OVERVIEW OF

BIOLOGICS
PRODUCTION
CH4306: Bioanalytical Techniques

School of Chemical and Biomedical Engineering

Nanyang Technological University, Singapore
Definition of Biologics
• Biological products include a wide range of products such as
vaccines, blood and blood components, allergenics, somatic
cells, gene therapy, tissues, and recombinant therapeutic
proteins. (In this class, we will use insulin as an example)

• Biologics can be composed of sugars, proteins, or nucleic

acids or complex combinations of these substances, or may be
living entities such as cells and tissues.

• Biologics are isolated from a variety of natural sources -

human, animal, or microorganism - and may be produced by
biotechnology methods and other cutting-edge technologies.
Gene-based and cellular biologics, for example, often are at
the forefront of biomedical research, and may be used to treat
a variety of medical conditions for which no other treatments
are available.
Biologics vs. Conventional Drugs
• In contrast to most drugs that are chemically synthesized
and their structure is known, most biologics are complex
mixtures that are not easily identified or characterized.
Biological products, including those manufactured by
biotechnology, tend to be heat sensitive and susceptible
to microbial contamination. Therefore, it is necessary to
use aseptic principles from initial manufacturing steps,
which is also in contrast to most conventional drugs.

• Biological products often represent the cutting-edge of

biomedical research and, in time, may offer the most
effective means to treat a variety of medical illnesses and
conditions that presently have no other treatments
available.
Biosimilars
• Many patents for biologics are running out.

• A biosimilar product is a biological product that is

approved based on a showing that it is highly similar to
an FDA-approved biological product, known as a
reference product, and has no clinically meaningful
differences in terms of safety and effectiveness from the
reference product. Only minor differences in clinically
inactive components are allowable in biosimilar products.

• First US approved biosimilar (2015):

• Zarxio (Sandoz; a Novartis company).
• Reference drug: Neupogen (Amgen)
The Biologics Market
The Path to Commercialization
In-class Exercise
• Who are the market leaders in biologics?
• Names of companies
• Where their locations are

• What are their products?

• Names of products
• Indication of the products

• How many of them are in Singpapore?

• Check: SciEx and A*STAR

• https://biotechin.asia/2016/03/07/sciex-and-astars-bioprocessing-
technology-institute-enter-multi-year-collaboration-to-advance-
biologics-rd-in-singapore/
The Body’s Response to Blood Sugar
 PDB ID: 4INS

Insulin
• Hormone produced by beta cells of the pancreas PDB ID: 1AI0

islets of Langerhans
• Main function is to regulate the blood sugar level

• When insulin binds to insulin receptor it will trigger

the cells (liver & muscle) to take-up glucose from
the blood stream.

PDB ID: 4ZXB

• Human insulin is 110 amino acids, 5808 Da.
• Dimer linked by disulfide bond.

• Primary structure determined by Frederick Sanger

(1951) – Nobel in Chemistry 1958
• Quaternary (crystal) structure by Dorothy Hodgkin
(1969) – Nobel in Chemistry 1964
Diabetes Mellitus (DM)
• There are 2+1 types:
• Type 1 (early onset; juvenile diabetes) – no insulin production; 5% population
• Type 2 (late onset) – insulin is produced but the body is ‘resistant’
• Gestational – only happens during pregnancy
• Long-term effects of high blood sugar: eye, kidney, nerve, heart

• Symptoms:
• Frequent urination, thirsty, hungry, extreme fatigue, blurry vision, cuts/bruises
hard to heal

• Treatments:
• Type 1: insulin injections
• Type 2: oral medications (ê glucose release from liver or é insulin production
by pancreas) or by insulin injections

• Glucose monitoring:
• AccuCheck (Roche)
• Continuous glucose monitoring (Medtronic)
Discovery of Insulin
• 1869 – Paul Langerhans showed that some clusters of cells
in the pancreas have unknown function.
• 1889 – Oskar Minkowski & Joseph von Mering showed that
removing pancreas from a dog caused it to suffer from
diabetes; however, tying the duct does not.
• 1920 – Frederick Banting developed an idea that tying the
duct will cause the pancreas to degenerate and he would
be able to recover the cells responsible for the juice
production.
• 1921 – John Macleod decided to give a small funding to
Banting and Charles Best was recruited; the experiment
began that summer.
• 1922 – Leonard Thompson, was chosen as the first person
with diabetes to receive insulin.
• 1923 – Frederick G. Banting and John Macleod were
awarded the Nobel Prize in Physiology or Medicine "for the
discovery of insulin."
Source: Nobel Prize
Biotech Production of Insulin
 Production of Insulin – Genetic Engineering

