Bridging size and charge variants of a therapeutic monoclonal antibody by two-dimensional liquid chromatography

Chen, Zhi; Zeng, Mingyong; Park, Sung‐Joo; Balakrishnan, Gurusamy; Zhou, Kaimeng; Pan, Duohai; Das, Tapan K.

doi:10.1016/j.jpba.2020.113178

Cited by 12 publications

(14 citation statements)

References 26 publications

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“…Other common modifications that have been identified in basic species include the presence of partial leader sequence [ 9 , 27 , 32 , 34 , 38 , 45 , 64 , 69 ], succinimide as intermediate for both Asp isomerization [ 26 , 57 , 58 , 59 ] or Asn deamidation [ 44 ] intermediates, and aggregates [ 9 , 10 , 33 ]. Using ion-exchange columns in tandem with the size-exclusion column, it was found that aggregates are eluted in the late fractions from both cation and anion-exchange columns [ 72 ]. The authors hypothesized that the later elution could be due to secondary hydrophobic interactions between aggregates and column resins.…”

Section: Modifications Of Acidic and Basic Speciesmentioning

confidence: 99%

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

et al. 2022

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Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of information about mAbs regarding their structure, stability, post-translation modifications, and the relationship between modification and function has been reported. Yet, substantial resources are still required throughout development and commercialization to have appropriate control strategies to maintain consistent product quality, safety, and efficacy. A typical feature of mAbs is charge heterogeneity, which stems from a variety of modifications, including modifications that are common to many mAbs or unique to a specific molecule or process. Charge heterogeneity is highly sensitive to process changes and thus a good indicator of a robust process. It is a high-risk quality attribute that could potentially fail the specification and comparability required for batch disposition. Failure to meet product specifications or comparability can substantially affect clinical development timelines. To mitigate these risks, the general rule is to maintain a comparable charge profile when process changes are inevitably introduced during development and even after commercialization. Otherwise, new peaks or varied levels of acidic and basic species must be justified based on scientific knowledge and clinical experience for a specific molecule. Here, we summarize the current understanding of mAb charge variants and outline risk-based control strategies to support process development and ultimately commercialization.

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Section: Modifications Of Acidic and Basic Speciesmentioning

confidence: 99%

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

et al. 2022

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“…With 96-well plate fractionation and on-column preconcentration by multi-injection, 11 charged species were able to be detected at low abundance (>1.5%). Compared with traditional preparative CEX chromatography which consumed more than 100 mg of sample and took weeks in method optimization and characterization, this platform finely located PTMs with potential risks on immunogenicity, binding capacity, PK and ADCC potency with about 1.25 mg of sample in 72 h. This process enabled charge variants analysis of mAbs at the discovery and process development stages. In summary, MDLC-MS can play an important role in R&D, meeting the accelerated timeline of bringing a mAb from the DNA sequence to Investigational New Drug (IND) filing.…”

Section: Discussionmentioning

confidence: 99%

“…To avoid these issues, CEX fractions shall be desalted and concentrated prior to further LC–MS analysis. The traditional approaches of peak collection, purification, and subsequent characterization of fractions are labor-intensive and time-consuming, taking days and weeks . Instability of fractions over this long period has also been a concern for some mAbs.…”

Section: Introductionmentioning

confidence: 99%

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry

et al. 2023

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In-depth characterization of charge heterogeneity is a pivotal step desired in the therapeutics antibody development.To this end, a novel on-line multidimensional liquid chromatography-mass spectrometry (MDLC-MS) method for charge variant characterization was developed to dig out potential risks on safety and efficacy. This method implemented 96-well plate fractionation and on-column preconcentration by multi-injection, thereby facilitating detection of charged species at low abundance. Eleven charge variants of mAb-A were preliminarily characterized by 2DLC(CEX × RP-C 4 )-MS. TRVHS and RVHS signal peptide variants of mAb-A were found in basic peaks of the CEX profile. The results supported process development in a timely manner, and the signal peptide-containing variants with potential immunogenicity were successfully removed by an optimized purification process. The retained seven charge variants of mAb-A were further characterized by 4DLC(CEX × RP-C 4 × Trypsin×RP-C 18 )-MS. Post-translational modifications including deamidation, cyclization of N-terminal glutamine, C-terminal lysine truncation as well as proline amidation, and methionine oxidation were identified, and their potential risks were evaluated. Biological activity of the seven charge variants was evaluated by 2DLC (CEX × FcγRIIIa). Increased FcγRIIIa receptor binding affinity was observed in the acidic variants. The MDLC-MS detection can be completed in 72 h with 1.25 mg of mAb, demonstrating to be sample-economic, timeeffective, and labor-saving. It provided a powerful and timely tool for charge variant characterization and met the aggressive timeline desired for antibody development.

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“…Cation-exchange chromatography (CEX) is the most commonly used analytical tool for the characterization and quantification of protein charge variants . Charge variants are predominantly caused by glycosylation, lysine glycation, asparagine deamidation, aspartate isomerization, and further post-translational modifications (PTMs). , In addition, these may include degradations and higher order structure alterations, such as fragments and aggregates . The variety of species poses a substantial challenge for state-of-the-art chromatography and identification of specific product variants.…”

mentioning

confidence: 99%

“…6 In addition, these may include degradations and higher order structure alterations, such as fragments and aggregates. 8 The variety of species poses a substantial challenge for state-of-theart chromatography and identification of specific product variants. Moreover, insufficient chromatographic separation of multiple charge variants complicates their identification and subsequent functional characterization.…”

mentioning

confidence: 99%

Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

Grunert

Heinrich

Hingar

et al. 2022

J. Am. Soc. Mass Spectrom.

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Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium-and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

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Bridging size and charge variants of a therapeutic monoclonal antibody by two-dimensional liquid chromatography

Cited by 12 publications

References 26 publications

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry

Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

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