Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations
Melanie Maier,
Linus Weiß,
Nikolas Zeh,
Valerie Schmieder-Todtenhaupt,
Alireza Dehghani,
Marius Nicolaus Felix,
Daniel Heinzelmann,
Benjamin Lindner,
Moritz Schmidt,
Joey Studts,
Patrick Schulz,
Bernd Reisinger,
Kerstin Otte,
Matthias Franzreb,
Daniel Lakatos,
Simon Fischer
Affiliations
Melanie Maier
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Linus Weiß
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Nikolas Zeh
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Valerie Schmieder-Todtenhaupt
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Alireza Dehghani
Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Marius Nicolaus Felix
Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Daniel Heinzelmann
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Benjamin Lindner
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Moritz Schmidt
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Joey Studts
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Patrick Schulz
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Bernd Reisinger
Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Kerstin Otte
Institute for Applied Biotechnology, University of Applied Sciences Biberach, Biberach an der Riss, Germany
Matthias Franzreb
Institute of Functional Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany
Daniel Lakatos
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Simon Fischer
Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.