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Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions

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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.

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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

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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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Authors and Affiliations

  1. Pharma Research and Early Development, Roche Diagnostics GmbH, Nonnenwald 2, 82372, Penzberg, Germany

    Stefan Dengl, Friederike Hesse & Kurt Lang

  2. Department of Chemistry, Technical University Munich, 85748, Garching, Germany

    Marc Wehmer

  3. Pharma Research and Early Development, Roche Diagnostics GmbH, 82372, Penzberg, Germany

    Florian Lipsmeier & Oliver Popp

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  1. Stefan Dengl
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Correspondence to Kurt Lang.

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Dengl, S., Wehmer, M., Hesse, F. et al. Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions. Pharm Res 30, 1380–1399 (2013). https://doi.org/10.1007/s11095-013-0977-8

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  • DOI: https://doi.org/10.1007/s11095-013-0977-8

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