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Pharmaceutical Research

, Volume 30, Issue 5, pp 1380–1399 | Cite as

Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions

  • Stefan Dengl
  • Marc Wehmer
  • Friederike Hesse
  • Florian Lipsmeier
  • Oliver Popp
  • Kurt Lang
Research Paper

Abstract

Purpose

To investigate antibody stability and formation of modified species under upstream processing conditions.

Methods

The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC).

Results

Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations.

Conclusions

Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

KEY WORDS

aggregation antibodies deamidation fermentation stress models 

Abbreviations

Tagg

aggregation temperature

cIEX

cation exchange chromatography

DSC

differential scanning calorimetry

DSP

downstream processing

DLS

dynamic light scattering

F-Buffer

fermentation-like buffer

HMW

high molecular weight

Tm

melting point temperature

MAb

monoclonal antibody

NTU

nephelometric turbidity units

Re

Reynold’s number

SEC

size exclusion chromatography

USP

upstream processing

CHO

chinese Hamster Ovary

IgG

Immunoglobulin G

xMAB

CrossMab

PTFE

polytetrafluoroethylene

PVDF

polyvinylidenefluoride

UV

ultraviolet

PBS

phosphate-buffered saline

Notes

Acknowledgments and Disclosures

We would like to thank Carolin Lucia and Katharina Didzus from Roche Penzberg for their support with bioreactor stress experiments, Sabrina Mahlack for her support with MAb4.2 purification, and Hubert Kettenberger, Holger Kley and Xaver Reiser for their support with DLS and DSC measurements. We would also like to thank Alexandra Schindl for carrying out the shaking stress experiments and Robert Puskeiler for support with calculations of physical parameters of the stirring stress models.

We are also grateful to Jörg Hörnschemeyer and Jonas Fast of Roche Basel for sharing information about the identity of cIEX peaks.

S.D, F.H. F.L. O.P. and K.L are all employees of Roche at the time of publication. The remaining authors declare no competing financial interests.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Stefan Dengl
    • 1
  • Marc Wehmer
    • 2
  • Friederike Hesse
    • 1
  • Florian Lipsmeier
    • 3
  • Oliver Popp
    • 3
  • Kurt Lang
    • 1
  1. 1.Pharma Research and Early DevelopmentRoche Diagnostics GmbHPenzbergGermany
  2. 2.Department of ChemistryTechnical University MunichGarchingGermany
  3. 3.Pharma Research and Early DevelopmentRoche Diagnostics GmbHPenzbergGermany

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