Non-enzymatic hinge region fragmentation of antibodies in solution

Cordoba, Armando J.; Shyong, Bao-Jen; Breen, Deirdre; Harris, Reed J.

doi:10.1016/j.jchromb.2004.12.033

Cited by 177 publications

(148 citation statements)

References 11 publications

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“…The result may be due to the fact that these antibodies share the same framework and only vary in the CDRs, while fragmentation is an intra-molecular self-cleavage reaction within the hinge region. Previous studies showed that the sequence SCDKTHTC [33][34][35] promoted the cleavage of peptide bonds in the hinge region. Possible cleavage sites include S/C, C/D, D/K and H/T.…”

Section: Methodsmentioning

confidence: 99%

The impact of glycosylation on monoclonal antibody conformation and stability

Zheng

Bantog

Bayer

2011

mAbs

281

210

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Section: Methodsmentioning

confidence: 99%

The impact of glycosylation on monoclonal antibody conformation and stability

Zheng

Bantog

Bayer

2011

mAbs

281

210

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“…As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2,3]; profile N-linked carbohydrate heterogeneity [3,4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions.…”

mentioning

confidence: 99%

Molecular mass analysis of antibodies by on-line SEC-MS

Brady

Valliere-Douglass

Martinez

et al. 2008

J. Am. Soc. Mass Spectrom.

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Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required. M ass analysis of recombinant proteins is a key characterization assay used to evaluate the entire amino acid sequence of the molecule and the presence of post-translational modifications. As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2,3]; profile N-linked carbohydrate heterogeneity [3,4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions. This method is highly resolving and advantageous for the detection of minor product impurities and the resolution of different amino acid sequences or heterogeneous post-translational modifications. However, rpHPLC of antibodies has several distinct disadvantages. Due to the large size and relatively hydrophobic nature of antibodies, high temperatures are typically employed to improve elution and peak shape profiles [1,4]. However, high temperatures may lead to artifactual degradation of the sample during analysis. Inclusion of TFA as a mobile phase additive in rpHPLC is often necessary to obtain good chromatography [1], but may result in decreased ionization [10,11]. Additionally, reduction of antibodies and analysis of the constituent light and heavy chains often results in tailing of the heavy-chain component and the potential for carry-over problems. These issues can lead to lengthened run times or the inclusion of blank runs between samples to increase confidence in the results.We optimized the use of a commercially available polyhydroxyet...

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“…A mixture of protease inhibitors including E64, leupeptin, benzamidine, E-amino caproic acid, pepstatin A, and EDTA was tested. It did not stop the peptide bond cleavage 187 . An exhaustive review of human protease inhibitors in the frame of protease-targeted drugs research provides a great number of small molecules either approved for clinical use or in development 188 .…”

Section: Low-molecular-weight Speciesmentioning

confidence: 95%

“…Both the disruption of these bonds and the cleavage of a covalent peptide bond by coexistent spontaneous and enzymatic reactions have been evoked to be at the core of LMW formation 68 . The sites in the flexible hinge region of mAbs within the constant heavy 1 and 2 domains (CH1 and CH2) and around domain interfaces are most prone to fragmentation 179,187 . Known for its ability to provide oxidizing potential in redox reactions, copper plays a pivotal role in non-enzymatic cleavage 185 .…”

Section: Introductionmentioning

confidence: 99%

Tailoring recombinant protein quality by rational media design

Brühlmann

Jordan

Hemberger³

et al. 2015

Biotechnology Progress

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Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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Non-enzymatic hinge region fragmentation of antibodies in solution

Cited by 177 publications

References 11 publications

The impact of glycosylation on monoclonal antibody conformation and stability

The impact of glycosylation on monoclonal antibody conformation and stability

Molecular mass analysis of antibodies by on-line SEC-MS

Tailoring recombinant protein quality by rational media design

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