Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5

Liu, Shanshan; Moulton, Kevin R.; Auclair, Jared R.; Zhou, Zhaohui Sunny

doi:10.1007/s00726-015-2166-z

Cited by 52 publications

(37 citation statements)

References 58 publications

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“…Endoprotease Glu-C that shows activity at low pH was reported to be used in peptide mapping to reduce artificial deamidation. 28 This study used a 17,000 Dalton small protein, calmodulin, and digestion was at pH 4.5 under native condition. This condition is highly unlikely work for antibody molecules and this paper did not show any data on succinimide formation.…”

Section: Introductionmentioning

confidence: 99%

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping

Cao

Niu

Kabundi

et al. 2019

Journal of Pharmaceutical Sciences

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a b s t r a c t mAbs undergo several post-translational modifications, including the formation of succinimide from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of mAbs, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here, we report the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient mAb digestion equivalent to conventional tryptic peptide-mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a good manufacturing practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness.

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Section: Introductionmentioning

confidence: 99%

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping

Cao

Niu

Kabundi

et al. 2019

Journal of Pharmaceutical Sciences

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“…It is therefore reasonable to expect that MI can accelerate the deamidation of proteins. Liu, Moulton, Auclair, and Zhou18 developed a novel method for the study of protein deamidation using mass spectrometry, and it is very likely that their approach is also suitable for the study of milk proteins. In this study, milk samples incubated with MTGase at 30 °C for 3 h presented a reduction in the quantity of α S -CN, κ-CN, and β-CN (Fig.…”

Section: Resultsmentioning

confidence: 99%

Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase

Chen

Hsieh

2016

Sci Rep

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We investigated the combined effects of microbial transglutaminase (MTGase, 7.0 units/mL) and microwave irradiation (MI) on the polymerization of milk proteins at 30 °C for 3 h. The addition of MTGase caused the milk proteins to become polymerized, which resulted in the formation of components with a higher molecular-weight (>130 kDa). SDS-PAGE analysis revealed reductions in the protein content of β-lactoglobulin (β-LG), αS-casein (αS-CN), κ-casein (κ-CN) and β-casein (β-CN) to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. The use of MTGase in conjunction MI with led to a 3-fold increase in the rate of milk protein polymerization, compared to a sample that contained MTGase but did not undergo MI. Results of two-dimensional gel electrophoresis (2-DE) indicated that κ-CN, β-CN, a fraction of serum albumin (SA), β-LG, α-lactalbumin (α-LA), αs1-casein (αs1-CN), and αs2-casein (αs2-CN) were polymerized in the milk, following incubation with MTGase and MI at 30 °C for 1 h. Based on this result, the combined use of MTGase and MI appears to be a better way to polymerize milk proteins.

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“…At this temperature, the pH of the phosphate buffer is very close to 7.40. At the end of the incubations, 4.5 μL of 1 M hydrochloric acid was added to each tube, lowering the pH to about 4 to minimize any additional deamidation of the asparaginyl residue (Liu et al 2016). These aged samples were then stored at −20 °C prior to analysis by HPLC.…”

Section: Methodsmentioning

confidence: 99%

Deuteration protects asparagine residues against racemization

et al. 2016

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Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age.

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Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5

Cited by 52 publications

References 58 publications

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping

Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase

Deuteration protects asparagine residues against racemization

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