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Conventional manufacturing of protein biopharmaceuticals in generally require unique customized processes for each molecule
centralized, large-scale, single-product facilities is not well-suited from expression to purification, constraining commercial facilities
to the agile production of drugs for small patient populations or to a single class of product.
individuals. Previous solutions for small-scale manufacturing New technologies to manufacture many different pharmaceutical-
are limited in both process reproducibility and product quality, quality biologics in small quantities with efficiency and agility are
owing to their complicated means of protein expression needed to make precision biologic medicines both available and eco-
and purification1–4. We describe an automated, benchtop, nomically feasible7. Technologies such as automated laboratory-scale
multiproduct manufacturing system, called Integrated Scalable batch processes, in vitro transcription and translation, and microfluid-
Cyto-Technology (InSCyT), for the end-to-end production of ics can rapidly produce limited quantities of different biomolecules
hundreds to thousands of doses of clinical-quality protein on demand1–4. While some of the products generated show biological
biologics in about 3 d. Unlike previous systems, InSCyT includes activity, they lack sufficient quality attributes for clinical use, includ-
fully integrated modules for sustained production, efficient ing identity, purity, safety and potency as required by regulatory agen-
purification without the use of affinity tags, and formulation cies. To address this need, we developed an automated multiproduct
to a final dosage form of recombinant biopharmaceuticals. We manufacturing system capable of rapidly producing clinical-grade
demonstrate that InSCyT can accelerate process development recombinant proteins and requiring only minimal reconfiguration
from sequence to purified drug in 12 weeks. We used integrated to make different biopharmaceuticals. Unlike previous solutions,
design to produce human growth hormone, interferon a-2b InSCyT comprises fully integrated and automated modules for sus-
and granulocyte colony-stimulating factor with highly similar tained production, efficient purification of the native protein, and
processes on this system and show that their purity and potency final formulation for parenteral use. The yeast-based production
are comparable to those of marketed reference products. module allows both rapid production of tens to hundreds of doses in
under 80 h and sustained production for >100 h of up to thousands
Biologic medicines, such as recombinantly expressed cytokines, hor- of doses as needed, whereas cell-free solutions1–3 provide only short-
mones, replacement enzymes, blood factors or antibodies, are rou- term expression. The multistage purification module (and associ-
tinely used to treat cancer, autoimmune disorders and rare diseases. ated methods for rapid process development of suitable purification
Increasingly, protein biologics are tailored to small groups of patients sequences) enables purification of multiple clinical-grade products
based on an understanding of the underlying biology of their disease5. using highly similar processes without requiring affinity tags that alter
The need for small numbers of doses of many products poses a chal- the drug sequence and present risks of immunogenicity. In contrast,
lenge to conventional manufacturers, who produce drugs in large previous approaches used affinity tags for purification or had no inte-
volumes to achieve economies of scale6. Furthermore, different classes grated purification step. Finally, the integrated formulation module
of biopharmaceuticals (for example, enzymes, hormones, vaccines) prepares protein biopharmaceuticals in a final dosage form. These
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2Department of Chemical Engineering,
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 3Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy,
New York, USA. 4Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA. 5GlaxoSmithKline, King of Prussia,
Pennsylvania, USA. 6Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA. 7Pall Life Sciences,
Westborough, Massachusetts, USA. 8Biogen, Cambridge, Massachusetts, USA. 9Department of Biochemical Engineering, University College London, London, England.
10Sanofi, Framingham, Massachusetts, USA. 11Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
12BioAnalytix Inc., Cambridge, Massachusetts, USA. 13These authors contributed equally to this work. Correspondence should be addressed to J.C.L. (clove@mit.edu).
