Tailoring recombinant protein quality by rational media design

Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé

doi:10.1002/btpr.2089

Cited by 65 publications

(55 citation statements)

References 281 publications

(483 reference statements)

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“…Finally, numerous previous studies have demonstrated the impact of media on glycosylation (Brühlman et al, 2015; Hossler et al, 2014; Surve and Gadgil, 2015). Thus, it will be of great value to integrate our model with models of other metabolic pathways.…”

Section: Discussionmentioning

confidence: 91%

“…Experimental control strategies, including genetic modifications and media additives, have been used to manipulate glycosylation (Brühlman et al, 2015; Shi and Goudar, 2014; Sou et al, 2014; St Amand et al, 2014), but so far a systematic understanding of these strategies is largely lacking. Therefore, computational models of glycosylation carry a great potential in elucidating control strategies in a rational and cost-efficient manner.…”

Section: Introductionmentioning

confidence: 99%

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A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

Spahn

Hansen

et al. 2016

Metabolic Engineering

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Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks underlying glycosylation and the vast number of different glycans that can be synthesized in a host cell. Computational modeling offers an intriguing option to rationally guide glycoengineering, but the high parametric demands of current modeling approaches pose challenges to their application. Here we present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles on EPO, IgG as well as the endogenous secretome following glycosyltransferase knock-out in different Chinese hamster ovary (CHO) cell lines. Our approach offers a flexible and user-friendly platform that can serve as a basis for powerful computational engineering efforts in mammalian cell factories for biopharmaceutical production.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

Spahn

Hansen

et al. 2016

Metabolic Engineering

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“…The increasing evidence for product alterations through chemical conjugations or modifications ascribable to cell culture medium reaction products supports the need for novel approaches to define and monitor CDM quality . At present, Raman and fluorescence spectroscopy in combination with chemometric tools represent the most commonly used methods to examine alterations in cell culture media batches .…”

Section: Introductionmentioning

confidence: 99%

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Krattenmacher

Heermann

Calvet

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Chemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations. RESULTS In order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC‐MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l‐cysteine, l‐proline, vitamin B6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products. CONCLUSION The successful development of a new LC‐MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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“…The achievable cell density, titer, metabolism and culture duration is dependent on the compositions of cell culture media [3]. For the predominantly used Chinese hamster ovary (CHO) cells, research shows that individual components of mammalian cell culture media (CCM), even in trace amounts, influence the post translational modifications crucial to the function of the recombinant proteins produced [4][5][6]. The formulation of these media is done in small scale in liquid using stock solution of ingredients.…”

Section: Introductionmentioning

confidence: 99%

Compaction of chemically defined cell culture media increases its dissolution rate through an increase of solvent accessible surface area

et al. 2016

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Cultivation of mammalian cells for the generation of therapeutics requires the use of cell culture media which is a defined mixture of nutrients including carbohydrates, amino acids, lipids, vitamins, and inorganic salts. Its composition influences the cell density, yield, and quality of the molecule expressed. These chemically defined cell culture media are manufactured as powders since they offer advantages of storage, handling and stability. To prevent de-mixing of the powders and maintain homogeneity, these powders are milled down to small particle size ranges. However, this small particle size allows for inter-particle forces to impact powder flow properties, and decreases the surface area available to dissolution. Hence a method is required that improves these powder parameters, without altering their composition. A deeper understanding of the dissolution mechanism of multicomponent mixtures like cell culture media is also needed. Here, cell culture media granules, generated from homogeneous powders by roller compaction are evaluated. The granulation process did not alter the composition of the media and properties of solutions generated thereof. The analysis and comparison of the dissolution profile of powder and compacted media enabled the calculation of the actual surface area available to dissolution. The improvement in granule dissolution can be attributed to dispersion and disintegration of the granules on wetting increasing available surface area. Decrease in inter-particle forces in granules also improves flow-ability properties. Surface character and pores on the granules further augments the improvement in dissolution.

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Tailoring recombinant protein quality by rational media design

Cited by 65 publications

References 281 publications

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Compaction of chemically defined cell culture media increases its dissolution rate through an increase of solvent accessible surface area

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