Anion exchange chromatography – Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals

Schaick, Guusje van; Gstöttner, Christoph; Büttner, Alexander; Reusch, Dietmar; Wuhrer, Manfred; Domínguez‐Vega, Elena

doi:10.1016/j.aca.2020.11.027

Cited by 23 publications

(27 citation statements)

References 26 publications

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“…This allows for separation of charge variants according to surface charge at intact proteoform level [17]. An alternative approach employs a pH gradient using volatile buffers (e.g., formic acid/formate, acetic acid/acetate, ammonium/ammonia), eluting the different proteoforms depending on their isoelectric point (pI), hence their acidic properties [16,[18][19][20]. In contrast to the conventional non-volatile salt gradient, a pH gradient involving volatile mobile phases facilitates direct hyphenation to mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

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

Marco

Berger

Esser‐Skala

et al. 2021

IJMS

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Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

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Section: Introductionmentioning

confidence: 99%

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

Marco

Berger

Esser‐Skala

et al. 2021

IJMS

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“…By means of buffer exchange to a volatile, MS compatible buffer, the individual fractions could be analyzed with MS. Direct coupling of cation [ 50 ] and anion exchange chromatography [ 12 ] with MS has been reported. The employed strategies could also be applied for further developments of the first dimension if MS compatibility is desired.…”

Section: Resultsmentioning

confidence: 99%

“…Such combinations provide a unique selectivity and may offer an alternative to other approaches. Even though, both capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and ion exchange chromatography (IEX) are frequently employed for the characterization of proteins [9][10][11][12], the coupling of both techniques has not received much attention [13].…”

Section: Introductionmentioning

confidence: 99%

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

et al. 2021

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Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. Graphical abstract .

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“…The mobile phases were optimized to provide good separation power and enable online MS detection while maintaining enzymatic activity. Recently, it was shown that elution with pH gradient (using volatile salts) allows good and robust separation without compromising on the quality of the MS data [16][17][18]. The start pH of the gradient was chosen to be able to retain the charged proteoforms on the column while staying within the active pH range of the enzyme leading to an optimal pH of 5.5.…”

Section: Charge Variant Characterization Of With Iex−uv−msmentioning

confidence: 99%

“…Ion exchange chromatography (IEX) has been widely established to characterize proteoforms with a difference in charge due to, e.g., deamidation, sialylation of glycans, and phosphorylation [14]. In particular, IEX methods eluting with pH gradients have recently been shown to provide robust separation of proteoforms and enable online MS coupling for identification [15][16][17][18]. Another interesting native LC approach is boronate affinity chromatography (BAC), which can be employed to selectively enrich glycated species.…”

Section: Introductionmentioning

confidence: 99%

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Schaick

Hajjouti

Nicolardi

et al. 2022

IJMS

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Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.

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Anion exchange chromatography – Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals

Cited by 23 publications

References 26 publications

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

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

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

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