Sources: abpischools, nlm.nih

Production of Insulin –
Fermentation to Formulation

Source: DiabetesVoice (2011) v56(2):40-43

How to Make Other Biologics
• In the lab

• In the industry
Knowledge Requirements
• Cell biology
• Molecular biology
• Protein science and engineering
• Cell metabolism

• Basic fermentation
• Various engineering disciplines:
• Bio/Chemical
• Electrical
• Industrial
• Mechanical
BASIC CELL BIOLOGY
RE2005: Engineering Science II

Renaissance in Engineering Program (REP)

Nanyang Technological University, Singapore
18
 19

Further reading
de Duve, C., “The birth of complex cells”, Scientific American; Apr96, Vol. 274 (4), p50
Life Defined
• According to dictionary
• “The state or quality that distinguishes living beings or organisms
from dead ones and from inorganic matter, characterized chiefly by
metabolism, growth, and the ability to reproduce and respond to
stimuli”

• According to biology textbook

• Are highly organized compared to natural inanimated objects
• Display homeostasis, maintaining a relatively constant internal
environment
• Reproduce themselves
• Grow and develop from simple beginnings
• Take energy and matter from the environment and transform it
• Respond to stimuli
• Show adaptation to their environment
Cells: The Fundamental Unit of Living Matter

• Various appearances • Similar basic chemistry

and functions

Is virus alive?
What We
Can See

Source: Genetic Engineering News

 23

01_09_Scale.jpg
Visualizing Cells, Organelles:
Optical microscope – 200 nm
• Light microscopy

• Fluorescence microscopy
Robert Hooke's microscope.
From Scheme I. of his 1665
Micrographia.
Image source: Wikipedia

• Confocal microscopy
Visualizing Organelles, Protein:
Electron microscope – 0.2 nm

• Transmission electron • Scanning electron

microscopy (TEM) microscopy (SEM)
Visualizing Proteins, Atoms:
Scanning probe microscope
• Atomic force microscopy • Scanning tunneling
(AFM) – nm (dry), 10s nm microscopy (STM) – Å
(in soln.)

AFM tip
www.xintek.com

http://ralfseidel.de/Dateien/afm.jpg http://hermes.phys.uwm.edu/projects/hleed/stm.jpg

Image obtained from a microscope is called micrograph

History in Cell Structure Determination
Domains of Life

• Introduced by Carl Woese in early 1990-s

• Based on 16S rRNA sequence

• pro = before
• eu = well, truly Greek words
• karyon = kernel, nucleus
Archaea vs. Eubacteria vs. Eucarya
• Archaea are distinct from Eubacteria and Eucarya:
• Lipid is ether linked
• Some species live in environment hostile to most other cells
(resemblance of the primitive earth)

• Similarity to Eubacteria:
• Metabolic enzymes, proteins in cell division, singular
chromosome, 5’-terminal cap, Shine-Dalgarno site

• Similarity to Eucarya:
• Cell wall lacks peptidoglycan, RNA polymerase, protein
synthesis, and replication machinery
Procaryotic Cells
• Simple structure

• No organelles
• Tough protective coat (cell wall)
• Surrounds plasma membrane
• Single compartment containing cytoplasm and DNA
• Matrix with no obvious organized internal structures
• No nucleus (fundamental classification of all living things)

• Diverse
• Mostly single cell
• Some join together forming chain, cluster, multicellular structure

• Divided into two domains: Eubacteria & Archaea

Eucaryotic Cells
• Cells are more elaborate compared to
procaryotes

• Life style:
• Single cell, i.e.: amoebae, yeast, protozoan
• Multi cell, i.e.: plants, animals, fungi

• By definition, all eucaryotic cells have a nucleus

• Posses other organelles
Generalized Cell Structures
• Plasma membrane = cell membrane

• Nucleus = genetic material of cell

• Cytoplasm = everything between the membrane and the

nucleus
• cytosol = intracellular fluid
• organelles = subcellular structures with specific functions
The Typical Cell

• Not all cells contain all of these organelles.

