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



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


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).


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.


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.


aggregation antibodies deamidation fermentation stress models 



aggregation temperature


cation exchange chromatography


differential scanning calorimetry


downstream processing


dynamic light scattering


fermentation-like buffer


high molecular weight


melting point temperature


monoclonal antibody


nephelometric turbidity units


Reynold’s number


size exclusion chromatography


upstream processing


chinese Hamster Ovary


Immunoglobulin G










phosphate-buffered saline


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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