(GC)
to 500
ROX™ Each
GC/10 sample
µl.
channel
reactions
concentrations
generated and
(Ct<40;
without
of 5 GC/10Non-Template
with Microsart
data μLnot
ATMPControl
shown).
®
cross-contamination
to 500 GC/10 (NTC)
Mycoplasma.
A success-
was showed an
μL indicated. Mycoplasma
amplifi-
Table 2: Average oraleGC to CFU synoviae
ratio of 93.5 × 10 GC/CFU
different 0.9 × 10 GC/CFU
mycoplasma species
an amplification of the internal control DNA and cation of the internal control DNA and consequently fluorescence Mycoplasma fermentans
Mycoplasma arginini 1.1××10
1.2 10GC/CFU
GC/CFU CFU can successfully be demonstrated and easily be
fully PCR without
generated inhibitionATMP
with Microsart ® was indicated.
Mycoplasma. The NTC did not show Mycoplasma gallisepticum 1.7 × 10 GC/CFU
consequently fluorescence signal in the ROX® channel a fluorescence signal signalininthe theFAM™ROX™channel,
channel as (Ct<40;
expected data(seenot Fig.
shown).2 A success- Mycoplasma hyorhinis
Mycoplasma orale 3.5××10 10GC/CFU
GC/CFU implemented during validation. Furthermore, detection
Mycoplasma
Mycoplasma Species
pneumoniae Average
4.3 × 10 GC/CFU GC 0.9to CFU ratio
(Ct<40; data not shown). A successful PCR without Each sample and fully PCR
Non-Template without inhibition
Control
and 3). Consequently a mycoplasma free preparation of the PCR (NTC) was indicated.
showed an The
amplifi- NTC did not show Mycoplasma gallisepticum 1.7 × 10 GC/CFU of GC by PCR shows a more realistic result of the
Mycoplasma
Mycoplasma Acholeplasma laidlawii1.1
arginini
synoviae 0.9 ×
× 10
10 GC/CFU
GC/CFU5.6 × 10 GC/CFU
inhibition was indicated. The NTC did not show a cation of the a
internalfluorescence
control DNA
reactions without cross-contamination was indicated.signal and in the FAM™
consequently channel, as
fluorescence expected (see Fig. 2 real contamination level in the respective sample and
Mycoplasmacitri
Spiroplasma pneumoniae 4.3××10
6.8 10GC/CFU
GC/CFU
signal in the ROX™ andchannel
3). Consequently
(Ct<40; data a mycoplasma
not shown).free preparation of Mycoplasma
A success- the PCR
Mycoplasma orale
fermentans 3.5
1.2 × × 1010 GC/CFU
GC/CFU
fluorescence signal in the FAM® channel, as expected Mycoplasma synoviae 0.9 × 10 GC/CFU therefore directly contributes to drug safety.
(see Figures 2 and 3). Consequently a mycoplasma-
fully PCR withoutreactions inhibition without cross-contamination
was indicated. The NTC didwas not indicated.
show Mycoplasma hyorhinis
Mycoplasma gallisepticum 1.7 ×
0.9 × 1010 GC/CFU
GC/CFU
a fluorescence signal in the FAM™ channel, as expected (see Fig. 2 Mycoplasma fermentans 1.2 × 10 GC/CFU
free preparation of the PCR reactions without cross- Mycoplasma
Acholeplasma pneumoniae
The study indicated that
laidlawii 4.3
5.6 ×
the
× 10 GC/CFU
GC/CFU
10 GC/CFU ratio varied from species to
and 3). Consequently a mycoplasma free preparation of the PCR Mycoplasma hyorhinis 0.9 × 10 GC/CFU
contamination was indicated. Mycoplasma
Spiroplasma citri Discussion
species
synoviae and lies within0.9 a range
× 10 of 9 GC/CFU to 68 GC/CFU after
GC/CFU References
reactions without cross-contamination was indicated. Acholeplasma
DNA extraction. laidlawii6.8 × 10 GC/CFU 5.6 × 10 GC/CFU
3 Mycoplasma fermentans In this study, the correlation1.2 × 10 GC/CFU between genome copies 1. Drexler HG, Uphoff CC. Mycoplasma contamination
Spiroplasma citri 6.8 × 10 GC/CFU
20 CFU/mL and 40 CFU/mL of each Mycoplasma Mycoplasma hyorhinis (GC) and colony forming 0.9 × 10units GC/CFU (CFU) of 9 different of cell cultures: Incidence, sources, effects, detection,
species have been detected successfully in all samples The study indicated that the GC/CFU
Mycoplasma species ratio
has varied
beenfrom species to using
investigated elimination, prevention. Cytotechnology. 2002;39(2):75-90.
