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Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

ABSTRACT: With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

SUBMITTER: Boddapati S

PROVIDER: S-EPMC7595273 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

Boddapati Shanta S Gilmore Jason J Boone Kyle K Bushey John J Ross Jonathan J Gfeller Brian B McFee William W Rao Romesh R Corrigan Greg G Chen Aaron A Clarke Howard H Valliere-Douglass John J Bhargava Swapnil S

PloS one 20201029 10

With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proli ...[more]

PMID: 33119652

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Project description:Background: Cell line specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) too. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing in all genome-scale metabolic models of CHO cell lines. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions.Results: We report protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line and condition specific biomass functions for usage in cell line specific, genome-scale metabolic models of CHO. Finally, flux-balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis.Conclusions: Our study reveals a strong variability of the total protein content and cell dry mass across cell lines. Yet, the relative amino acid composition is independent of the cell lines and conditions and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and needs to be determined for each case. These cell line specific variations in the biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are typically dominated by inaccuracies in the exchange rate spectra. Cell specific biomass variations become only important if the experimental errors in the exchange rate spectra drop below twenty percent. Links:All data and code for data processing are available at <a href='http://dx.doi.org/10.17632/g3wz7spyc9.1' rel='noopener noreferrer' target='_blank'>Data Mendeley</a>.Cell line specific metabolic models were deposited in <a href='https://www.ebi.ac.uk/biomodels/(Chelliah et al, 2014)' rel='noopener noreferrer' target='_blank'>BioModels</a>, identifiers are listed in the study sample table.

Dataset Information

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

Publications

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

Similar Datasets

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