Structural Characterization of Monoclonal Antibodies - The author describes techniques that can be used to provide the analytical data required by ICH Q6B for characterization of monoclonal

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Structural Characterization of Monoclonal Antibodies
The author describes techniques that can be used to provide the analytical data required by ICH Q6B for characterization of monoclonal antibodies.


BioPharm International Supplements
Volume 24, Issue 8, pp. s15-s22

ISOFORM PATTERN

In a similar way, imaging capillary isoelectric focusing (cIEF) has revolutionized analysis of the isoforms of mAbs. cIEF is free solution isoelectric focusing in a capillary column. Dedicated systems such as the iCE280 analyser (Convergent Biosciences) detect focused protein zones using a whole column UV absorption detector that avoids disturbing these focused zones. This technology is unique in that it has the comparable resolution of traditional gel IEF, but incorporates the advantages of a column-based separation technology, including quantitation (using UV at 280nm) and automation.


Figure 7: UV (280 nm) response across the capillary obtained following capillary isoelectric focusing of a mAb.
The raw data obtained from cIEF profiling of a mAb is shown in Figure 7. The data obtained allows an accurate determination of the pI of each isoform and quantitation of each isoform based on the UV peak area.

ELECTROPHORETIC AND LIQUID CHROMATOGRAPHIC PATTERNS

Analysis of mAbs using isoelectric focusing (as discussed above), SDS–PAGE (prior to and following reduction), and capillary electrophoresis are routinely used to provide product distributions based on size and charge.

Chromatographic analysis using size-exclusion chromatography (SEC), RP–HPLC and ion-exchange liquid chromatography provide product distributions based on size, hydrophilicity/hydrophobicity and charge distribution.

Both electrophoresis and chromatography provide information relating to identity, homogeneity, and purity of the product and are often used to purify impurities for identification and structural characterization using the analytical techniques outlined above.

PROTEIN AGGREGATION

For mAbs in particular, it is essential to assess each product for the presence of multimers and aggregates using a combination of methods. Currently, it is usual for the regulatory authorities to request SEC with multi-angle laser-light scattering (SEC–MALS) and a column free technique such as analytical centrifugation (AUC) to cross check the data obtained from SEC analysis.


Figure 8: Raw data obtained from size-exclusion chromatography with multi-angle laser-light scattering (SEC–MALS) analysis of a mAb.
Because aggregates can potentially be lost by non-specific binding to the SEC column or, indeed, dispersed by the dilution of the product on the column and the shearing forces during the chromatography, confirmation of SEC results using a column-free technique is required.


Figure 9: Raw data obtained from analytical centrifugation (SV-AUC) analysis of a mAb.
The raw data obtained from SEC–MALS and AUC analysis of a mAb are shown in Figures 8 and 9 respectively. The data obtained from SEC–MALS analysis of the mAb suggests, in this case, the presence of low levels of aggregates (approximately 2.6%). The data obtained from AUC analysis suggests the presence of approximately 5.0% aggregates. It is usual to observe higher levels of aggregation using AUC over SEC–MALS, probably due to the factors potentially reducing the levels of aggregate in the column-based analysis data outlined above.


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