Amino acid misincorporation in recombinant biopharmaceutical products

Harris, Richard; Kilby, Peter M.

doi:10.1016/j.copbio.2014.05.003

Cited by 29 publications

(21 citation statements)

References 40 publications

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“…In the production of biopharmaceutical proteins, including antibodies and antibody fragments, product‐related variants can result from both post‐translational (Harris and Kilby, ) and biochemical modification during cell cultivation as well as during subsequent processing, formulation, and storage (Goswami et al, ). Deamidation is a commonly occurring chemical modification in biopharmaceuticals (Goswami et al, ).…”

Section: Resultsmentioning

confidence: 99%

QSAR models for prediction of chromatographic behavior of homologous Fab variants

Robinson

Karkov

Woo

et al. 2017

Biotech & Bioengineering

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While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc.

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Section: Resultsmentioning

confidence: 99%

QSAR models for prediction of chromatographic behavior of homologous Fab variants

Robinson

Karkov

Woo

et al. 2017

Biotech & Bioengineering

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“…The most frequently observed case is arginine to lysine substitution, where the arginine rare codon AGA is erroneously recognized by [12–14]. Mis-incorporation of glutamine (CAG) for arginine (CGG) has also been reported [15, 16], and the impact in areas such as biopharmaceutical production has been discussed [17]. A simple explanation for this phenomenon is that the lack of arginine tRNA's for these codons allows other more abundant amino-acyl-tRNA complexes (EF-Tu:GTP:tRNA) to out compete for the ribosomal A site and accomodate a near-cognate tRNA.…”

Section: Introductionmentioning

confidence: 99%

Mistakes in translation: Reflections on mechanism

Liu

Sharp

et al. 2017

PLoS ONE

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Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.

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“…Although the aim of our study was to identify impurity KR14, we were also curious about what would be a possible cause of such misincorporation. It is known that frequency of translation errors increases during overexpression of heterologous proteins in E.coli due to stressed conditions, amino acid starvation and translation from nonoptimal codons ( 20 , 39 , 40 ). Infidelity of the genetic code translation on the molecular level can result from the shift of the mRNA reading frame, tRNA mischarging or codon misreading.…”

Section: Resultsmentioning

confidence: 99%

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli

et al. 2019

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Purpose Identification of human insulin analogs’ impurity with a mass shift +14 Da in comparison to a parent protein. Methods The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing. Results The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs. Conclusions Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing. Electronic supplementary material The online version of this article (10.1007/s11095-019-2601-z) contains supplementary material, which is available to authorized users.

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Amino acid misincorporation in recombinant biopharmaceutical products

Cited by 29 publications

References 40 publications

QSAR models for prediction of chromatographic behavior of homologous Fab variants

QSAR models for prediction of chromatographic behavior of homologous Fab variants

Mistakes in translation: Reflections on mechanism

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli

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