Figure 2: Exemplary amplification plot of Acholeplasma laidlawii, generated Acholeplasma
species and lies
laidlawii
within a range of 9
5.6 × 10 GC/CFU
GC/CFU to 68 GC/CFU after
(Assay LOD is ≤ 10 CFU/mL; determined during kit Sartorius’
The quantified
study indicated that GCGC/CFUand CFU ratiostandards.
varied from A doi:10.1023/A:1022913015916.
6.8the species to
with Microsart ATMP Mycoplasma qPCR. Fluorescence signals in FAM™
®
Spiroplasma
DNA extraction.citri × 10 GC/CFU
validation). channel. Black Lines: Non template Control (NTC). Blue Lines: 20 CFU/ml of correlation
species and lies between
within a CFU range and GC is required
of 9 GC/CFU to 68 GC/CFU by after 2. Olarerin-George AO, Hogenesch JB. Assessing the
A. laidlawii. Red Lines: 40 CFU/ml of A. laidlawii.
different
DNA authorities, i.e., the Korean Food and Drug
extraction. prevalence of mycoplasma contamination in cell culture via
Based on the linear equation of the standard curve, The study indicated Administration
that the GC/CFU (KFDA) ratioorvaried
the Pharmaceuticals
from species to and a survey of NCBI’s RNA-seq archive. Nucleic Acids Research.
Figure 2:2:Exemplary
Figure Exemplary amplification
amplification plot of Acholeplasma
plot of Acholeplasma laidlawii, generated
the software calculated the GC concentrations of the with Microsart ATMP Mycoplasma qPCR. Fluorescence
®
signals in FAM™ Medical Devices Agency
species and lies within a range of 9 GC/CFU to 68 GC/CFU after (PMDA), Japan, for assay 2015;43(5):2535-2542. doi:10.1093/nar/gkv136.
laidlawii, generated Figure with Microsart
2: Exemplary ®
ATMP Mycoplasma
amplification plot of Acholeplasma qPCR. laidlawii, generated
mycoplasma samples (20 CFU/mL and 40 CFU/mL channel. Black Lines: Non template® Control (NTC). Blue Lines: 20 CFU/ml of DNA extraction.
Fluorescence signals in FAMATMP
with Microsart ®
channel.
Mycoplasma Black Lines:
qPCR. Fluorescence signals in FAM™
extracts). In Table 2, the average GC to CFU ratios of A. laidlawii. Red Lines: 40 CFU/ml of A. laidlawii.
Non-Template Control (NTC). Blue Lines: 20 CFU/mL of
channel. Black Lines: Non template Control (NTC). Blue Lines: 20 CFU/ml of
9 different Mycoplasma species are shown. A. laidlawii. Red A. Lines:
laidlawii40. RedCFU/mL
Lines: 40of A. laidlawii.
CFU/ml of A. laidlawii.
Figure 2: Exemplary amplification plot of Acholeplasma laidlawii, generated
36 with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™ 37
channel. Black Lines: Non template Control (NTC). Blue Lines: 20 CFU/ml of
A. laidlawii. Red4 Lines: 40 CFU/ml of A. laidlawii.
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES Centrifugal Ion Exchange Membrane Absorbers AP P L I C AT I O N NO T E S

C. Naumann and N. Kashani-Poor

In contrast to traditional column chromatography
methods, Vivapure IEX centrifugal columns allow
Introduction In the first step of this protocol, binding con- Buffers used
scouting of
For separation and purification ofseveral
proteins chromatography conditions
ditions are evaluated in the sample
by loading Buffer A: 25 mM citrate, pH 4
parallel,
from biological samples, leading
different quickly to
character- ondifferent
Vivapure Qfractions which
and S columns at various pH-
istics of the target protein
can bee.g. its size,analyzed
further charge, forvalues, eluting or
enriched bound
even proteins
already with a high Buffer B: 25 mM potassium phosphate,
hydrophobicity or specifically engineered tags salt concentration buffer and analyzing all pH 6
purified target protein.