Received 16 November 2017; accepted 27 August 2018; published online 1 October 2018; doi:10.1038/nbt.4262
features, combined with the use of integrated design principles to host-cell DNA. Minimal high-molecular-weight species were present
create simplified processes, facilitate the production of recombinant in the InSCyT hGH product (<0.5%), and levels of process-related
proteins with quality attributes sufficient for clinical use. impurities were each below typical values for clinical-stage develop-
We selected Pichia pastoris as our expression host because it can ment (1,000 p.p.m. for HCPs and 10 ng/dose for DNA)14–16. Regulatory
grow quickly to high cell densities and efficiently secrete recombinant agencies usually consider limits for host-cell proteins on a case-by-case
proteins8. Other advantages of P. pastoris include low levels of secreted basis, although in vitro studies using peripheral blood mononuclear
host-cell proteins; little to no risk of viral contamination; validated cells from both healthy and diseased individuals have shown that HCP
expression of myriad proteins, including therapeutics approved by levels up to 4,000 p.p.m. from Chinese hamster ovary (CHO) cells do
the US Food and Drug Administration (FDA) and the European not pose a higher immunogenicity risk than a highly purified mono-
Medicines Agency (EMA); and the capability for human-like post- clonal antibody (<50 p.p.m.)15. A small percentage (average: 6%) of
translational modifications in engineered strains9,10. a proteolytically cleaved form of hGH was also observed (two-chain
InSCyT uses fluidically connected modules for fermentation, multi- variant); this natural form is both highly potent and previously has been
stage chromatography, and ultrafiltration and/or diafiltration, as well as determined as clinically irrelevant17. Further assessment for product-
integrated sensors and system controllers for system-wide programmed specific impurities by tandem liquid chromatography/mass spectrom-
operations (Fig. 1a,b and Supplementary Fig. 1). We implemented etry (LC–MS), however, showed that InSCyT hGH was not comparable
continuous fermentation by perfusion to reduce the volume of the bio- to marketed products because of increased levels of deamidation and
reactor and enable high space–time yields11. To this end, we adapted oxidation (Fig. 2c and Supplementary Fig. 5).
a sub-liter benchtop bioreactor for in-tank perfusion and equipped Given the consistency of both the operation of individual InSCyT
it with sensors to control input and output flows, pH, temperature, systems and the hGH products produced by each system, we attrib-
impeller speed and dissolved oxygen (Fig. 1c). The bioreactor was con- uted the deamidation and oxidation observed in our product to process
nected to a module for inline pH adjustment of the cell culture fluid parameters, rather than the InSCyT system specifically. We therefore
before chromatographic separations; this module allowed the balanc- modified our process for hGH without any significant hardware changes
ing of flow rates between those for production and purification. An by changing to a defined cultivation medium, adjusting the set point for
integrated module for purification was designed to enable either two dissolved oxygen and eliminating agitation in the surge tank. Using this
or three stages of chromatographic separation (Fig. 1c). This module adjusted process, a single InSCyT system produced nearly 50 maximum
allowed straight-through processing with no intermediate holding weight-based doses of hGH in 75 h (Supplementary Fig. 6). Oxidation
tanks or adjustments between purification steps. Our design simpli- and deamidation were reduced to below 1% at each residue, while all
fies the operation of the module relative to traditional purifications, other quality attributes were maintained (Fig. 2c,d and Supplementary
wherein multiple intermediate procedures are often required to adjust Fig. 6). Process yield also increased to nearly 80%. Together, these data
pH, conductivity, concentration and composition of fluids between demonstrate the capability of our manufacturing system to rapidly and
steps of purification. The final module in the system is a tangential reproducibly produce tens of doses of a potent and pure form of a bio-
flow-filtration system for buffer exchange and formulation to a final liq- logic drug in an automated, short production cycle.
uid dosage form of the product (Fig. 1c). A custom integrated software We next sought to demonstrate on-demand production of hGH. The
architecture unified operation of all three modules with appropriate upstream perfusion process was operated fully automated for 240 h.
controls as a fully automated single system. In addition to the initial purification and formulation cycle on day 3
We built and used three independent InSCyT systems to demon- (described above), two more cycles of purification and formulation were
strate consistent operational performance for both production and performed on demand during days 6 and 10 (Supplementary Fig. 6).
