Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by <i>Escherichia coli</i> cells

Huang, Yunping; O’Mara, Brian W.; Conover, Matthew; Ludwig, Richard; Fu, Jinmei; Tao, Li; Li, Zheng Jian; Rieble, Siegfried; Grace, Michael J.; Russell, Reb J.

doi:10.1002/pro.2046

Cited by 16 publications

(9 citation statements)

References 32 publications

(39 reference statements)

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“…For the WT and CC genes, this was probably due to the very low protein yield, but in the case of the NF2B gene, it is likely that the optimization, which removed repeated nucleotides, reduced the level of frame-shifting and thus the probability of synthesis of truncated proteins. Only a few studies have investigated amino acid mis-incorporations in proteins using various methods, and quantification of these mis-incorporations has not been performed [3,[37][38][39][40][41][42][43][50][51][52][53][54][55][56][57]. We observed 71 misincorporation sites over the entire length of the PfLys-RS protein (584 amino acids), with a mean level of amino acid mis-incorporation of 0.1% (Tables 2 and 3).…”

Section: Discussionmentioning

confidence: 99%

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies

et al. 2015

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Heterologous protein production is a key technology for biotechnological, health sciences and many other research fields. Various approaches have been developed for its optimization, but the research emphasis has been on optimization of protein yield rather than protein quality. In this study, we have established a workflow for synthetic gene optimization for heterologous protein expression that combines bioinformatics, laboratory experiments, mass spectrometry and statistical analysis. Two gene primary structure analysis platforms, Anaconda and EuGene, and multivariate optimization methods were employed to re-design the Plasmodium falciparum lysyl-tRNA synthetase gene for optimal expression in Escherichia coli. Synthetic genes were expressed from common vectors, and amino acid misincorporations in the expressed proteins were detected and quantified using mass spectrometry. The association between the identified amino acid misincorporations and 23 gene variables was then analysed. The synthetic genes yielded significantly higher levels of protein relative to the wild-type gene, but 71 amino acid mis-incorporation sites were observed along the whole protein and across the synthetic genes that were statistically associated with specific codons and protein secondary structures. The optimization method that led to production of the most accurate protein was based on a multivariate approach that combined variables that are known to influence mRNA translation.

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Section: Discussionmentioning

confidence: 99%

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies

et al. 2015

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“…Incorrect amino acids can be mis-incorporated into proteins during translation due to aminoacyl tRNA mis-charging or due to wobble in aminoacyl tRNA-mRNA recognition. [29][30][31][32][33][34][35][36] Single amino acid sequence variants may also derive from genetic sources as point mutations in one or more copies of the protein coding cassette in the host cell genome. [37][38][39][40] Whether non-enzymatic or biosynthetic in origin, the aforementioned forms of heterogeneity are all relatively simple and frequently easy to identify.…”

Section: Introductionmentioning

confidence: 99%

Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody

Rehder

Wisniewski

Liu

et al. 2018

mAbs

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While characterizing a therapeutic IgG4 monoclonal antibody (mAb), we observed a variant with a mass 1177 Da larger than the predominant mAb form that could not be ascribed to previously described modifications. Through successive rounds of experimentation, we localized the mass addition to the C-terminus of the heavy chain (HC). During this process we observed that when the mAb was broken down into separate domains, the Fc and the 1177 Da-modified Fc could be chromatographically separated. Separation allowed collection of native and modified Fc fractions for LC/MS peptide mapping. A unique peptide present in the modified fraction was de novo sequenced and demonstrated to be a modified form of the HC C-terminus lacking two native residues (GK) and gaining twelve additional nonnative residues (EAEAASASELFQ). Aware of other mAb variants with genetic origins, we sought to understand whether this modification too had a genetic basis. In silico translation of the expression vector encoding the mAb demonstrated that a normally non-coding section of nucleotides in the + 1 reading frame relative to the HC C-terminal coding region could have led to a transcript with the nonnative C-terminal extension. Two potential mechanisms for how this nucleotide sequence might have fused to the native HC coding region and led to expression of the extension product are presented. ARTICLE HISTORY

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2015

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The development of sensitive techniques to detect sequence variants (SVs), which naturally arise due to DNA mutations and errors in transcription/translation (amino acid misincorporations), has resulted in increased attention to their potential presence in protein-based biologic drugs in recent years. Often, these SVs may be below 0.1%, adding challenges for consistent and accurate detection. Furthermore, the presence of false-positive (FP) signals, a hallmark of SV analysis, requires time-consuming analyst inspection of the data to sort true from erroneous signal. Consequently, gaps in information about the prevalence, type, and impact of SVs in marketed and in-development products are significant. Here, we report the results of a simple, straightforward, and sensitive approach to sequence variant analysis. This strategy employs mixing of two samples of an antibody or protein with the same amino acid sequence in a dilution series followed by subsequent sequence variant analysis. Using automated peptide map analysis software, a quantitative assessment of the levels of SVs in each sample can be made based on the signal derived from the mass spectrometric data. We used this strategy to rapidly detect differences in sequence variants in a monoclonal antibody after a change in process scale, and in a comparison of three mAbs as part of a biosimilar program. This approach is powerful, as true signals can be readily distinguished from FP signal, even at a level well below 0.1%, by using a simple linear regression analysis across the data set with none to minimal inspection of the MS/MS data. Additionally, the data produced from these studies can also be used to make a quantitative assessment of relative levels of product quality attributes. The information provided here extends the published knowledge about SVs and provides context for the discussion around the potential impact of these SVs on product heterogeneity and immunogenicity.

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Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

Cited by 16 publications

References 32 publications

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies

Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody

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

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