Comparative Assessment of mAb C1q Binding Using a Sensitive SPR Assay
21st January 2019
By: Daniel O'Loughlin - Scientist, Technical Services,
Developing a Method for a Complex Molecular Interaction
Effector functions such as complement dependent cytotoxicity (CDC) are important modes of action for many therapeutic monoclonal antibodies (mAbs). The degree of C1q binding and resultant CDC activity can influence the safety and efficacy of a mAb and therefore requires characterization during the development process. In addition, demonstrating analytical equivalence of C1q binding by biosimilar candidates to the Reference Medicinal Product (RMP) is a critical step towards approval of new biosimilar drugs.
The complement system is a proteolytic cascade that is part of the innate immune system and complements the activity of adaptive immunity. Therapeutic mAbs can induce CDC by recruiting the multivalent C1q molecule to the surface of cells upon binding of the mAb to the target antigen. This binding event causes localized activation of the complement cascade by C1q resulting in opsonization of the antibody bound target cell, deposition of membrane attack complexes and recruitment of additional immune cells. This ‘classical’ pathway of complement activation is the most likely pathway used by antibodies to induce CDC however the lectin dependent and ‘alternative’ pathways can also lead to complement activation. The ability of mAbs to induce CDC is often critical to their mechanism of action and is strongly affected by their ability to bind C1q (1).
C1q is a large multimeric protein of around 460kDa composed of six copies each of three different proteins. The structure of C1q is six globular heads which oligomerise via a collagen-like domain – it is these globular heads that bind to a variety of molecular targets and give C1q a variety of functions (2). Scientific literature indicates that the affinity of each individual C1q globular head domain for a single IgG is quite low at around 100μM (3). C1q therefore relies on avidity effects of multiple globular heads binding simultaneously to multiple IgG molecules to form a stable, higher affinity binding complex. This multivalent binding mechanism, weak affinity of each individual C1q globular head and the fact that C1q is a large, highly charged molecule raised challenges during assay development. Despite these challenges, Sartorius Stedim BioOutsource has successfully developed an SPR (surface plasmon resonance) assay on the Biacore platform which can be used to assess C1q binding similarity between innovator and biosimilar molecules using relative binding and sensorgram comparison. The complexity of the C1q interaction means that no kinetic model is currently available to accurately determine the affinity of C1q for mAbs.
Confirming our Data
The sensitivity of this assay has been investigated by comparing adalimumab to a candidate biosimilar with increased galactosylation – confirmed by our released N-glycan assay (See figure 1). The level of terminal galactose is known to affect binding to C1q and subsequently CDC activity (3). Our Biacore C1q assay was able to detect a measurable difference in C1q binding between these two versions of adalimumab both in relative binding and sensorgram comparison (Figure 2). The SPR data for this candidate biosimilar of adalimumab was confirmed with data from an orthogonal C1q binding assay available at BioOutsource: a cell-based CDC assay (See figure 3).
Several therapeutic IgG1 antibodies available in the clinic have been tested during the development of this assay indicating that the assay can be used for a broad range of IgG1 mAbs. The assay can also be used for assessing a reduced or absence of C1q binding as is seen for antibodies of the IgG2 and IgG4 isotypes when compared to the IgG1 isotype. Figure 4 shows the difference in C1q binding between antibody isotypes.
BioOutsource has developed a specific and sensitive C1q assay using the Biacore SPR platform that can be used to assess binding to C1q. The data from this assay can be combined with C1q ELISA and C1q bioassay data to give a more comprehensive package of data on C1q binding. The assay also has the potential for up to 8 samples per run with a short run time and can be used for positive and negative binders of C1q.
For more information about this SPR C1q binding assay, orthogonal C1q activity assays or any potential testing enquiries, contact our expert scientists.
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