How to characterise omalizumab

25th June 2020

Category: Bioanalytical / Biosimilars

By: Lisa Blackwood, PhD - Senior Scientist, Product Development,

Immunoglobulin E (IgE) functions to recognise the presence of foreign particles, and in combination with mast cells, form part of the adaptive immune response in charge of mounting powerful inflammatory reactions. Under normal circumstances, IgE forms a very small proportion of circulating immunoglobulins, however, atypical abundance results in hypersensitivity. Examples of related pathologies include asthma, dermatitis and several allergies.

Xolair® (Omalizumab) is a humanised murine monoclonal antibody (mAb) targeting the Fc region of IgE. Thus, Xolair® specifically recognises free, unbound IgE in circulating interstitial blood and IgE expressing B-cells. Once IgE has been bound by Xolair it can no longer bind to the Fcε receptor this results in sequestration and reduces illicit activation of the immune response to non-threatening stimuli.

Prior to any functional assessment the structural properties of Xolair® were assessed. At Sartorius our platform methods for Glycan Analysis, Intact mass, Reduced and Non-reduced Peptide Mapping and SoloVPE for concentration analysis were utilised as Xolair® is a standard IgG1 molecule.

Development of a biosimilar to Xolair® requires characterisation of the Fab and Fc binding regions of the molecule. There are various methods employed at Sartorius to examine the Fab binding of the molecule to IgE. Firstly, a binding ELISA using IgE bound to the plate surface and detected using an anti-human IgG specific for the Fc region, and secondly utilising the power of SPR where Xolair® is immobilised and IgE is flowed across the molecule. Both the orthogonal methods demonstrate accurate and reproducible results. In addition to target binding by SPR the binding of omalizumab to the Fc receptors can be assessed using the platform SPR binding methods which exist for IgG1 molecules. Lastly, it is also important to assess IgE binding using live cells, a more biologically relevant model. We employed a suspension cell line expressing low levels of membrane bound IgE to which we are able to demonstrate accurate and reproducible binding using the iQue Screener PLUS.

Characterisation of the Fc region of the molecule can be tested in a number of different methods. The simplest is without cells being present and looking at the binding interactions between the receptors using an ELISA based method. C1q and FcγRIIIa binding using HRP-conjugated receptors is a quick method to determine if binding to the receptors responsible for ADCC and CDC is possible. For Xolair®, binding to the receptors does occur as shown by the ELISAs, however, using our platform cell based ADCC and ADCP assays we have shown there is no induction of target cell death of phagocytosis. Utilising the iQue screener PLUS we demonstrated that IgE expressing target cells and primary cells isolated from blood were unable to perform ADCC or ADCP however a positive control confirms that death or phagocytosis of the target cells is possible.

As a mAb which decreases histamine release a bioassay for Xolair® to demonstrate this mechanism of action however this is a challenging assay requiring mast cells and a source of pollen and blood from a donor allergic to the same pollen. Therefore, the potency ELISA was developed which demonstrates the interaction between the Fcε receptor, IgE and Xolair®. Due to the Fcε receptor binding to Xolair® the IgE can no longer bind to the Fcε receptor, as would occur in the patient. This method provides a quicker, more controlled and reproducible approach replacing the requirement for a long, variable and complex cell based potency assay.

Figure 1: Potency ELISA demonstrating the inhibition of IgE and Fcε receptor binding in the presence of increasing concentrations of omalizumab.

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