Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability

Dillon, Thomas M.; Bondarenko, Pavel V.; Rehder, Douglas S.; Pipes, Gary D.; Kleemann, Gerd R.; Ricci, Margaret Speed

doi:10.1016/j.chroma.2006.01.016

Cited by 172 publications

(165 citation statements)

References 41 publications

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“…Previous studies showed that intact mAbs, as well as mAb subunits, show poor recovery in RPLC conditions when working at moderate temperature (e.g. ≤80 • C) [31,40,41], thus demonstrating the need to work at 80-90 • C to avoid adsorption issues and reach acceptable recovery (e.g. above 90% of the injected protein amount).…”

Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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a b s t r a c tHydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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“…However, it has recently been reported that IgG2 antibody preparations are not homogeneous and instead consist of distinct isoforms. [19][20][21][22][23] The isomeric structures are due to disulfide shuffling between cysteine residues within the light (LC) and heavy chains (HC) with the upper hinge sequence. Three IgG2 isomers (A, B, and A/B) have been characterized and shown to be structurally and, in at least some instances, functionally distinct.…”

Section: Introductionmentioning

confidence: 99%

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

et al. 2010

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Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild-type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.

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“…Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.…”

mentioning

confidence: 99%

“…The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on-line RP-HPLC and mass spectrometry (MS) [9,24]. However, high temperature and low pH are normally employed for elution, which can cause antibody degradation [9].…”

mentioning

confidence: 99%

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Liu

Gaza-Bulseco

Chumsae

2009

J. Am. Soc. Mass Spectrom.

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Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly. (J Am Soc Mass Spectrom 2009, 20, 2258 -2264) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry is one of the most indispensable techniques for the characterization of recombinant monoclonal antibodies because most of the modifications that result in heterogeneity and degradation are involved in molecular weight differences [1,2]. Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on...

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Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability

Cited by 172 publications

References 41 publications

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

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