 Distinguishing cell
features
• Bacterial cells
• Cell wall
• DNA in cytosol

• Plant cells
• Cell wall
• Vacuole + membrane
• Chloroplast

• Animal cells
• Vesicles
• Extracellular matrix (ECM)
Nucleus: Information store

• Enclosed within 2 concentric membranes forming nuclear

envelope
• Segregated from the rest of the cell contents
• Procaryote does not keep their DNA inside a nuclear envelope

• Contains DNA molecules

• As cells prepare to divide, DNA molecules become compact and
visible as chromosomes
 36

Mitochondria: Power house

• Present in all eucaryotic cells
• Sausage- or worm-shape
• One to few µm long
• Each enclosed in two separate membranes
• Inner membrane forms folds that project into the interior

• Contains its own DNA

• Reproduce by dividing in two

• Generates chemical energy for the cell

• Harness energy from oxidation of food molecules, i.e. sugar
• Produce adenosine triphosphate (ATP)
• Basic chemical fuel also known as the ‘energy currency’

• Consume O2 and release CO2 à cellular respiration

• Use O2 to extract maximum amount of energy from food
• Without it, O2 would be poisoneous
 Chloroplasts: Captures energy from sunlight
• Possess chlorophyll
• Internal stacks of membranes
containing green pigments

• The green color is light

dependent
• Fades in the dark, returns in
the light

• Performs photosynthesis
Large green organelle;
Found only in plants & algae
Contains its own DNA
Reproduce by dividing in two
• Trap energy from sunlight à
use to manufacture energy-
rich sugar molecules à
release O2
• Extract the stored chemical
energy by oxidizing in
mitochondria
• Able to generate both food
molecules & oxygen
 38

Other organelles
• Endoplasmic reticulum (ER)
• An irregular maze of interconnected spaces enclosed by folded membrane
• Continuous with the membrane of the nuclear envelope
• Site at which most cell membrane components are made
• Particular regions that are coated with ribosomes are often called “rough ER”
• Golgi apparatus
• Stacks of flattened membrane enclosed sacs
• Receive and chemically modify molecules made in the ER
• Directs them to the various locations, in/out of the cell

• Lysosomes
• Small, irregularly shaped
• Where intracellular digestion occurs
• Release of nutrients from food particles
• Break down of unwanted molecules for recycling or excretion
• pH dropped from physiological to ~ 5

• Peroxisomes
• Small, membrane enclosed vesicles
• Provides contained environment for reactions generating H2O2

• Vesicles
• Mediate exchange of materials between ER, Golgi apparatus, lysosome, the
outside of the cell
• Pinch off from one membrane and fuse with another
 39

Other organelles: Vesicles

• Mediate exchange of materials between
• ER
• Golgi apparatus
• Lysosome
• The outside of the cell

• Pinch off from one membrane and fuse with another

• Endocytosis
• Portion of the plasma membrane tuck inward forms vesicle
• Carry captured material into the cell
• Fuse with lysosome where digestion occurs
• Exocytosis
• Vesicle from inside the cell fuse with plasma membrane
• Release content to the external medium
• Transport of hormones, neurotransmitters, signaling molecules
 Tortora & Grabowski 9/e ã2000 JWS

Plasma Membrane
• Flexible but sturdy barrier that surround cytoplasm of
cell

• Fluid mosaic model describes its structure

• “sea of lipids in which proteins float like icebergs”
• membrane is 50 % lipid & 50 % protein
• held together by hydrogen bonds
• lipid is barrier to entry or exit of polar substances
• proteins are “gatekeepers” -- regulate traffic

• 50 lipid molecules for each protein molecule

 Tortora & Grabowski 9/e ã2000 JWS

Lipid Bilayer of the Cell Membrane

• Two back-to-back layers of 3 types of lipid molecules

• Cholesterol and glycolipids scattered among a double row of
phospholipid molecules
Each molecule is amphipathic (has both a polar &
nonpolar region)
• polar parts (heads) are hydophilic and face on
both surfaces a watery environment
• nonpolar parts (tails) are hydrophobic and line
up next to each other in the interior
Phospholipids