are exploit. fractions for the target protein. This step Buffer C: 25 mM HEPES, pH 8
results in the optimal binding pH and the best
Scouting Protein
How to Avoid Purification
Contamination in PipettingConditions separation is achieved
Here, we demonstrate the
With ion exchange chromatography,
on the basis of
ionperformance
exchange chemistry
Mini spin columns for evaluation of optimal purification
of Vivapure IEX
for the purification. Buffer D: 25 mM sodium bicarbonate,
pH 10
different charges of biomolecules. This makes In a second step, the best elution method is Buffer E: 25 mM citrate, pH 4, supple-
Using Vivapure Centrifugal Ion Exchange conditions
it to a versatile method often used
in a two-step
fractionation or purification
offor
cloned
pre- SH2evaluated
procedure.
of a target pro-
domains byfrom
This protocol
trations
applying
to columns
an increasing
can
E. coli lysate
generally
which
salt concen-
be to
were shown
mented with 1 M NaCl.
25 mM potassium phosphate, pH 6,
tein from crude protein mixtures.for Tofinding bind the target
optimizea purification proteinbased
in stepon leading to Buffer F:
one,ion
#05
Membrane Absorbers
employed method supplemented with 0.2 M, 0.4 mM,
the purification procedure for an individual a complete purification protocol in less than
exchange
target, several binding and elution chromatography
conditions onefor a given target protein, as
hour.
0.6 mM, 0.8 mM,
it is fast and only uses up small amounts of the sample. & 1 M NaCl, respectively.
have to be tested on cation and anion
Application #01 exchange matrices. Experiment Buffer G: 25 mM HEPES, pH 8,
Using the described scouting procedure, supplemented with 1 M NaCl
In the first step of this protocol, binding conditions are
Note In contrast to traditional column chromatog-
evaluated
a purification method for a SH2 domain
by loading the expressed
sample on Buffer H: 25 mM sodium bicarbonate,
C. Naumann and N. Kashani-Poor raphy methods, Vivapure IEX centrifugal in E.Vivapure Q and S In a first
coli was developed. pH 10, supplemented with
columns allow scoutingcolumns at various
of several chro- pH values, eluting
step, proteins bound
were bound proteins
to the Vivapure IEX
Practical methods #02 matography conditions withinaparallel,
high salt leading membranes
concentration buffer at and
different pH values, then
analyzing
1 M NaCl
for avoiding quickly to different fractions which can be eluted with high-salt buffer. In Step Two a
contamination in further analyzed forall fractions
enriched for already
or even the targetfresh
protein.
sampleThis
was step results
adjusted to the respective
in the optimal binding pHpH
purified target protein. and the best
elucidated ion exchange
previously as the best choice
pipetting. #03 chemistry for the purification. for binding the protein and was loaded onto
Introduction Here, we demonstrate the performance of
Vivapure IEX Mini spin columns for evaluation conditions.
a new column for refining optimal elution
Procedure
In the
of optimal purification second
conditions of step,
clonedthe best elution method is evaluated
#04
For separation and purification of proteins from biological samples, different SH2 domains from an byE.applying
coli lysateincreasing
in a two salt concentrations to columns
Materials Step One: Scouting for Binding Conditions to
characteristics of the target protein, e.g., its size, charge, hydrophobicity, or step procedure. Thiswhich
protocol can generally be – Vivapure Mini
were shown to bind the target protein in step
employed for finding a purification method
Q H spin columns
– Vivapure Mini S H spin columns
the Appropriate Ion Exchange Chemistry
one, leading to a complete purification protocol in less
specifically engineered tags, are exploited. based on ion exchange chromatography for a – Minisart syringe filter (0.45 µm CA,
Expression of Target Protein
given target proteinthan one
as it is fasthour.
