Monoclonal antibodies (mAbs) are successful biotherapeutics in the treatment of various diseases. During manufacturing of mAbs higher molecular weight (HMW) aggregates can be formed during upstream (USP) and downstream (DSP) processing, which negatively influence product yields, reduce the therapeutic efficacy of the mAbs and trigger immunogenic responses upon administration. Reducing the level of aggregates during USP could improve the production of biopharmaceuticals and reduce the burden on expensive DSP removal of the HMW species. However, the lack of analytical tools to detect mAb aggregates in USP restricts understanding the origin of the aggregates and identifying cell culture conditions influencing product quality to reduce the level of mAb aggregates. We present a high-throughput compatible method which allows quantification of mAb aggregate formation directly in cell culture samples of Chinese hamster ovary (CHO) cells replacing falsifying, laborious and time-consuming chromatographic methods. Using this new methodology, we have screened for different culture conditions effecting mAb aggregate formation in a non-producing and a mAb producing CHO cell line. Finally, we have identified important process parameters to influencing protein aggregation in mammalian cell culture. Hence, our work demonstrates that the formation of mAb aggregates can be assessed directly in mammalian cell culture and product quality can be controlled by the selection of certain cell culture process parameters.

1. Identification of Process Parameters influencing
Product Quality in Mammalian Cell Culture
Albert Paul
Institute of Applied Biotechnology (IAB) Biberach
24th ESACT Meeting Barcelona, Spain

2. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 2
Monoclonal antibodies (mAbs)
• Most successful biopharmaceuticals
Protein aggregation
• Quality, safety and efficacy issues
• Occurs in all steps of manufacturing
• Aggregates are removed in DSP
 Reduced process yields
• Upstream reduction of aggregation
 Reduced burden on DSP
 Increased process yield
 Relative little known
Introduction
M
DSP
Protein
Aggregation

3. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 3
USP analysis of mAb aggregates is difficult
• No single analytical method to cover entire size
range of aggregates
• Contaminants complicate analysis in cell culture
 DNA, lipids, and host cell proteins
• Evaluation of aggregate levels in cell culture
 Protein A purification step
 SEC analysis
• Capture step exposes antibodies to pH-shifts,
which influences the aggregation itself
[Joubert et al., J Biol Chem 2010]
[Paul et al., Pharm Res 2012]
Introduction
10nm

4. Methods for USP analysis of
mAb aggregate formation

5. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 5
SE-HPLC analysis
• NaCl induction
– mAb1 (1 mg/ml)
– NaCl: 0-1.5 M
Concentration-dependent formation
of mAb dimer
• Freeze-thawing (FT)
– mAb2 (1 mg/ml)
– FT cycles: -80°C/25°C for 15 min
MAb dimer and tetramer formation
USP analysis of mAb aggregates

6. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 6
SE-HPLC analysis
• MAbPac SEC-1 (A) and Yarra S4000 (B)
• NaCl-induced mAb in SFM4CHO (A)
• FT-induced mAb in CHO DG44 supernatant (B)
Results
 Host cell/medium components elute later
 Monomer and aggregates detectable
 Quantification possible
[Paul et al., BMC Biotechnol 2014]
USP analysis of mAb aggregates

7. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 7
SE-HPLC analysis
• Aggregate formation during batch cultivation
− CHO mAb producer cell line
− Supernatant analysis
 Directly after inoculation
 After 144 h cultivation
• Results
− After inoculation
 No signals for mAb
− After 144 h cultivation
 Aggregates and monomer detectable
− Quantification
 Monomer 23% ± 0.4%
 Dimer 10% ± 0.3%
 Oligomers 67% ± 0.7%
[Paul et al., BMC Biotechnol 2014]
USP analysis of mAb aggregates

8. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 8
Analysis using fluorescence dyes
• Extrinsic fluorescence dyes
− Thioflavin T (ThT)
− 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS)
• Fluorescence dye-based aggregation (FDBA) assay
− Fluorescence spectroscopy
 Soluble aggregates
• Fluorescence microscopy
− NyONE : Fully automated cell imager
 Large HMW species
USP analysis of mAb aggregates
ThT
Bis-ANS

9. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 9
FDBA-assay
– CHO mAb cells cultivated for 120 h in SFL,
TFL and TPP
– Analysis: SE-HPLC (A), 100 µM ThT (B) and
10 µM Bis-ANS (C)
– Controls: SFM4CHO medium, untreated
(negative ctr) and stressed mAb2 in
medium (positive ctr)
Results
 Aggregate content (TPP<SFL<TFL)
successfully determined using both dyes
 ThT lower signal for cell culture samples
than controls
 Bis-ANS suitable for cell culture samples
[Paul, Schwab et al., Anal Bioanal Chem 2015]
USP analysis of mAb aggregates

10. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 10
• CHO DG44: 4x105 cells/mL
• 1M NaCl stressed mAb
• 25 µg/mL mAb
• 10 µM Bis-ANS
USP analysis of mAb aggregates
Fluorescence microscopy
CHO cells (BF) CHO cells + mAb aggregates (BF) CHO cells + mAb aggregates (UV/Green)
• Results
 Large HMW species visible
 HMW species distinguishable from
CHO cells
 MAb aggregates detectable

11. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 11
• CHO mAb cells
• 4x105 cells/mL
• SFM4CHO medium
• 118 h cultivation
• 0.2 µM Bis-ANS
• Results
 Particles of different sizes
detected
 Increase of particles over
cultivation time
USP analysis of mAb aggregates
Fluorescence microscopy

12. Identification of Process Parameters
influencing Protein Aggregation

13. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 13
Process parameters influencing protein aggregation
Parameters
• pH value: pH 6.8-7.4
• Osmolality: 333-533 mOsm/Kg
• Agitation: 100-160 rpm
• Antifoam: 0.02-0.04%
• Valproic Acid: 0-4 mM
Responses
• Cell concentration
• LDH level
• MAb concentration
• FDBA assay
• Fluorescence Microscopy
Screening using DoE
TubeSpin Bioreactors®
• CHO Mock cell line
• CHO mAb producing cell line
• Seed 4x105 cells/mL
• SFM4CHO medium
10 mL

14. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 14
Process parameters influencing protein aggregation
CHO Mock cell line
Cell concentration
CHO Producer cell line
Cell concentration MAb Productivity
Effect of Osmolality Effect of Osm and VPA Effect of Osm and VPA

15. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 15
Process parameters influencing protein aggregation
CHO Mock cell line - Fluorescence CHO Producer cell line - Fluorescence
Effect of Agitation Effect of Agitation

16. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 16
Process parameters influencing protein aggregation
CHO Mock cell line – Particle Count CHO Producer cell line – Particle Count
Effect of Agi Effect of Osm Effect of Agi
Effect of VPA
Effect of Osm

17. Effect of VPA on
product quality

18. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 18
Influence of VPA (Valproic acid)
• HDAC (histone deacetylase) inhibitor
• MAb productivity and aggregate
formation
• CHO producer cells
• After 120 h cultivation
• Protein A and SE-HPLC
• Results
 Increased specific productivity using 2 mM
VPA
 VPA dose-dependent formation of mAb
aggregates
[Fischer, Paul et al., Biotechnol Bioeng 2015]
Effect of VPA on mAb aggregation

19. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 19
SE-HPLC analysis
• CHO mAb supernatant (96h)
• 0, 72, 96 and 120 h
• With 0, 1, 2 or 4 mM VPA
Results
 MAb aggregates in supernatant
increase with incubation time
 VPA no significant impact in
supernatant
 Two levels
 Cellular level
 Bioprocess level
[Fischer, Paul et al., Biotechnol Bioeng 2015]
Two differerent levels of aggregate formation

20. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 20
N-Glycan analysis
• CHO mAb cells
• 144h cultivation
• Biological triplicates
• HILIC-analysis
Results
 VPA also influences N-
Glycan profile
 Less G1F and G2F
 More G0F
[Fischer, Paul et al., Biotechnol Bioeng 2015]
VPA - Impact on Glycosylation
w/o VPA
2 mM VPA

21. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 21
Methods for USP analysis of mAb aggregates
Identification of process parameters
Agitation and VPA induce aggregate formation
Osmolality decreases aggregate formation
Aggregate formation in USP occurs on two levels
Cellular level
Bioprocess level
VPA: Increased level of aggregates and impaired
N-glycosylation
Summary

22. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 22
Influence of Temperature
Influence of Agitation
Influence of Osmolality
 Increase of specific productivity
 Reduced aggregate formation
Transfer of results on bioreactor cultivation
Outlook

23. 1st June 2015 Albert Paul - 24th ESACT Meeting Slide 23
• Prof. Dr. Friedemann Hesse
• Prof. Dr. Boris Mizaikoff
• PhD Eva Herold
• Dipl.-Biol. Karen Schwab
• M.Sc. Fabian Stiefel
• M.Sc. Alina Handl
• Nina Prokoph
• Elena Haas
• Franziska Schandock
• Melanie Leitte
• Heidi Schulze
• Martin Domnowski
• Jörg Zimmermann
• IAB members
Acknowledgements

24. Thank you for your attention!
Questions?