• Comprises 75% of lipids

• Phospholipid bilayer = 2 parallel

layers of molecules
 Tortora & Grabowski 9/e ã2000 JWS

Glycolipids within the Cell Membrane

• Comprises 5% of the lipids of the cell membrane

• Carbohydrate groups form a polar head only on the side of the
membrane facing the extracellular fluid
 Tortora & Grabowski 9/e ã2000 JWS

Cholesterol within the Cell Membrane

• Comprises 20% of cell membrane lipids

• Interspersed among the other lipids in both layers
• Stiff steroid rings & hydrocarbon tail are nonpolar and hide in
the middle of the cell membrane
 Tortora & Grabowski 9/e ã2000 JWS

Types of Membrane Proteins

• Integral proteins
• extend into or completely across cell membrane
• if extend completely across = transmembrane proteins
• all are amphipathic with hydrophobic portions hiding among the
phospholipid tails
• glycoproteins have the sugar portion facing the extracellular fluid to
form a glycocalyx
• gives cell “uniqueness”, protects it from being digested, creates a
stickiness to hold it to other cells or so it can hold a fluid layer creating a
slippery surface

• Peripheral proteins
• attached to either inner or outer surface of cell membrane and are
easily removed from it
 Tortora & Grabowski 9/e ã2000 JWS

Integral vs. Peripheral Membrane Proteins

Functions of Membrane Proteins
• Formation of Channel
• passageway to allow
specific substance to pass
through

• Transporter Proteins
• bind a specific substance,
change their shape & move
it across membrane

• Receptor Proteins
• cellular recognition site --
bind to substance

Tortora & Grabowski 9/e ã2000 JWS 3-

48
Functions of Membrane Proteins
• Cell Identity Marker
• allow cell to recognize other
similar cells

• Linker
• anchor proteins in cell
membrane or to other cells
• allow cell movement
• cell shape & structure

• Act as Enzyme
• speed up reactions

Tortora & Grabowski 9/e ã2000 JWS 3-

49
 Tortora & Grabowski 9/e ã2000 JWS

Membrane Fluidity
• Membranes are fluid structures (oil layer)
• self-sealing if punctured with needle

• Explanation -- a compromise of forces

• membrane molecules can rotate & move freely
• need to stay in one half of lipid bilayer
• difficult for hydrophilic parts to pass through hydrophobic core of bilipid
layer
• fluidity is reduced by presence of cholesterol
• increases stiffness of membrane it forms hydrogen bonds with
neighboring phospholipid heads
 Tortora & Grabowski 9/e ã2000 JWS

Selective Permeability of Membrane

• Lipid bilayer
• permeable to nonpolar, uncharged molecules -- oxygen, CO2,
steroids
• permeable to water which flows through gaps that form in
hydrophobic core of membrane as phospholipids move about

• Transmembrane proteins act as specific channels

• small and medium polar & charged particles

• Macromolecules unable to pass through the

membrane
• vesicular transport
 Tortora & Grabowski 9/e ã2000 JWS

Gradients Across the Plasma Membrane

• Membrane can maintain difference in concentration of a
substance inside versus outside of the membrane
(concentration gradient)
• more O2 & Na+ outside of cell membrane
• more CO2 and K+ inside of cell membrane

• Membrane can maintain a difference in charged ions

between inside & outside of membrane (electrical gradient
or membrane potential)

• Thus, substances move down their concentration gradient

and towards the oppositely charged area
• ions have electrochemical gradients
 Tortora & Grabowski 9/e ã2000 JWS

Gradients Across Membrane

• Concentration
gradient

• Electrical gradient
 Tortora & Grabowski 9/e ã2000 JWS

Electrolyte and protein anion concentrations

 Tortora & Grabowski 9/e ã2000 JWS

Transport Across the Plasma Membrane

• Substances cross membranes by a variety of processes:
• Mediated transport moves
materials with the help of a
transporter protein
• Non-mediated transport does
not use a transporter protein

• Active transport uses ATP to

drive substances against their
concentration gradients
• Passive transport moves substances
down their concentration gradient with only their kinetic energy

• Vesicular transport moves materials across membranes in small

vesicles -- either by exocytosis or endocytosis
 Tortora & Grabowski 9/e ã2000 JWS

