Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Marco, Fiammetta Di; Berger, Thomas; Esser‐Skala, Wolfgang; Rapp, Erdmann; Regl, Christof; Huber, Christian G.

doi:10.3390/ijms22169072

Cited by 15 publications

(19 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The A2 peak in our study has a mass difference of 162 Da, indicating it to be the glycated form present in mAb B. mAb B is prone to asparagine deamidation, with the peptide map revealing four sites in the heavy chain and three on the light chain. Similar to mAb A, the deamidation in mAb B is resolved as an acidic variant in the form of peak A3, in line with published and present data …”

Section: Resultssupporting

confidence: 92%

“…Similar to mAb A, the deamidation in mAb B is resolved as an acidic variant in the form of peak A3, in line with published and present data. 56 Cysteinylation in mAbs has been reported in mAbs when the cysteine residue forms a disulfide bond with the terminal cysteine molecule and is often resolved as a basic variant (Table S1). mAb B is also susceptible to cysteinylation as identified by WCX-MS analysis.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

2022

View full text Add to dashboard Cite

Charged heterogeneity of monoclonal antibody (mAb) products is regarded as a critical quality attribute (CQA) depending on its impact on the safety and efficacy profile of the product. Hence, manufacturers are expected to perform a comprehensive characterization of the charge heterogeneity to ensure that the manufactured product meets its specifications. Further, monitoring is also expected during the product lifecycle to demonstrate consistency in product quality. However, conventional analytical methods for characterization of hydrophobic and charge variants are nonvolatile salt-based and require manual fraction collection and desalting steps before analysis through mass spectrometry can be performed. In the present study, a workflow of a two-dimensional liquid chromatography method using mass spectrometry (MS)-compatible buffers coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without any need for manual fractionation. This novel two-dimensional (2D) hydrophobic interaction chromatography (HIC)-weak cation-exchange chromatography (WCX)-MS workflow identified 10 variants in mAb A, out of which 2 variants are exclusive to the 2D orthogonal method. Similarly, for mAb B, a total of 11 variants are identified, including 5 variants exclusive to the 2D orthogonal workflow. When compared to stand-alone, HIC resolved only 4 variants for both mAbs and WCX resolved 7 variants for mAb A and 6 variants for mAb B. In addition, the proposed method allows direct characterization of hydrophobic/charge variant peaks through native mass spectrometry in a single-run workflow.

show abstract

Section: Resultssupporting

confidence: 92%

Section: ■ Results and Discussionmentioning

confidence: 99%

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

2022

View full text Add to dashboard Cite

show abstract

“…This eluent, however, is not suitable for MS analysis, due to its high salt concentration. Furthermore, due to denaturation of tertiary structure and exposure of charged residues, highly charged mAb ions are produced, which results in the generation of complex MS [11].…”

Section: Introductionmentioning

confidence: 99%

Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies

Bhatt

Alok

Kulkarni

2022

BioTech

View full text Add to dashboard Cite

Post-translational modifications (“PTMs”) in monoclonal antibodies (mAbs) contribute to charge variant distribution, which will affect biological efficacy and safety. For the characterization of mAbs, charge variants are used as a critical quality attributes for product quality, stability consistency and effectiveness. Charge variants in mAbs are characterized by a time-consuming and a multistep process starting from cation/anion exchange chromatography, acidic/basic fractions collection and subsequent reverse phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis. Hence, an alternative characterization approach that would be highly selective for ion exchange chromatography-based charge variant analysis, which is compatible with on-line MS detection, is needed in the biopharma industry. Against this backdrop, multiple studies are being conducted to develop a simple straight on-line charge variant analysis method. In this regard, we apply the current study, which aims to develop a charge variant analytical method, based on volatile buffers with low ionic strength that can be used for on-line MS detection of charge variants of mAbs. This would enable the detection on “PTMs” using low ionic strength mobile phase compatible with MS. Hence, fruitful data can be obtained with a single chromatography run without any test sample preparation, eliminating the need for multiple steps of analysis, time-consuming process and multiple sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the characterization of charge-related PTMs on the intact protein stage. In this regard, this study is about development of a method having combination of chromatography and volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native form. The method is qualified considering pharmacopeia guidelines because the ultimate aim is to transfer this method for Quality Control (QC) release testing of a monoclonal antibody, which is critical for batch release and the regulatory point of view. Acidic and basic variants have been separated with high resolution peak profile. Furthermore, there was no matrix interference and good separation selectivity in terms of specificity was obtained using this method. The experimental data suggested for the linearity of the method are 2.4 mg/mL to 3.6 mg/mL with % RSD below 2.0%. Additionally, Limit of Quantitation is found to be 0.15 mg/mL, which is 5% of loading amount. Consistently, the data show that the method is precise under the same operating conditions with a short time interval. Overall a simple, accurate, robust and precise pH gradient cation exchange chromatography method was developed and qualified for the characterization of a therapeutic native mAb. Additionally, this method can be used to claim a biosimilar product profile of an in-house product compare to an innovator.

show abstract

“…5,16 Recently, novel semi-automated approaches applying native separation techniques such as strong cation-exchange (SCX) high-performance liquid chromatography coupled to MS (HPLC-MS) have been applied for the characterisation of biopharmaceutical proteoforms. [17][18][19][20][21][22] In contrast to established SCX methods using a gradient of non-volatile salt in the mobile phase, a pH gradient based on volatile buffering components is compatible with MS. Separation of distinct proteoforms is predominantly based on differences in isoelectric points (pI), resulting in the elution of the protein variant at a pH close to its pI.…”

Section: Introductionmentioning

confidence: 99%

“…Hitherto, this approach has been used to study intact monoclonal antibody (mAb) charge variants. [17][18][19][20][21][22] However, for proteins exhibiting an acidic pI, strong anionexchange (SAX) HPLC-MS is better suited to separate glycoforms on the basis of negative charge, e.g. the number of sialic acid residues.…”

Section: Introductionmentioning

confidence: 99%

Glycoproteomics of a single protein: revealing hundreds of thousands of Myozyme® glycoforms by hybrid HPLC-MS approaches

Marco

Blöchl

Esser‐Skala

et al. 2022

Preprint

View full text Add to dashboard Cite

Characterisation of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of co-existent glycoforms, i.e. heterogenous glycoprotein variants. Here, we report the use of an array of HPLC-MS-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme®, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion-exchange (SAX)-HPLC-MS approach involving a pH gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. The large set of interdepend data acquired at different structural levels was integrated using a set of bioinformatics tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 different intact glycoform structures. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms revealing the strengths but also intrinsic limitations of this approach for fully characterising such highly complex glycoproteins by mass spectrometry.

show abstract

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Cited by 15 publications

References 58 publications

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies

Glycoproteomics of a single protein: revealing hundreds of thousands of Myozyme® glycoforms by hybrid HPLC-MS approaches

Contact Info

Product

Resources

About