and only uses Sartorius AG)
up small amounts of the sample. – Centrifuge, 45°-fixed-angle rotor; 2000 300 + g mL LB media were inoculated with 4 mL of an
With ion exchange chromatography, separation is achieved on the basis of Experiment overnight culture and incubated at 37°C, shaking at
Using the described scouting procedure, a purification 150 rpm until an OD600 of 1.0 was reached. IPTG was
different charges of biomolecules. This makes it a versatile method often method for a SH2 domain expressed in E. coli was added to a final concentration of 1 mM and incubated for
used for prefractionation or purification of a target protein from crude protein developed. In Step One, proteins were bound to further 4 h with shaking at 150 rpm. Cells were harvested
mixtures. To optimize the purification procedure for an individual target, the Vivapure IEX membranes at different pH values, by centrifugation at 4000 x g for 30 min at 4°C. The
then eluted with high-salt buffer. In Step Two, a fresh pellet was resuspended in 35 mL PBS (150 mM KPi, pH
several binding and elution conditions have to be tested on cation and anion sample was adjusted to the respective pH elucidated 7.3) and cells were lysed by addition of lysozyme to a
exchange matrices. previously as the best choice for binding the protein final concentration of 0.1 mg/mL and incubation for 1 h
and was loaded onto a new column for refining optimal at 37°C. Insoluble particles as cell debris were removed
elution conditions. by centrifugation at 10000 x g for 30 min at 4°C.

Materials Sample Preparation

– Vivapure Mini Q H spin columns
– Vivapure Mini S H spin columns
– Minisart syringe filter (0.45 μm CA, Sartorius AG)
– Centrifuge, 45°-fixed-angle rotor; 2000 x g
4 x 200 μL of the cell lysate were diluted with 1.8 mL
binding buffer A to D each, to adjust the sample to the
respective pH conditions. In order to avoid clogging
of the membranes in the Vivapure Mini spin columns,
samples were clarified by passage through Minisart
syringe filters.

38 39
 Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES AP P L I C AT I O N NO T E S

re Step Two: Optimizing elution conditions

Column Equilibration pH = 6 Loosely bound proteins were washed away by
e: Scouting pH = 6 pH = 8 pH =Sample
10 preparation
4 x for binding
Q and 4 x Sconditions
Vivapure Taking account of the results of Step One, application of 400 μL binding buffer to the column and
ppropriate ion exchange
Mini spin columnschemistry.
were 200 µl cell lysate were diluted with 1.8 ml spun for 5 min at 2000 x g. Flow-through and wash
binding buffer B (25 mM KPi, pH 6). In order
labeled 4, 6, 8, and 10, to avoid clogging of the membrane in the fraction were saved for analysis.
on of target protein Vivapure Mini spin column, the pH adjusted 66 kDA
corresponding
B media were inoculated to withthe4 pH of
ml of 66 kDA sample was clarified by passage through a
theand
ight culture buffer to be used.
incubated To
at 37°C, Minisart syringe filter. Stepwise Elution
45 kDA
at 150 rpm untilspin
each an OD600
column, of 1.0 was Target 100 μL elution buffer F, supplemented with 0.2 M NaCl,
Column equilibration
IPTG was400added to a
μL of the final concentra- 45 kDA 400 µl binding buffer B were applied to one protein were applied to the Vivapure S Mini spin column and
mM and corresponding
incubated for further 4h Target
Vivapure S Mini spin column and spun for
binding protein
5 minutes at 2000 + g.
spun for 3 min at 2000 x g. The eluate was collected. In
king at 150 rpm. Cells were harvested 31 kDA
ifugation buffer
at 4000were added and
+ g for 30 min at Binding and washing
the next step, 100 μL of elution buffer F, supplemented
pellet was resuspended inat352000
spun for 5 min x g.
ml PBS 31 kDA 400 µl of the clarified sample were applied with 0.4 M salt, were applied and again spun for 3 min at
M KPi, pH 7,3) and cells were lysed by to the equilibrated Vivapure S column and 22 kDA 2000 x g. Elution was continued until the entire gradient
spun for 5 min at 2000 + g. Afterwards, the
Binding
of lysozyme and
to a final Washing
concentration Vivapure S Mini spin column was reloaded had been tested, saving the eluates from each step.
g/ml and incubation
400 μL of the 1clarified
for h at 37°C. with 400 µl sample and spun again for 5 min
e particlessamples
as cell debris were
adjusted to
removed at 2000 + g. Sample Sample Volume loaded
Analysis
22 kDA volume (L) on the gel (L)
ifugation at 10000 + g for 30 min at M = Broad range marker
pH values 4, 6, 8, and Loosely bound proteins were washed away by 4 μL of flow-through, wash, and elution
application of 400 µl binding buffer to the s = Sample before application 800 16
10 were applied each column and spinning for 5 min at 2000 + g. f = Flow-through 800 16 fractions from each column were analyzed on reducing
to the correspondingly
preparation Flow-through and wash fraction were saved w = Wash fraction 400 16 SDS-PAGE, followed by silver staining.
