Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry

Woodall, Daniel W.; Dillon, Thomas M.; Kalenian, Kevin; Padaki, Rupa; Kuhns, Scott; Semin, David; Bondarenko, Pavel V.

doi:10.1080/19420862.2021.2004982

Cited by 12 publications

(14 citation statements)

References 78 publications

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“…The binding kinetics study by Woodall et al indicated that affinity measurements from FcγRIIIA affinity chromatography with aglycosylated FcγRIIIA receptor were synonymous with earlier studies using SPR with aglycosylated FcγRIIIA. However, these observations were made in studies with fucosylated and afucosylated antibodies 23 . In our study, there were only minor differences between the k d values obtained with glycosylated FcγRIIIA (SPR) and aglycosylated FcγRIIIA (affinity chromatography).…”

Section: Resultscontrasting

confidence: 56%

“…Carbohydrate interactions between the glycans of FcγRIIIA and Rituximab are known to result in optimum binding and affinity to execute the effector function. It was also observed by Hayes et al that mammalian expression system (HEK 293, CHO, mouse [NS0] cell lines), growth conditions, nutrient availability used to produce the receptor protein also result in differing23 In our study, there were only minor differences between the k d values obtained with glycosylated FcγRIIIA (SPR) and aglycosylated FcγRIIIA (affinity chromatography). We speculate that these observations were most likely because of non-deleterious effect of glucose stress of 24 h and intact glycan structure of Rituximab.…”

supporting

confidence: 43%

“…Each peak corresponds to a glycoform mixture. The predominant glycans present in each peak of Rituximab glycoform mixture have been indicated 23 …”

Section: Resultsmentioning

confidence: 99%

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FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab

Narvekar

Puranik

Kulkarni

et al. 2022

Biotechnology Progress

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Analytical and functional characterization of batches of biologics/biosimilar products are imperative towards qualifying them for pre-clinical and clinical investigations.Several orthogonal strategies are employed to characterize the functional attributes of these drugs. However, the use of conventional techniques for online monitoring of functional attributes is not feasible. Liquid chromatography is one of the crucial unit operations during the downstream processing of biopharmaceuticals. In this work, we have demonstrated the utility of FcγRIIIA affinity chromatography as an independent quantitative functional characterization tool. FcγRIIIA affinity chromatography aided in sequential elution of Rituximab glycoform mixtures, based on varying levels of galactosylation, and thereby the affinity for the receptor protein. The predominant glycans present in the three Rituximab glycoform mixture peaks were G0F, G1F, and G2F, respectively. Dissociation rate constants were derived from the chromatographic elution profiles by the peak profiling method, for the control and glucose stress conditions. The glucose stress conditions did not result in unfavorable binding kinetics of Rituximab and FcγRIIIA. The dissociation rate constants of the glycoform mixture 2, predominantly consisting of G1F, were similar to the dissociation rate constants obtained by surface plasmon resonance. Moreover, the glycosylation profiles obtained from chromatographic estimation can be corroborated with the ADCC activity. However, the ex vivo ADCC reporter assay indicated that there was an increase in the effector activity with increasing glucose stress. Thus, FcγRIIIA affinity chromatography permitted three independent assessments via a single analysis. Such approaches can be utilized as potential process analytical technology (PAT) tools in the biosimilar development process.

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Section: Resultscontrasting

confidence: 56%

supporting

confidence: 43%

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FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab

Narvekar

Puranik

Kulkarni

et al. 2022

Biotechnology Progress

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“…MAb-A can facilitate tumor cell killing by initiating antibody-dependent cellmediated cytotoxicity (ADCC). As FcγRIIIa binding affinity generally shows good correlation with ADCC potency, 39 and the FcγRIIIa binding capacity can be reflected on the FcγRIIIa column, 40 the charge variants' biological activity of mAb-A was indirectly monitored by 2DLC(CEX × FcγRIIIa) analysis. Seven charge variants were fractionated into a 96-well plate by 1 D CEX and then each variant was concentrated on a 2 D FcγRIIIa column by multi-injection and separated.…”

Section: Characterization Of Charge Variants At the Subunit Level By ...mentioning

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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“…Affinity chromatography (AC) is a promising technique for separating functionally different mAb proteoforms by using relevant interaction partners immobilized on the column matrix. , Moreover, the combination of AC and mass spectrometry (AC-MS) allows for unprecedented molecular resolution in structure–function studies. , Current reports have focused on separating fragment crystallizable (Fc) modifications of mAbs, such as glycosylation and methionine oxidation, based on relevant Fc receptor interactions (e.g., FcγRIII, FcRn). However, the Fc part of most antibodies is highly similar, and the criticality of individual PTMs on relevant Fc receptor interactions has been mostly well understood.…”

Section: Introductionmentioning

confidence: 99%

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function–Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants

et al. 2023

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T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-ofconcept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob−knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

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Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry

Cited by 12 publications

References 78 publications

FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab

FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab

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

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function–Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants

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