purification processes (Fig. 1d,e). We first produced and purified the Each on-demand cycle produced between 50 and 75 doses of hGH within
common biologic medicine human growth hormone (hGH), used to 12 h, with product quality and yields similar to those of the batch pro-
treat growth deficiencies12. The extensive knowledge available about duced on day 3 (Fig. 2c,d and Supplementary Fig. 6). (We attributed the
this drug’s quality, safety and potency aided our assessment of InSCyT reduced yield (50%) and increased host-cell protein levels observed dur-
and the novel process we developed for production of hGH. Our ing the day 6 cycle to overloading the capture column, which we adjusted
production process used two straight-through stages of chromato- before the day 10 cycle.) Further optimization of the process and column
graphic purification and required fewer than half as many operations sizing could improve the consistency from batch to batch. Nonetheless,
as the Innovator process (Supplementary Fig. 2). We implemented the ability to produce small lots of this product on demand shows the
our process on three separate InSCyT systems. After inoculation, potential for manufacturing medicines as needed and highlights the sta-
each process ran ‘hands-free’ and produced >100 doses of formulated bility of yeast-based bioprocesses in continuous operations.
product in less than 1 week, with initial yields for the process ranging Biopharmaceuticals typically require custom manufacturing proc-
from 27–31% on each system (Fig. 2a). esses that vary widely, especially for proteins other than monoclonal
We compared the biophysical and biochemical attributes of our puri- antibodies, and require unique facility designs11. This constraint
fied product from multiple time points on each system to a marketed limits facility flexibility to provide additional products, which would
drug substance using multiple analytics commonly used to establish be essential for on-demand supply. We therefore assessed whether
identity, potency, safety and purity of therapeutic proteins13 (Fig. 2b,c our modular manufacturing system and choice of host could readily
and Supplementary Fig. 3). We confirmed the protein sequence (100% produce other molecules with no substantial hardware alterations. We
coverage) by mass spectrometry (Supplementary Fig. 4). Potency of selected interferon-α (IFN-α)-2b as a second example. This potent
InSCyT hGH was comparable to the National Institute for Biological 19.2-kDa cytokine is used in both monotherapies and drug combina-
Standards and Control World Health Organization (WHO) 98/574 ref- tions to treat cancer and hepatitis, and is produced commercially in
erence standard (96–104%) using a cell-based proliferation assay. Key a specialized 13-step process using E. coli18 (Supplementary Fig. 2).
contributors to product safety include the levels of potentially immuno- Owing to the ease of targeted transgene insertion and simplicity of
genic product-related impurities, such as aggregates, and the presence upstream process development in P. pastoris, we developed a draft fer-
of process-related impurities, including host-cell proteins (HCPs) and mentation process for producing secreted IFNα-2b less than 4 weeks
a b 10 cm
Production Purification Formulation Purification
(USP) (DSP) (TFF) (DSP)
Final
Production product Formulation
(USP) (TFF)
FER WASTE
BUFFER BUF
DIA
MEDIA ME
Air
Off gas Hollow fiber
(O2, CO2) Pump filter
To DSP Mixing
valve Pressure
regulator
pH UV UV
pH Capture Polish
DO column column(s)
T
Inoculum
UV UV
To TFF
RPM Product
To waste g
USP Dilution buffer
Perfusion vessel DSP Retentate reservoir TFF
d Production (USP)
400
System 1 System 2 System 3
0.3
30
4
25
(°C)
Reactor
20
Block
15
7.0 0.1
(mL/min) (% contact)
pH Acid addition
6.5 0.05
pH
Base addition
6.0 0
1.0 100
Level Feed A
(g/min)
Flow
Feed B
0.5 50
0.0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Time (h) Time (h) Time (h)
e Purification (DSP)
System 1 System 2 System 3
300
Column 1 UV Cycle 1
200
Column 2 UV
100
0
300
Cycle 2
200
A280 (mAU)
100
0
300
Cycle 3
200
100
0
300 Cycle 4
200
100
0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (h) Time (h) Time (h)
Figure 1 Schematic of the InSCyT system for on-demand biomanufacturing and demonstration of consistent operation across three distinct InSCyT
systems. (a) To-scale rendering of the InSCyT system. Human figure is approximately 5 feet 7 inches (170 cm) tall. (b) Photograph of an operational
InSCyT system. (c) Detailed schematic of the InSCyT system, including interactions between modules and key control points for the production
(upstream processing, USP), purification (downstream processing, DSP) and formulation (tangential flow filtration, TFF) modules. DO, dissolved oxygen;
T, temperature. (d,e) Process parameter profiles collected by the control software from the production (USP) module (d) and the purification (DSP)
module (e) of three separate InSCyT systems during hGH fermentation. SLPM, standard liters per minute.