Sample Sample Volumefor
loaded
analysis. e1 = 25 mM KPi, pH 6, 200 mM NaCl 100 8
µl of the cell lysate wereVivapure
equilibrated diluted with e2 = 25 mM KPi, pH 6, 400 mM NaCl 100 8
nding buffer A toSDspin
each,columns.
to adjust volume (L) on the gel (L)
Q and Stepwise elution e3 = 25 mM KPi, pH 6, 600 mM NaCl 100 8 Result of Step Two
ple to the respective pH conditions. In M = Broad range marker 100 µl elution buffer F, supplemented with e4 = 25 mM KPi, pH 6, 800 mM NaCl 100 8
Columns were spun for s = Sample before application 800 4 0.2 M NaCl were applied to the Vivapure S e5 = 25 mM KPi, pH 6, 1 M NaCl 100 8 The target protein started to elute with 200 mM NaCl,
avoid clogging of the membranes in
pure Mini5spin
mincolumns,
at 2000samples
x g. were however the main fraction eluted with 400 mM NaCl.
f = Flow-through 800 4 Mini spin column and spun for 3 min at
2000 + g. The eluate was collected. In the next Figure 2: Scouting for optimal elution conditions of a SH2 domain
by passage through Minisart syringe w = Wash fraction 400 4 step, 100 µl of elution buffer F, supplemented Fig. 2 Scouting for optimal elution conditions of a SH2 domain expressed in E. coli. SDS gel (reducing, 12 %), silver stained.Traces of the target protein were also found in the next
e = Elution with 1 M NaCl 200 4 expressed
Sample before loading, E. coli. SDS
inflow-through, gel
wash, and (reducing,
elution fractions from12%),
Vivapure Ssilver
Mini spinstained.
column at pHSample
6 are shown.
Afterwards, Vivapure with 0.4 M salt were applied and again spun elution step with 600 mM NaCl, but this might be dueto
for 3 min at 2000 + g. Elution was continued before loading, flow-through, wash, and elution fractions from
Mini spin columns were until the entire gradient had been tested, Vivapure S Mini spin column at pH 6 are shown. the low elution volume.
equilibration
reloaded with 400 μL Figure 1: Scouting for optimal binding conditions of a SH2 domain expressed in E.
saving the coli.from
eluates SDSeach step.
d 4 + S Vivapure Mini spin columns gel1:(reducing,
Fig. 12%), binding
Scouting for optimal silver stained. Shown
conditions of are sample
a SH2 domain expressedbefore
in E. coliloading, flow-through,
. SDS gel (reducing, wash,
12%), silver stained.
sample and spun again
eled 4, 6, 8 and 10 corresponding to Shown are sample
and elution before loading,
fractions (1 Mflow-through,
NaCl) fromwash, and elution
Vivapure Q andfractions
S Mini(1 Mspin
NaCl)columns,
from Vivapureat Qthe
Analysis andvarious
S Mini spin
pH
Discussion
for 5 min at 2000 x g.
f the buffer to be used. To each spin
columns, at the various pH values tested.
values tested.
4 µl of flow-through, wash, and elution Step Two: Optimizing Elution Conditions A two-step procedure was used to rapidly scout optimal
fractions from each column were analyzed
400 µl of Loosely bound proteins
the corresponding binding on reducing SDS-PAGE followed by silver purification conditions for a target protein (a SH2 domain
ere addedwere washed
and spun for 5away withatthe application of 400 μL of
minutes in this experiment. As can be seen on the SDS gel in
staining. Sample Preparation from E. coli lysate) with ion exchange chromatography. In
g. the respective binding buffer to each of the columns Figure 1, the target protein was present Result in the Two
of Step eluate Taking account of the results of Step One, 200 μL cell the first step, the most suited buffer pH for binding the target
and spun for 5 min at 2000 x g. Flow-through and wash of the Vivapure Q Mini spin column The at target
all pH protein started to elute with
values lysate were diluted with 1.8 mL binding buffer B (25 mM protein to the most adequate ion exchanger was verified.
