Discovery and Investigation of Misincorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in Chinese Hamster Ovary Cells

Abstract

Misincorporation of amino acids in proteins expressed inEscherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA Asn rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions. Many recombinant proteins have been approved as therapeutic drugs by the Food and Drug Administration, and many more are undergoing clinical trial (1). For economic and practical reasons, considerable effort has been made to increase product yield and process efficiency for proteins made in mammalian cell culture. Nowadays, large amounts of proteins can be expressed efficiently in optimized expression systems, with yields from bioreactors having improved more than 100-fold during the past two decades (2). Yields as high as 10 g/liter have been reported for production of monoclonal antibodies in CHO 2 cells (3). These yields are due mainly to improvements in host cell engineering, cell line selection, and culture medium optimization (4). However, it is well known that overexpressing recombinant proteins can lead to nutritional stresses in the host cells and that these stresses can markedly increase the frequency of random translational errors, resulting in a heterogeneous mixture of proteins (5-11). A variety of translational errors have been observed during overexpression of proteins in Escherichia coli, including frame shifts, premature truncation, read-through, leaky stop codons, and amino acid misincorporation (12-16). Nevertheless, there are few such reports for proteins made in mammalian cells, and it is commonly believed that the fidelity of translation in mammalian cells is higher (8, 17). Here we report for the first time that misincorporation, namely of Ser for Asn, can occur in proteins overexpressed in CHO cells under normal recombinant protein production conditions. Further investigation showed that supplementation of the medium with Asn can overcome this problem. Our work demonstrates that protein products should be closely monitored for misincorporation, for example, by molecular mass determination and peptide mapping during optimization of culture conditions. EX...

Tailoring recombinant protein quality by rational media design

“…As a result of using modern analytical technologies including intact mass measurement, peptide mapping, and tandem mass spectroscopy sequencing, the well documented misincorporation of amino acids occurring in proteins expressed in Escherichia coli was detected during recombinant protein production in mammalian hosts at high protein expression levels also 189 . Specifically, when cells are starved for Asn, the frequency of serine (Ser) incorporation at Asn positions increases during translation 189 .…”

“…Specifically, when cells are starved for Asn, the frequency of serine (Ser) incorporation at Asn positions increases during translation 189 . In spite of the fact that the underlying mechanisms have yet to be fully understood 190 , it is widely believed that the error rate during protein translation depends on factors such as the type of organism, the genotype and phenotype of the transfected cell lines, the codon usage, and the cell culture conditions 191 .…”

“…When Asn abundance in the media is decreasing, Ser and Asn competition intensifies, leading to misacylation of tRNA Asn by Ser. Serine substitution can be prevented by supplementing the culture medium with Asn 189,190 and regularly feeding Asn (6 g/L) throughout the culture to circumvent depletion of the latter 190 . Nonetheless, even at low levels of Asn in the medium, Ser misincorporation can be prevented through controlled feeding 190 , which may be a useful strategy to avoid important ammonia production as Asn synthetase is able to convert Asn to Gln, and eventually liberate ammonia to replenish the tricarboxylic acid cycle.…”

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

“…For example, 3 variants have been verified by polymerase chain reaction (PCR) analysis to be a genomic nucleotide mutation at the DNA level. 6 Although peptide mapping with UV detection is still a simple but powerful method to detect sequence variants in mAbs, the sequence variants can only be detected while the percentage is 1 to 5% or above. Because sequence variants in the therapeutic mAbs are generally very rare, detecting and characterizing sequence variants is a substantial challenge.…”

“…3 Sequence variants, which result from unintended amino acid substitutions, are protein isoforms containing undesired amino acid sequences that may cause concern during the production of mAbs and other therapeutic proteins. [2][3][4][5][6][7] Therefore, it is necessary to detect potential sequence variants to confirm the purity and safety of the product during clone selection and bioprocess development. To ensure product safety, efficacy and consistency, manufacturers who produce the therapeutic mAbs are increasingly using advanced analytical methods and technologies during product characterization and development.…”