This document summarizes the Selexis-KBI Integrated (SKI) workflow for expediting the development and delivery of bispecific antibodies to the clinic. The SKI workflow integrates cell line development, process development, and clinical manufacturing. Key aspects include: - Using stable pools of clones early in development to assess product quality and titer with a platform process designed for bispecifics. - Completing the first clinical manufacturing batch within 12 months by performing clone selection and process development in parallel. - Demonstrating success with over 10 bispecific molecules, including increasing titer over 2-fold in one case and reducing homodimer levels by over 85% in another. -

1. 12
Titer(g/L)
Day
12
Viability(%)
Day
Peak Day
PeakVCC(x106
cells/mL)
100 L
50 L
12
Lactate(g/L)
Day
Dane A. Grismer, Yogender K. Gowtham, Srivatsan Gopalakrishnan, David. W. Chang,
Niket Bubna, and Sigma S. Mostafa
KBI Biopharma, Durham, NC
10th Annual World Bispecific Summit
17 Sep – 19 Sep 2019 • Waltham, MA
www.bispecific.com
Integration of Cell Line and Process Development to Expedite
Delivery of Bispecifics to the Clinic
Abstract
Challenges with low productivity and product quality for bispecific antibodies often lead to lengthy
timelines for generating clinical material. Implementation of the Selexis-KBI Integrated (SKI)
workflow—cell line development, process development, and clinical manufacturing— addresses
these hurdles. Stable pools of clones are used to assess product titer and product quality using a
platform process designed for bispecifics. Clone selection is performed in parallel with other
process development activities. The SKI workflow results in completion of the first FIH clinical
manufacturing batch in 12 months. We have demonstrated successful use of the SKI project
paradigm for more than 10 bispecifics. Three case studies, each highlighting an improvement in
titer or product quality will be presented. The first molecule successfully employed a platform cell
culture process with minor modifications, leading to over 2-fold increase in product titer. High
titers during development enabled reduction of the manufacturing scale from 2,000 L to 1,000 L.
The second case study will demonstrate successful remediation of product degradation in clarified
harvest. The third case study will show results for significant homodimer reduction (more than
85%). Together, these case studies establish that a strong foundational platform shortens process
development timelines, which allows focus to be placed on resolution of product challenges.
Preventing Homodimer Generation
High Titer Leading to Scale Reduction
Inhibiting Product Fragmentation
Acknowledgements
Selexis-KBI Integrated (SKI) Workflow
Conclusions
A strong foundational cell culture and harvest platform process has removed the need for
extensive development and optimization studies. Focus was maintained on resolving product
challenges that were known before the start of the project or appeared during process
development activities. Experience on numerous SKI projects and overcoming an array of product
challenges provides an extensive toolkit to develop robust and scalable manufacturing processes
for a variety of bispecifics.
• Partnership between Selexis and KBI
provides synergies that shorten the
time between transfection and BDS fill.
• Selexis has an established track record
in cell line development and variant
screening.
• KBI Biopharma brings process and
analytical development and clinical
and commercial cGMP manufacturing
expertise to the partnership.
We would like to thank Stewart McNaull and Tim Kelly for their guidance. Additional thanks to Carl
Zhang, James Hamlin, and Jake Kim for contributions to analysis and discussions.
• A platform cell culture process
with minor modifications
performed consistently well
across numerous scales.
• A more than 2-fold increase in
product titer during process
development enabled reduction
of the manufacturing scale from
2,000 L to 1,000 L while still
achieving project benchmarks.
0 2 4 6 8 10 12 14
Day
ambr15 (n=6) 3 L Scale 50 L Clone PD
200 L PD 1000 L cGMP
0 2 4 6 8 10 12 14
Day
ambr15 (n=6) 3 L Scale 200 L PD
1000 L cGMP
6 8 10 12 14
Day
ambr15 (n=6) 3 L Scale 50 L Clone PD
200 L PD 1000 L cGMP
Acidic Basic Main
Product Quality
ambr15 (n=6) 3 L Scale 50 L Clone PD 200 L PD
• The generation of significant levels of scFV homodimer
was present at the beginning of the project.
• A more than 85% reduction of homodimer levels was
achieved during process development.
• Three approaches were utilized together to reduce
homodimer generation:
1. Optimization of the feeding scheme
2. Temperature downshift triggered by a VCC
threshold
3. Reduction of the culture duration from 14 to 12
days
• High titer for SKI processes allows for modulation of
product quality attributes through a variety of methods,
including shortening the process to ensure low levels of
homodimers and high levels of heterodimers at harvest.