and washing Analysis Differences could be detected
200 mMinNaCl,
thehowever
binding the main fraction
fractions were collected for subsequent detection of the tested together with most of the E. coli elutedproteins
with 400 mM NaCl. Traces of the target KPi, pH 6). In order to avoid clogging of the membrane In the second step, the elution condition was optimized,
f the clarified samples adjusted to pH 4 µl of flow-through, wash, and elution efficiency of the target protein as at pHfound
8
target
6, 8 and 10 wereprotein.
applied each to the (Lanes Q “e”). traces
fractions from each column were analyzed In contrast, theprotein
usingprotein
of the target
were also
Vivapure S Miniin the next elution in the Vivapure Mini spin column, the pH adjusted building on the results gained in Step One of this protocol
stepwere already
with 600 mM NaCl, but this might be due
ndingly equilibrated Vivapure Q and S on reducing SDS-PAGE followed byspin column, at
silver all pH-values
found tested,
in the flow-through, most
with
to the E. coli
low slightly
elution high-
volume. sample was clarified by passage through a Minisart (elution optimization after optimal binding of the target to the
Complete
umns. Columns were spun Elution of at
for 5 min Bound Proteins
staining. proteins did not bind to the
er amounts membrane
at pH and At
10 (Lane S "e"). werepH 6,found
the syringe filter. proper ion exchanger). With the scouting procedure described
g. 200 μL of elution buffer E, F, G, and H were applied to most efficient binding of the target
in the flow-through (Lane S “f”), thus resulting in pure protein here, it was possible to quickly and conveniently purify the
the washed columns and spun Result
for 3of Step
min at One
2000 x g. target protein to in the S membrane
all elution was observed.
fractions (Lane SNow “e”).that Column Equilibration target protein to homogeneity. The results obtained in this
rds, Vivapure Mini spin columns were Dilution of the E. coli lysate with binding the binding conditions, i. e. binding pH and
with 400Eluates
µl samplewere saved for subsequent analysis.
and spun again buffer A (25 mM Citrate, pH 4) lead to com- the best suited ion exchange chemistry, were 400 μL binding buffer B were applied to one experiment can be used for various ends, e.g.:
n at 2000 + g. Loosely bound proteins plete precipitation of sample proteins.Differences
Thus, could
found,bethe
detected in the binding
elution protocol of the target Vivapure S Mini spin column and spun for
– polishing a specific protein after a first chromatography step
Analysis
shed away with the application of efficiency
pH 4 could not be tested in this experiment. of the target protein,
protein was optimized asinata pH 8, traces
second step. 5 min at 2000 x g.
f the respective binding buffer to As can be seen on the SDS gel in figure 1, the with another chemistry
4 μL of flow-through, wash, and elution fractions from of the target protein were already found in the flow-
the columnseachandcolumn
spinningwerefor 5analyzed
min at on target proteinSDS-PAGE,
reducing was present in the eluate
through,of with slightly higher amounts at pH 10 (Lane Binding and Washing – establishing quickly a FPLC method for a new protein
g. Flow-through and wash fractions
followed by silver staining.
the Vivapure Q Mini spin column at all pH
S “e”). At pH 6, the most efficient binding of the target 400 μL of the clarified sample were applied to the
lected for subsequent detection of the values tested together with most of the – finding a purification method for a new protein for
rotein. protein to the S membrane was observed. Now that
E. coli proteins (Lanes Q "e"). In contrast, equilibrated Vivapure S column and spun for 5 min at upscaling with Vivapure Maxi or Mega.
Result of Step One using the Vivapure S Mini spin column, the binding
at conditions, i.e., the binding pH and the 2000 x g. Afterwards, the Vivapure S Mini spin column
For these purposes, Vivawell 96-well plates, Vivapure Maxi,
te elutionDilution
of boundofproteins
the E. coli lysate with bindingtested,
all pH-values buffermost
A E. coli proteins
best suited ion exchange chemistry, were found, the was reloaded with 400 μL sample and spun again for
f elution buffer
(25 mM E, F,citrate,
G and H, pHwere did not bindprecipitation
4) led to complete to the membrane andelution
were protocol of the target protein was optimized in 5 min at 2000 x g. and Sartobind membrane adsorber units with FPLC
o the washed columnsproteins.
of sample and spun Thus,
for pHfound in the
4 could flow-through
not be tested (Lane Lane S "f"), step.
a second connectors are available.
2000 + g. Eluates were saved for thus resulting in pure target protein in all
ent analysis.
40 elution fractions (Lane S "e"). 41
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