Principles of Diffusion
• Random mixing of particles in a solution as a result of the
particle’s kinetic energy
• more molecules move away from an area of high concentration to
an area of low concentration
• the greater the difference in concentration between the 2 sides of the
membrane, the faster the rate of diffusion
• the higher the temperature, the faster the rate of diffusion
• the larger the size of the diffusing substance, the slower the rate of
diffusion
• an increase in surface area, increases the rate of diffusion
• increasing diffusion distance, slows rate of diffusion

• When the molecules are evenly distributed, equilibrium

has been reached
 Tortora & Grabowski 9/e ã2000 JWS

Diffusion
• Crystal of dye placed in
a cylinder of water

• Net diffusion from the

higher dye
concentration to the
region of lower dye

• Equilibrium has been

reached in the far right
cylinder

Tortora & Grabowski 9/e ã2000 JWS 3-

57
 Tortora & Grabowski 9/e ã2000 JWS

Osmosis
• Net movement of water through a selectively permeable
membrane from an area of high water concentration to
an area of lower water concentration
• diffusion through lipid bilayer
• aquaporins (transmembrane proteins) that function as water
channels

• Only occurs if membrane is permeable to water but not to

certain solutes
 Tortora & Grabowski 9/e ã2000 JWS

Osmosis of Water Through a Membrane

• Pure water on the left side & a membrane impermeable to

the solute found on the right side
• Net movement of water is from left to right, until hydrostatic
pressure (osmotic pressure ) starts to push water back to 3-
the left 59
 Tortora & Grabowski 9/e ã2000 JWS

Affects of Tonicity on RBCs in Lab

• Normally the osmotic pressure of the inside of the cell is
equal to the fluid outside the cell
• cell volume remains constant (solution is isotonic)

• Effects of fluids on RBCs in lab

• water enters the cell faster than it leaves
• water enters & leaves the cell in equal amounts
• water leaves the cell
 Tortora & Grabowski 9/e ã2000 JWS

Effects of Tonicity on Cell Membranes

• Isotonic solution
• water concentration the same inside & outside of cell results in no
net movement of water across cell membrane
• Hypotonic solution
• higher concentration of water outside of cell results in hemolysis
• Hypertonic solution
• lower concentration of water outside of cell causes crenation

3-
Tortora & Grabowski 9/e ã2000 JWS 61
 Tortora & Grabowski 9/e ã2000 JWS

Vesicular Transport of Particles

• Endocytosis = bringing something into cell
• phagocytosis = cell eating by macrophages & WBCs
• particle binds to receptor protein
• whole bacteria or viruses are engulfed & later digested
• pinocytosis = cell drinking
• no receptor proteins
• receptor-mediated endocytosis = selective input
• mechanism by which HIV virus enters cells

• Exocytosis = release something from cell

• Vesicles form inside cell, fuse to cell membrane
• Release their contents
• digestive enzymes, hormones, neurotransmitters or waste products
• replace cell membrane lost by endocytosis
 Tortora & Grabowski 9/e ã2000 JWS

Pinocytosis and Phagocytosis

• No pseudopods form • Pseudopods extend to form

• Nonselective drinking of
phagosome
extracellular fluid • Lysosome joins it
 Tortora & Grabowski 9/e ã2000 JWS

Receptor-Mediated
Endocytosis
• Mechanism for uptake of specific
substances – ligands

• Desired substance binds to receptor

protein in clathrin-coated pit region of
cell membrane causing membrane to
fold inward

• Vesicles become uncoated &

combine with endosome
• Receptor proteins separate from
ligands and return to surface

• Ligands are digested by lysosomal

enzymes or transported across cell --
epithelial cell crossing accomplished
 Tortora & Grabowski 9/e ã2000 JWS

Cytosol = Intracellular fluid

• 55% of cell volume

• 75-90% water with other components

• large organic molecules (proteins, carbos & lipids)
• suspended by electrical charges
• small organic molecules (simple sugars) & ions
• dissolved
• inclusions (large aggregates of one material)
• lipid droplets
• glycogen granules

• Site of many important chemical reactions

• production of ATP, synthesis of building blocks
Learning Points
• Biologics production

• Insulin

• Cell structure and organelles

• Cell membrane compositions

• Cellular transport

• Cytosol compositions