Assay Packages: Interlot and Biosimilar Comparability

Assay families that satisfy testing needs include:

• Molecular weight comparison

  Molecular weight is a major indicator of mAb identity, influenced by the nature of the light and heavy chains as well as by post-translational modifications (PTM). Providing an indication of similarity to reference material, intact mass (IM) analysis is supported by studies to evaluate the molecular weight of individual chains, or to assess levels of glycosylation.

• Amino acid analysis

  Used to determine the amino acid composition of a mAb, amino acid analysis is a popular way to establish product identity. It is often performed alongside an extinction coefficient measurement, a method routinely employed to establish titer between different productions.

• Sequence mapping

  Sequence mapping varies considerably in its level of complexity. To gain an indication of the identity of your mAb, comparison of simple, single enzyme peptide maps may be sufficient, whereas to better inform product engineering, N- or C-terminal sequencing may be performed. Mass spectrometry provides additional structural information.

• Higher order structure (HOS) comparison

  Many factors influence HOS – the 3D structure of a mAb – ranging from the choice of cell line for mAb production to bioprocessing conditions such as temperature, pH and light exposure. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a method that allows for a detailed insight into the tertiary structure of a mAb.

• Modification analysis

  Many different post-translational modifications can occur during the mAb manufacturing process, which are are greatly influenced by process parameters such as media, temperature, etc. To produce a consistent product, it is important these modifications are reproduced during every round of mAb synthesis. Modification analysis includes disulfide bridge mapping, and evaluation of glycan basic structure. It is also wise to quantify sialylation, since sialic acid can have a negative effect on your mAb.

• Product purity / impurity testing

  The presence of impurities in your mAb product can present a serious risk, preventing regulatory approval from being granted. Size and charge variants can be identified using techniques such as dynamic light scattering (DLS) and UHPLC ion exchange. It is also wise to monitor the presence of residuals such as detergent, surfactant, protein or DNA.

• Potency / binding

  The affinity of your mAb for its target is key to its clinical efficacy and a critical attribute that needs to be assessed in early process development. Measurement of binding using Surface Plasmon Resonance (SPR) can provide rapid determination of binding, with the added benefit of kinetic information regarding association and disassociation rates which is more informative than measurement of affinity using ELISA. Beyond Fab mediated biological activity, the success of many mAb therapeutics is dependent on biological activities mediated by the Fc region, where applicable, involving recruitment of immune system cells and components. To thoroughly evaluate the potency of your mAb, relevant binding and cell-based assays should be implemented early in the drug development process to understand the biological functionality of all regions of the mAb molecule.