after identifying the product sequence (Fig. 3a). We have found that Next we produced granulocyte colony stimulating factor (G-CSF),
P. pastoris routinely secretes a consistent set of host-cell proteins used to stimulate blood cell proliferation and reduce infections in can-
along with the heterologous product during fermentation to yield cer patients treated with myelosuppressive chemotherapy24. This drug
a high level of initially pure product (>80%)19. This feature made it has manufacturing challenges due to complex folding and a propensity
possible to develop an in silico tool to predict draft multistage puri- for aggregation and oxidation at specific amino acid residues25,26. We
fication processes20. With this in silico tool, we selected a process to designed a new process for production of G-CSF and implemented it
purify IFNα-2b within another 4 weeks. The procedures developed on three InSCyT systems (Supplementary Fig. 2). After inoculation,
for both production and purification did not require modifications each automated process yielded more than 165 doses of formulated
to the InSCyT system itself. drug in less than 100 h (Fig. 4a and Supplementary Fig. 9). Typical
Initial biophysical analyses of IFNα-2b produced in our first run on process yields for each cycle ranged from 70 to 90% (average 77%). We
the InSCyT system indicated minimal high molecular weight species assessed biophysical and biochemical attributes of the product using
(0.34%) and process-related impurities below typical values for clini- multiple analytics to establish its identity, purity, safety and potency
cal development (Fig. 3c). A cell-based viral replication assay dem- (Fig. 4a and Supplementary Fig. 9). InSCyT G-CSF was comparable
onstrated that the potency of our IFNα-2b was the same as or greater to a drug substance from a licensed product. We confirmed the pro-
than that of a reference drug substance. InSCyT-generated IFNα-2b tein sequence (100% coverage) by mass spectrometry (Supplementary
was highly potent (134%) in part owing to a naturally occurring C-ter- Fig. 10). Minimal high-molecular-weight species were present in the
minal truncation known to increase potency21. Assessment for purity product (0.33–0.65%). Levels of process-related contaminants in our
by LC–MS and reversed-phase liquid chromatography (RPLC), how- formulated G-CSF were each below values typical for early-stage clinical
ever, showed that our product quality was not sufficient due to the development (1,000 p.p.m. for HCPs and 100 pg/dose for DNA)14–16.
presence of oxidized forms that could potentially promote aggregation Potency of the InSCyT G-CSF was comparable to that of the NIBSC
and immunogenicity22 (Fig. 3c). These data show that rapid produc- WHO 09/136 reference standard (89.6–141.1%) in a cell-based prolif-
tion of biomolecules with acceptable bioactivity is necessary, but not eration assay. Our product contained a minor variant comprising an
sufficient, to define a clinical-quality biologic product. N-terminal truncation and was a mixture of aglycosylated and glyco-
To address these attributes, we optimized process conditions on sylated forms (Supplementary Fig. 9). Neither of these variants are
InSCyT, performing experiments on individual modules simulta- likely to be clinically meaningful, as both truncations and glycosylation
neously with fully integrated experiments comprising connected have been observed in licensed products without impact on product
modules (Fig. 3a and Supplementary Fig. 7). After 27 d of proc- activity or safety27,28. Overall, these data demonstrate that InSCyT can
ess development, a final run showed that oxidation was reduced to rapidly and consistently produce therapeutic proteins that are compa-
<1.5% at all residues (Fig. 3b,c). During this run the system pro- rable to currently marketed products.
