Characterization of Site-Specific Glycation During Process Development of a Human Therapeutic Monoclonal Antibody

Miller, Amanda; Hambly, David M.; Kerwin, Bruce A.; Treuheit, Michael J.; Gadgil, Himanshu S.

doi:10.1002/jps.22504

Cited by 79 publications

(84 citation statements)

References 28 publications

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“…37,38 In comparison, a therapeutic mAb in circulation is typically at 0.2/mg/ml, with an overall glycation level at 10-25%. 5,8,33,37,[39][40][41][42] The average lysine residue glycation rate is similar among the therapeutic antibodies, endogenous human IgG and human serum albumin. 37 If the IgG does not contain a highly reactive glycation site, then the glycation will be spread across the whole molecule with low level glycation on all susceptible glycation sites under endogenous conditions.…”

Section: Causes Of Therapeutic Antibody Glycationmentioning

confidence: 92%

“…For a highly glycated recombinant monoclonal antibody, the total glycation level decreased from 42% to 20% when it was incubated at 37 C for 100 hours in a pH 7 phosphate buffer without glucose, which confirmed the reversible nature of the glycation. 8 Although positively charged primary amines are generally located on the surface of protein molecules, only some of these accessible sites will be specifically reactive toward reducing sugar molecules. No specific sequence that signals a potential glycation site has been identified.…”

Section: Introductionmentioning

confidence: 99%

“…29 However, in the manufacturing of therapeutic recombinant antibody products, AGEs formation has not been observed at significant amounts, even with low 21 or high levels of glycation. 8 For therapeutic mAbs, the potential effects of glycation, such as blocking the biologically functional site or further degradation that induces aggregation, make glycation a potential critical quality attribute (CQA). Comprehensive studies are required to characterize and understand the structure and function relationship of glycation in mAbs.…”

Section: Introductionmentioning

confidence: 99%

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Glycation of antibodies: Modification, methods and potential effects on biological functions

Wei¹,

Berning²,

Quan³

et al. 2017

mAbs

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Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

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Section: Causes Of Therapeutic Antibody Glycationmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Glycation of antibodies: Modification, methods and potential effects on biological functions

Wei¹,

Berning²,

Quan³

et al. 2017

mAbs

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“…LC-MS MAM can also be used to monitor lysine glycation of mAb in bioreactors. Lysine glycation is a non-enzymatic modification that can potentially affect conformation, efficacy, and immunogenicity [22]. The level of glycation at a particular site depends on primary sequence and higher order structures, bioreactor glucose concentration and feed strategy, pH and temperature of buffer streams, and hold times of in-process intermediates [22].…”

mentioning

confidence: 99%

“…Lysine glycation is a non-enzymatic modification that can potentially affect conformation, efficacy, and immunogenicity [22]. The level of glycation at a particular site depends on primary sequence and higher order structures, bioreactor glucose concentration and feed strategy, pH and temperature of buffer streams, and hold times of in-process intermediates [22]. The LC-MAM analysis allows for simultaneous monitoring of all site-specific lysine glycation within a protein expressed in a bioreactor over time, which provides timely information to adjust feed strategy during process development.…”

mentioning

confidence: 99%

LC-MS multi-attribute method for characterization of biologics

Xu¹,

Qiu²,

Li³

2017

J Appl Bioanal

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Biologics, such as therapeutic monoclonal antibodies (mAbs), are complex protein molecules produced from mammalian tissue culture cells through recombinant DNA technology. As a result of naturally-occurring molecular heterogeneity as well as chemical and enzymatic modifications during manufacture, process, and storage, there are many product quality attributes (PQAs) presenting in therapeutic proteins. These PQAs can potentially include: product-related structural heterogeneity related to glycosylation profile, disulfide bond pattern, and higher order structure; product-related degradants and impurities, such as deamidation, oxidation, sequence variants; and process-related impurities and residuals, such as host cell protein (HCP), host cell DNA, and residual protein A [1]. Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules [2][3][4][5], which requires in-depth understanding of these PQAs at the molecular level to ensure that the drug products meet the desired safety and efficacy profiles [6]. The QbD guidelines require development of a quality target product profile (QTPP) that identifies critical quality attributes (CQAs) and implementation of control strategies to ensure that the QTPP is achieved. QTPP is a prospective summary of the quality characteristics of a drug product to be achieved to ensure the desired quality, safety and efficacy [2]. QTPP describes the design criteria for the product and forms the basis for determination of the CQAs, critical process parameters (CPPs), and control strategy. A CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality [2]. A CQA is identified based on the severity of harm to a patient resulting from failure to meet that quality attribute. Analytical methods to identify and quantify these PQAs, especially CQAs, are essential for the development of QTPP and implementation of control strategies. Conventionally, a panel of analytical techniques such as size-exclusion chromatography (SEC), ion-exchange chromatography (IEX), hydrophobic-interaction chromatography (HIC), or capillary electrophoresis (CE) is typically used to monitor product quality consistency as well as product variants and impurities at the intact protein level [7][8][9]. Although these chromatographic and electrophoretic methods widely are used as release assays for biologics [10], they cannot directly monitor biologically relevant PQAs at the molecular level, which does not align with QbD principles. The complexity of biologics attributes and the implementation of QbD strategies demand a multi-attribute method (MAM) that can monitor multiple biologics PQAs at the molecular level in a single assay. Coupling liquid chromatography (LC) to high resolution and high accuracy mass spectrometry (MS) techniques, LC-MS based peptide mapping has become a MAM approach that can identify and quantify multiple ...

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