Identification of Process Parameters influencing Product Quality in Mammalian Cell Culture

Albert Paul

Institute of Applied Biotechnology (IAB) Biberach

24th ESACT Meeting Barcelona, Spain

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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

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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

Methods for USP analysis of mAb aggregate formation

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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

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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

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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

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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

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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

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• 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

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• 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

Identification of Process Parameters influencing Protein Aggregation

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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

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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

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Process parameters influencing protein aggregation

CHO Mock cell line - Fluorescence CHO Producer cell line - Fluorescence

Effect of Agitation Effect of Agitation

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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

Effect of VPA on product quality

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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

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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

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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

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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

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Influence of Temperature Influence of Agitation Influence of Osmolality Increase of specific productivity Reduced aggregate formation

Transfer of results on bioreactor cultivation

Outlook

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• 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

Thank you for your attention! Questions?