duced nearly 8,000 formulated doses of IFNα-2b in less than 1 week We performed further nonclinical studies with InSCyT-produced
(Supplementary Fig. 8). The identity and purity of the product was G-CSF to provide a framework for future clinical development. We
confirmed by multiple analytical methods at four time points dur- assessed the pharmacokinetics, pharmacodynamics and toxicology of
ing the campaign (Fig. 3c and Supplementary Fig. 8). RPLC, size the InSCyT-produced G-CSF by comparing our product to a licensed
exclusion chromatography and LC–MS showed levels of purity within product (Neupogen) in a rat model. We found InSCyT-produced
targeted specifications; other analytics showed the specifications for G-CSF to be comparable to Neupogen in neutrophil activation
safety and potency of our product were also achieved. Differences in during a single-dose administration study (Fig. 4b). InSCyT G-CSF
chromatographic behavior of our product were confirmed by matrix- and Neupogen showed no statistically significant difference in
assisted laser desorption and ionization mass spectrometry as the pharmacokinetic profile when administered at the same dose
naturally occurring C-terminal truncation mentioned previously21 (Kolmogorov–Smirnov test, P = 0.9963) (Fig. 4b). A 5-d repeat-dosing
(Supplementary Fig. 8). The low overall process yield (~11%) is study also showed our product was comparable to Neupogen in
attributed to removal of an N-terminal product variant, which arises toxicity as based on survival, clinical signs, body weight, quantitative
as a result of incomplete cleavage of the secretion leader sequence food consumption, hematology, serum chemistry, organ weights and
during expression by our host. Further engineering of the expression macroscopic findings (Fig. 4b and Supplementary Fig. 11). In all these
vector with alternative signal sequences could alleviate the expression studies, no abnormal signs of toxicity, including injection site inflam-
of this variant23. mation, were observed in any animals dosed with InSCyT G-CSF
Figure 2 Production of hGH on the InSCyT system. Dose size used was 1.75 mg (ref. 12). Center values and error bars represent the mean and range,
respectively, of technical triplicates unless otherwise indicated. (a) Process flow chart (left) and timeline and yields (right) for production of hGH using InSCyT.
Wet cell weight (black), unpurified (orange) and formulated (blue) doses of hGH produced are shown. Gray circles represent individual data points. (b) Product
quality analyses for InSCyT-produced hGH before optimization, alongside a reference drug substance from a licensed hGH product produced in E. coli.
SDS–PAGE (12% Tris-glycine) analysis of samples from the USP during biomass accumulation and production (perfusate samples), final formulated samples,
and the reference (Std); M, molecular mass marker. Activity of InSCyT hGH alongside the WHO international standard (NIBSC 98/574). The final formulated
sample (day 6) was analyzed from each system. Quantification of host-cell protein (HCP) and host-cell DNA impurities in formulated InSCyT hGH. Host-cell
protein limits are shown as a target range14,15. Host-cell DNA guidelines are based on 100 pg/dose (FDA) and 10 ng/dose (EMA)16,30. For host-cell protein
data, each point represents a unique sample (12 points total: 4 time points from each of three InSCyT systems). For host-cell DNA, each point represents
a single pooled sample from each system comprising equal volumes of samples from each time point (3 points total, 1 per system). (c) Analysis of product-
related variants in formulated InSCyT hGH before (top) and after optimization (bottom) alongside levels typically found in marketed products (Supplementary
Fig. 5). Each data point represents a unique sample; there are 12 data points for runs before optimization (four time points from each of three InSCyT systems)
and 3 data points for runs after optimization (three time points from a single InSCyT system). Black boxes represent the range of InSCyT hGH samples, with a
line at the mean. (d) Product quality analyses for InSCyT-produced hGH after optimization alongside reference drug substance from a licensed hGH product.
SDS–PAGE (12% Tris-glycine) analysis of samples from the USP during biomass accumulation and production (perfusate samples), final formulated samples,
and the reference (Std). Activity of InSCyT hGH alongside the WHO international standard (NIBSC 98/574). Secondary structure analysis of InSCyT hGH
(individual formulated samples from days 3, 6 and 10) and the reference hGH standard using circular dichroism; MRW, mean residue ellipticity.
