Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF)

Sutton, Haley; Hong, Feng; Han, Xuemei; Rauniyar, Navin

doi:10.1039/d4ay00836g

Anal. Methods

2024

DOI: 10.1039/d4ay00836g

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Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF)

Haley Sutton,

Feng Hong,

Xuemei Han

et al.

Abstract: Imaged capillary isoelectric focusing (icIEF) is a preferred analytical method for determining isoelectric points (pIs) and charge heterogeneity profiles in biotherapeutic proteins. In this study, we optimized the icIEF method...

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2024

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Cited by 2 publications

References 29 publications

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An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation

Bloderer,

Grassi,

Cabrele

et al. 2024

J of Separation Science

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Imaged capillary isoelectric focusing was successfully applied for separating an in‐house synthesized closely related peptide pair, that is, a linear 12‐mer (Rp5‐L) and its cyclic 15‐mer variant (Rp5‐C). Rp5‐L represents a mimotope, that is, an epitope mimicking peptide, of the CD20 antigen, which is over‐expressed in B‐cell‐related tumors. Peptide identity—including the successful disulfide bond formation in Rp5‐C—was confirmed with matrix‐assisted laser desorption ionization‐time of flight mass spectrometry. The purity of synthesized products was determined by a reversed‐phase high‐performance liquid chromatographic method with ultraviolet detection. The apparent isoelectric point (pI) of cyclic Rp5‐C and Rp5‐L was 5.99 and 6.47, respectively. An appropriate combination of carrier ampholytes allowed for their baseline separation with an analysis time of <20 min. Method validation was done for the synthesized peptides and three flanking pI markers covering, for example, repeatability and intermediate precision. Calibrations on different days resulted in identical slopes for Rp5‐L and Rp5‐C, respectively, as statistically confirmed by Welch's t‐test and pooled t‐test over 8 days. The calibration data of mimotopes and pI markers were evaluated for outliers, normality, homoscedasticity, and autocorrelation with complementary statistical procedures, which identified an otherwise unnoticed outlier for a pI marker. The linearity of calibration for Rp5‐L, Rp5‐C, and the pI markers was tested with Mandel's fitting test and lack‐of‐fit test. For Rp5‐L and Rp5‐C, the calculated limits of detection and limits of quantification were ≤0.31 and ≤0.96 µmol/L, respectively.

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An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation

Bloderer,

Grassi,

Cabrele

et al. 2024

J of Separation Science

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Characterization of Product Related Variants in Therapeutic Monoclonal Antibodies

Rauniyar,

Han

2024

LCGC Int.

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The characterization of product-related variants in monoclonal antibodies involves identifying and quantifying the size and charge of variants that can impact the activity, efficacy, and safety of the antibodies. These variants represent distinct molecular forms that may arise from processes such as fragmentation, dimerization, aggregation, or post-translational modifications. The characterization of variants typically involves isolating the relevant species using a semi-preparative scale HPLC and analyzing them using various analytical techniques and biological assays. The commonly used analytical techniques include size-exclusion and ion-exchange chromatography, light scattering, mass spectrometry, capillary isoelectric focusing, and capillary electrophoresis-sodium dodecyl sulfate with ultraviolet or laser-induced fluorescence detection, among others. Additionally, functional assessments are performed using cell-based assays and binding assays to assess the biological activities of the variants. Identifying product-related variants through characterization enables the recognition of impurities that compromise the quality and safety of the drug.

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Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF)

Abstract: Imaged capillary isoelectric focusing (icIEF) is a preferred analytical method for determining isoelectric points (pIs) and charge heterogeneity profiles in biotherapeutic proteins. In this study, we optimized the icIEF method...

Cited by 2 publications

References 29 publications

An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation

An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation

Characterization of Product Related Variants in Therapeutic Monoclonal Antibodies

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