Control of misincorporation of serine for asparagine during antibody production using CHO cells

Abstract

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…155 Misincorporation of amino acids due to amino acid starvation can be eliminated by providing sufficient amounts of the specific amino acids. 149,150,154 It should be mentioned that low levels of misincorporation can occur naturally, 156 and it should not be a surprise that endogenous IgG molecules also contain low levels of sequence variants. Low levels of sequence variation may never be completely eliminated.…”

An optimized approach to the rapid assessment and detection of sequence variants in recombinant protein products

“…Amino acid substitutions consistent with N>S have been reported before [8]. Seven N>S SVs with R 2 values above 0.9 were detected in the A/B mixing study and six in the B/C study; all were confirmed by manual inspection of the MS/ MS spectra (data not shown) and were unique to mAb B.…”

“…These SVs can take the form of Nterminal or C-terminal extensions due to DNA rearrangements [1,2], un-cleaved signal sequence [3], DNA point mutations [4,5], or amino acid substitutions [6][7][8]. In the case of amino acid misincorporations, SVs due to these events are likely to be ubiquitous in recombinant protein drugs [9], though challenging to detect.…”

Tailoring recombinant protein quality by rational media design

“…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.…”

“…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.…”