This document summarizes information from Polymun Scientific Immunbiologische Forschung GmbH, a company that develops and manufactures biopharmaceuticals and liposomal formulations. It discusses Polymun's core activities of contract development and manufacturing of biopharmaceuticals, as well as its own R&D projects. Specific reference projects and Polymun's product pipeline are also mentioned. The document then focuses on Polymun's work for the EAVI Horizon 2020 project, developing envelop glycoprotein variants and antibodies for HIV vaccine studies. It discusses considerations for optimizing work flows and presents proof of concept data using an existing cell line as a model protein.

1. Polymun Scientific Immunbiologische Forschung GmbH
Monoclonality - Challenge or Chance
Thomas Sterovsky

2. Developing and Manufacturing Biopharmaceuticals
and Liposomal Formulations for Human Application
Donaustraße 99, 3400 Klosterneuburg, Austria
Polymun Scientific Immunbiologische Forschung GmbH
Inspected by EMA (June 2015) and FDA (October 2013)

3. CONTRACT DEVELOPMENT OF BIOPROCESSES
for the production of bio-pharmaceuticals for human application with focus on
mammalian cell culture products
CONTRACT MANUFACTURING OF BIOPHARMACEUTICALS
production license according §63 of the Austrian pharmaceutical law
LIPOSOMAL FORMULATION OF DRUGS AND VACCINES
formulation development and production of GMP-material
RESEARCH REAGENTS
manufacturing and distribution, mainly HIV reagents and recombinant trypsin
OWN R&D PROJECTS
funded by revenues from contract development and contract manufacturing
Core Activities

4. Reference Projects Biopharmaceuticals
Baxter AG
manufacturing of monoclonal antibody for affinity purification of Protein C
GeNeuro SA
process development and manufacturing of humanized antibody GNbAC1
for treatment of multiple sclerosis
GlaxoSmithKline
process development and manufacturing of recombinant human soluble
ACE2 for clinical studies
Imperial College London
process development and manufacturing of recombinant HIV envelope
protein for vaccination studies

5. Polymun´s Own Product Pipeline
BIOSIMILARS
rhFSH: validated production process at Polymun, market authorisation in
Europe, registration ongoing in several other countries and regions
worldwide
Epo: validated production process at Polymun, out-licensed for
USA/Canada (phase III study ongoing)
LIPOSOMAL SOD – LIPOXYSANTM
Clinical studies
RECOMBINANT HUMAN AND PORCINE TRYPSIN
Process enzyme, cell culture enzyme

6. EAVI Horizon 2020
EAVI2020
Financed by the European Commison, the European AIDS Vaccine
Initiative brings together leading HIV researchers from public
organisations and biotech companies from across Europe, Australia,
Canada and the USA in a focused effort to develop protective and
therapeutic HIV vaccines.
The EAVI2020 consortium, which is led by Imperial College London,
unites scientists from 22 institutions, pooling their knowledge and
expertise to develop novel candidate vaccines that can be taken through
to human trials within five years. EAVI2020 is funded with an EU-grant
under the health program of Horizon 2020 for research and innovation.

7. EAVI Horizon 2020
GP140
Envelope glycoprotein of HIV (Env)
Truncated form of gp160
Image credit: US National Institute of Health
Tommi A. White et al.
J. Virol. 2011;85:12114-
12123

8. EAVI Horizon 2020
POLYMUN WORK PACKAGE
Production of GMP grade material of eight different variants of gp140
Production of GMP grade material of a h-mAb (PGT145)
CRITICAL ISSUES
Time line – fast and reliable/robust cell line development
Expression of stable and fully cleaved gp140 trimers
Fast transfer to GMP level
Scalable production system

9. Work Flow Optimization
Jayapal, K P., et al. Chemical Engineering Progress 103.10 (2007): 40.

10. Work Flow Optimization
POINTS TO CONSIDER
Host cell line evaluation
Genetic construct
Work flow (cloning/screening)
Media optimization
Process development
Regulatory requirements

11. Work Flow Optimization
GENETIC CONSTRUCT
~220
kbp
<10 kbp
Rosa 26 BAC
↑ Transcriptional efficiency
↑ Specific productivity
↓ Chromatin positional effects
• Many genes on one BAC possible
• No Gene amplification necessary
• Less screening effort
Common Vector

12. Work Flow Optimization
WORK FLOW
Transfection Selection Amplification
MCB
Process
Development
Cloning
Protein
productivity
assessment
Clone Selection
One step single cell
cloning supported by
imaging system

13. Imaging System
Clonality Confirmation
Cell Monitoring
Focus Assurance
Quality Imaging
Speed
Certainty
Reporting
Single cell confirmation image on Day=0
Cell is not stuck to another cell
Monitor cell division, viability and growth over
next few days to form single colony
100% focus assurance across the microplate
allows for qualification of your cloning method
Highest quality brightfield imaging required for
the routine clone screens
Rapid imaging, image capture and clonal
reporting allows for single round of cloning
Provides photo proof of clonally derived cell line
Clonality Report can be used as part of filing
package with regulator

14. Imaging System
Day 0 Day 1 Day4/5 Day10/14
Review wells, select the wells that are clonal

15. Imaging System
Day 0 Day 1

16. Imaging System
Step 1
Select Data
Step 2
Generate Report
Step 3
Distribute

17. Proof of Concept – Historical Data
CN54GP140 AS MODEL PROTEIN
Cell line established in the
mid 90s at Imperial College of
London
GMP – production for clinical
material
DNA sequence was cloned into a BAC vector
CN54gp140 perfect model protein to evaluate new cell line
development setting for EAVI requirements

18. Proof of Concept
CN54GP140 AS MODEL PROTEIN
Lead clone within 12 weeks
after transfection
Proven clonality
Product demand can be
covered easily
Day 1 Day 2 Day 12Day 5

19. Summary – Monoclonality Challenge AND Chance
BENEFITS OF THE ESTABLISHED SETTING
Time requirement was reduced from 20-30 weeks to 12 weeks
Productivity is easily feasible for project demand. (without the
need of further process development at this project stage)
Evaluation of product quality in early stage of the project
IMAGING SYSTEM
• Confirmation of clonality
• Simple implementation of relevant data in
different reports
• Additional information during clone selection

20. Thank you www.polymun.com