(58 in total). Together, these data suggest that the InSCyT-produced purification and formulation under a single control architecture. The
G-CSF has potency in vivo and that potentially immunogenic proc- efficient secretion of proteins by P. pastoris, combined with a holistic
ess-related impurities are appropriately minimized. design of purification sequences, enabled processes for hGH, IFNα-2b
InSCyT can produce a variety of clinical-quality recombinant thera- and G-CSF that reduced the total number of processing steps by 45%
peutic proteins in a liquid dosage form through integrated production, or more and did not require refolding, excursions in pH or other
a
hGH Inoculation Induction Production
Production (USP)
1) Inoculate with hGH- USP DSP TFF DSP TFF DSP TFF DSP TFF
expressing strain 250 150
2) Accumulate biomass (32 h) Wet cell weight
3) Induce expression
4) Production with perfusion System 1
200 System 2
hGH (doses)
Purification (DSP)
150 Unpurified doses
1) Adjust supernatant (pH)
2) Multimodal cation exchange System 1
(bind and elute) System 2
100 System 3
3) Multimodal anion exchange 50
(flow-through) Formulated doses
50 System 1
System 2
Formulation (TFF) System 3
1) Concentration (as needed) 0 0
2) Buffer exchange 0 50 100 150
Time (h)
b System #1
Perfusate Formulated*
#2
Perfusate Formulated*
#3
Perfusate Formulated*
sample day sample day sample day sample day sample day sample day
kDa M Std 0 3 4 5 6 3 4 5 6 M M Std 0 3 4 5 6 3 4 5 6 M M Std 0 3 4 5 6 3 4 5 6 M
180 180
130 130
100 100
70 70
55 55
40 40
35 35
25 25
15 15
10 10
Rx Std: 0.27, 0.13 and 0.07 mg/mL *4× diluted
Activity Process-related variants
System 1 System 2 System 3 10,000
3,000 3,000 3,000 1,000 InSCyT hGH
Host cell protein (p.p.m.)
c Product-related variants
d Perfusate Formulated
sample day* sample day** Circular dichroism
20 Before optimization kDa M Std 0 3 6 10 3 6 10 MP Std M 4,000
Marketed hGH 180 Marketed hGH
130 2,000 InSCyT hGH
InSCyT hGH 100
15 0
Percent variant
70
55 –2,000
ΦMRW
40 –4,000
10
35 –6,000
5 25 –8,000
–10,000
0 15 –12,000
10 200 210 220 230 240 250
20 Rx Std: 0.5 and 0.1 mg/mL *2× diluted
After optimization Wavelength (nm)
MP: master pool **20× diluted
15
Percent variant
Activity
3 Day 3 3 3
Day 6 Day 10
10
2 2 2
Response
0 1 1 1
InSCyT hGH
e
0
14
5
at
io
17
12
M
at
eg
0 0 0
am
Ag
Oxidation
de
hGH dose
N
c IFNα-2b
First at-scale
process development run Qualification run Process-related variants
Host cell protein Host cell DNA
M Std P F M Std P F
180 180 1,000 1,000,000
Host ccell protein (p.p.m.)
130 130
15
ΦMRW
drug substance
–10,000
10
–15,000
5 –20,000
0 –25,000
200 220 240 260
n
16
21
59
1
Ag nt al
io
14
11
ria in
M
at
Wavelength (nm)
M
M
va term
eg
gr
-
N
Oxidation
Figure 3 Accelerated process development using the InSCyT system and production of IFNα-2b. Dose size was 12 µg (ref. 18). Center values and
error bars represent the mean and range, respectively, of technical triplicates unless otherwise noted. (a) Process development timeline for new
manufacturing processes using the InSCyT system, including simultaneous unit operation development (comprising strain development and purification
development), at-scale process development (comprising simultaneous experiments on individual modules and on the fully integrated system) and
process qualification. (b) Timeline for at-scale process development for IFNα-2b. Horizontal colored bars represent the modules that were used in each
experiment (USP, orange; DSP, purple; TFF, blue). Each new bar represents a new set of experimental conditions on that module. (c) Product quality
for InSCyT-produced IFNα-2b from the first at-scale run after initial unit operation development (first at-scale process development run) and the final
qualification run alongside a reference drug substance produced in E. coli. SDS–PAGE (12% Tris-glycine) analysis of samples from the USP during
production (P), a final formulated sample (F) and a reference drug substance (Std); M, molecular mass marker. Analysis of process-related variants
in formulated InSCyT IFNα-2b (per Fig. 2b). Each data point represents a unique sample, including one data point from the first at-scale process
development run and four data points from the qualification run (four time points from a single InSCyT system). Product-related variants detected in
formulated InSCyT IFNα-2b alongside levels typically found in a reference drug substance (Supplementary Fig. 5). Black boxes represent the range of
InSCyT IFNα-2b samples, with a line at the mean. Secondary structure analysis of InSCyT IFNα-2b (triplicate analyses of an individual sample from the
qualification run) and reference drug substance (duplicate analyses of an individual sample) using circular dichroism; MRW, mean residue ellipticity.