References
• Brinkmann, U. & Kontermann, R. E. (2017). The making of bispecific antibodies. mAbs, 9(2), 182–212.
• Chung, W. K., et al. (2017) Effects of antibody disulfide bond reduction on purification process performance
and final drug substance stability. Biotechnol. Bioeng., 114(6), 1264-1274.
• Labrijn, A. F., et al. (2013) Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. P. Natl.
Acad. Sci. USA, 110(13), 5145-50.
• Liu, H., et al. (2017) Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds. Front.
Immunol., 8(38).
• Trexler-Schmidt, M., et al. (2010). Identification and prevention of antibody disulfide bond reduction during
cell culture manufacturing. Biotechnol. Bioeng., 106(3), 452-61.
• Wang, Q. (2019) Design and Production of Bispecific Antibodies. Antibodies, 8(3), 43.
Harvest operations at process temperature
LMW Main HMW
PeakArea(%)
100 L Day 12 Bioreactor
100 L Day 14 Bioreactor
100 L Day 14 Clarified Harvest
Bioreactor cooled prior to harvest operations
LMW Main HMW
%PeakArea
50 L Day 12 Bioreactor
50 L Day 12 Clarified Harvest
• Process was shortened from 14 to 12 days.
• Prior to harvest initiation:
• DO setpoint was increased to 60-70%,
• Bioreactor was cooled to 18-20 °C.
• Clarified harvest was well-mixed and aerated before
transfer to pre-inflated bags.
• Clarified harvest was stored at 2-8 °C and quickly
processed through downstream purification.
SKI Transfection to BDS
• The duration from
transfection at Selexis to
BDS fill at KBI is targeted
at 12 months for
bispecifics.
• Flexibility built into the
timeline allows for studies
to investigate challenges
unique to each molecule.
Drug Substance
GLP Tox Material in 11 mo
cGMP (up to 2,000 L)200 L IND Tox Run
MCB MCB Release
Research Cell BankTransfect DNA
Cell Line Development
Upstream Development
Downstream Development
Analytical Development & Qualification
Formulation Development
Stable Pools RCBs
1 2 3 4 5 6 7 8 9 10 11 12 13 14
SKI Performance Across Molecules
• Final cell culture processes
routinely achieve titers of 3-6
g/L for bispecifics.
• Cell viability at the time of
harvest is above 80% for
most molecules, maintaining
productivity through the end
of the process.0
20
40
60
80
100
HarvestViability(%)
Bispecific Molecules
0
2
4
6
8
10
Titer(g/L)
Bispecific Molecules
10+
g/L
88%
Avg.
Fab Homodimer Heterodimer scFv Homodimer
PeakArea(%)
3 L Day 10
3 L Day 12
3 L Day 14
50 L Day 10
50 L Day 12
50 L Day 14
cGMP - Day 12 ProA
Reduction in
homodimer
levels achieved
4 6 8 10 12 14
3 L
50 L
Titer(g/L)
Day
• During scale-up of harvest operations, reduction of the product led to high LMW species.
SCC2 Generation
0 weeks
1 week
3.5 weeks
7 weeks
Gene Synthesis &
Vector Construction
4.5 weeks
12.5 weeks
SCC1 Generation
Super-Transfection
SCC0 Generation
Transfection +
Pool Generation
2000 L SUB
cGMP
Manufacturing
Run
50 L SUB Harvest
Supply Run
+
ambr15 (24 vessels)
Screening Study
200 L SUB
Demonstration Run:
IND Tox
50 L SUB Harvest
Supply Run
3 L Glass
Bioreactors
(4-8 vessels)
Aging Study
ambr250 (12 vessels)
Clones Evaluation
DownstreamDevelopment,AnalyticalDevelopment,AnalyticalMethodQualification
MCBProduction
• Once available, SCC2 clones
are utilized to scale-up the
cell culture process.
• Upstream and Downstream
Process Development
studies occur in parallel
with Analytical
Development activities.
• Following tech transfer from
process development,
clinical manufacturing can
be performed at scales up
to 2,000 L.
Selexis Activities KBI Activities
• Process development at KBI starts with either stable
pools, SCC0 candidates, or an enriched pool, depending
on a project’s timeline.