Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature

Pace, Amanda L.; Wong, Rita; Zhang, Yonghua Taylor; Kao, Yung-Hsiang; Wang, Y. John

doi:10.1002/jps.23529

Cited by 120 publications

(102 citation statements)

References 27 publications

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“…Deamidation of asparagines and glutamines is the most prominent nonenzymatic chemical reaction that is significantly enhanced at higher temperatures and physiological pH. 5,6 Deamidation results in an addition of negative charge to the residue resulting in alterations in protein interactions, which could affect the activity or refolding. 7,8 Interest in deamidation has gained importance since the extent of deamidation was associated with aging, 9,10 cataract formation, 9,11,12 immune recognition, 13 shelf-life of protein pharmaceuticals such as antibodies, 9,13,14 and so on.…”

Section: Introductionmentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

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At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75 C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90 C. MutC has retained more than 50% activity even after incubation at 100 C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.

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Section: Introductionmentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

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“…Separate charge profiles of Fab and Fc domains have been obtained using limited Lys-C or papain proteolysis in combination with CEX separation. [57][58][59][60][61] It is known that the variable regions of both LC and HC could contribute to the overall charge heterogeneity of the antibody product, 55,56 and deamidation of LC and HC CDR asparagine residues could cause loss in bioactivity. 44,62 There is, however, no previous report on domain-specific charge profiling of LC and Fd domains.…”

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confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

123

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“…In addition to previously discussed factors, buffer pH is another parameter that affects asparagine deamidation levels. Higher pH tends to increase the rate of modification since more basic environments promote the deprotonation of the peptidebond nitrogen [26,37]. During the course of this study we measured the pH levels of the buffers used under each temperature condition.…”

Section: Resultsmentioning

confidence: 99%

“…The susceptibility of an asparagine residue to deamidation is affected by primary, secondary and tertiary structure [17][18][19][20][21] as well as buffer pH, ionic strength, concentration and temperature [22][23][24][25][26] which allows this modification to be controlled in a biopharmaceutical product to some extent through the use of appropriate buffer formulations. However, many analytical techniques used to assess the stability of a biotherapeutic are not performed in the optimal drug product buffer formulation and therefore run the risk of introducing artifactual modifications such as deamidation which are not indicative of the true properties of the drug.…”

Section: Introductionmentioning

confidence: 99%

Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody

2014

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The thorough characterization of biopharmaceuticals is essential for ensuring their quality and safety since many potential variations can cause changes to the properties of a drug that may be detrimental to the patient such as decreased efficacy, shorter half-life or increased immunogenicity. Prior to approval and release, protein-based drugs are subject to a battery of analyses to assess the nature of those parameters that are considered critical quality attributes. In some cases the analytical method used may itself cause modifications that are impossible to distinguish from those induced by the intended test conditions (e.g. storage time/temperature, light exposure) which are used to assess drug stability. It is therefore important to develop and utilize analytical methods which impose as few artifactual modifications as possible. Asparagine deamidation is a common protein modification and it is known to be induced during tryptic digestion. Therefore we examined common tryptic digestion protocols and compared their propensities towards asparagine modification. Since microwave assisted hydrolysis techniques are often used to shorten digestion times and the effect on deamidation is unknown we sought to compare this method against alternate digestion protocols.

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Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature

Cited by 120 publications

References 27 publications

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

A new tool for monoclonal antibody analysis

Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody

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