a G-CSF
SDS–PAGE IEF
Inoculation Induction Production Vials
Perfusate Formulated M Std G P F M Std InSCyT
USP DSP TFF 8.2
180
130 8.0
250 200 7.8
Wet cell weight 100
Unpurified doses 70 7.5
Wet cell weight (g/L) 200 Formulated doses 55
150 7.1
40
G-CSF (doses)
150 35 7.0
100
25 6.5
100
6.0
50 5.1
50 15
10 4.65
0 0 Rx Std: 0.5 and 0.1 mg/mL
0 20 40 60 80 G: USP sample during biomass accumulation
Time (h) P: USP sample during production
F: formulated InSCyT G-CSF (5× diluted)
Product-related variants Process-related variants Circular dichroism
5 Marketed G-CSF 1,000 100,000 20,000
InSCyT G-CSF InSCyT G-CSF
3 1,000 0
ΦMRW
500
2 100 –10,000
1 10 –20,000
0 0 1 –30,000
Host cell Host cell 200 210 220 230 240 250
e
8
at
io
12
12
13
protein DNA
at
eg
Wavelength (nm)
M
M
id
gr
am
Ag
Activity
4 Batch 1 4 Batch 2 4 Batch 3 4 Batch 4 4 Batch 5 4 Batch 6
InSCyT G-CSF
3 WHO 3 3 3 3 3
Response
Intl Std
2 2 2 2 2 2
1 1 1 1 1 1
0 0 0 0 0 0
0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.001 0.01 0.1 1
G-CSF dose G-CSF dose G-CSF dose G-CSF dose G-CSF dose G-CSF dose
control G-CSF
Relative neutrophil counts
40 2 Hematology
0.6
Neutrophils 9.15* 32.65** 39.61**
(10 cells/µL)
1
30
Lymphocytes 86.15* 59.78† 52.74
0.4 0
0 10 20 30 40 80 120 Monocytes 1.93* 4.42** 4.17**
20
3
Figure 4 Production of G-CSF on three identical InSCyT systems. Dose size 300 µg (ref. 24). Center values and error bars represent the mean and
range, respectively, of technical triplicates unless otherwise noted. (a) Timeline and yields for production of G-CSF using the InSCyT system for a single
representative sample (batch 1). Wet cell weight (black circles) and cumulative unpurified (orange) and formulated (blue) doses of G-CSF are shown.
Gray circles represent individual data points. Product quality for InSCyT-produced G-CSF alongside drug substance from a licensed product produced in
E. coli and Neupogen (produced by Amgen in E. coli). A photograph of vials comparing material sampled from the USP (perfusate) to final formulated
material (formulated). SDS–PAGE (12% Tris-glycine) analysis of batch 1 from the USP during biomass accumulation (G) and production (P), and a
final formulated InSCyT sample (F) alongside drug substance from a licensed product (Std); M, molecular mass marker. Analysis of product purity
by isoelectric focusing (IEF) for formulated batch 1. Gel analyses of batch 1 are representative of all six batches (Supplementary Fig. 9). Analysis of
product-related variants and process-related variants. Each data point represents a unique batch (two time points from each of three distinct systems).
Paired data points indicate analyses from a single batch. Product-related variants are shown alongside levels typically found in marketed products
(Supplementary Fig. 5). Black boxes represent the range of InSCyT G-CSF samples, with a line at the mean. Process-related variants are shown
alongside common guidelines (per Fig. 2b). Analysis of the secondary structures of InSCyT G-CSF (batches 1–6) and a reference drug substance from
a licensed product using circular dichroism. Activity of InSCyT G-CSF alongside that of the WHO international standard (NIBSC 09/136). (b) Analysis
of pharmacokinetics, pharmacodynamics and toxicology of InSCyT-produced G-CSF and a licensed product (Neupogen) in a rat model. Neutrophil
activation and pharmacokinetic profile of low dose (115 µg/kg, n = 3 animals, t1/2 = 2.1 h) and high dose (575 µg/kg, n = 3 animals, t1/2 = 4.6 h)
InSCyT G-CSF in rats compared to Neupogen (115 µg/kg, n = 3 animals, t1/2 = 1.4 h) (pharmacokinetics: P = 0.9963, Kolmogorov–Smirnov test). For
neutrophil activation, gray boxes represent the range of three individual animals, with a line at the mean. For pharmacokinetics, center points and error
bars represent the mean and range, respectively, of three individual animals. Summary of statistically significant results comparing the toxicology of
InSCyT G-CSF and Neupogen to a vehicle control. Values represent the mean; s.d. and sample size can be found in Supplementary Figure 11. Statistical
significance was determined by one-way ANOVA. ALP, alkaline phosphatase; MRW, mean residue ellipticity; M, male; F, female.
` Experimental design
1. Sample size
Describe how sample size was determined. The experiments presented in this work were representative of greater than 35
independent operational replicates performed under similar conditions to
determine optimal process parameters for production and their impact on quality
attributes of proteins. Sample sizes were not predetermined. Instead, replicate
experiments (min n=3) were initially performed for each condition or process.
Variations or quality deviations were noted. Process conditions were adjusted
accordingly and experiments were performed again in replicate to confirm new
conditions. Iterations were performed until variation and quality was acceptable.
Sample size for non-clinical studies was selected to be the minimum number of
animals required to obtain statistically significant results.
2. Data exclusions
Describe any data exclusions. Technical outliers were excluded from the cell-based potency assays for biologic
activity of hGH (as determined by a third-party CRO Bioassay GmbH based on their
Quality Assurance protocols for data generated in these assays and described in
Methods). No data were excluded from analysis in other experiments reported.
3. Replication
Describe whether the experimental findings were The production and characterization of G-CSF, IFNalpha-2b, and hGH presented in
reliably reproduced. this work were representative of more than 35 independent operational replicates
performed with similar parameters to map optimal production conditions. Quality
attributes of materials were consistent with expectations (for example, higher
dissolved oxygen led to higher levels of oxidation of protein). Materials produced
in this work were regularly generated in triplicate using three independent
automated production systems.
4. Randomization
Describe how samples/organisms/participants were For the non-clinical studies presented in this work randomization was performed
allocated into experimental groups. to allocate animals into experimental groups using Research Randomizer software,
version 4.0.
5. Blinding
Describe whether the investigators were blinded to For the non-clinical studies presented in this work blinding was not performed. The
group allocation during data collection and/or analysis. study employed methodology to minimize uncertainty and to control bias for data
collection and analysis, however, which included but was not limited to:
concurrent control data, system suitability assessment, randomization, and
method controls such as blanks and replicates.
Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.
June 2017
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Nature Biotechnology: doi:10.1038/nbt.4262
6. Statistical parameters
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
A statement indicating how many times each experiment was replicated
The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more
complex techniques should be described in the Methods section)
A description of any assumptions or corrections, such as an adjustment for multiple comparisons
The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted
A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars
See the web collection on statistics for biologists for further resources and guidance.
` Software
Policy information about availability of computer code
7. Software
Describe the software used to analyze the data in this Wonderware 2014 R2 was used as a human-machine interface to the integrated
study. system and as a data historian. GraphPad Prism v7 was used to plot and analyze all
data in the manuscript and supplemental material, except for the following.
MATLAB 2017a was used for plotting and analysis of the fermentograms and UV
traces presented. LightCycler software release 1.5.0SP4 was used for qPCR
analysis. Empower 3 was used for control in chromatographic analyses. Thermo
BioPharmaFinder 2.0 was used for analysis of LCMS data. Provantis 9.3.1 and
WinNonlin 6.3 was used for analysis in the non clinical studies.
For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made
available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for
providing algorithms and software for publication provides further information on this topic.
substrate. For AssayPro products, each lot of antibody was validated by ELISA using
biotinylated recombinant IFNaplha-2b. The Quantikine kit was validated by ELISA
using recombinant human G-CSF.
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10. Eukaryotic cell lines
b. Describe the method of cell line authentication used. K. phaffii NRRL Y-11430 and derivatives described in the methods were
authenticated by genome sequencing as reported in Love, K. R. et al. BMC
Genomics (2016) 17:550.
c. Report whether the cell lines were tested for Only yeast were used in this study and all cultivations used animal-free
mycoplasma contamination. components; mycoplasma contamination/testing is not applicable and was not
performed.
d. If any of the cell lines used are listed in the database No commonly misidentified cell lines were used.
of commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.
June 2017
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Nature Biotechnology: doi:10.1038/nbt.4262