Novel Analytical Techniques For Monoclonal Antibodies

Source: Catalent
Novel Analytical Techniques For Monoclonal Antibodies

How an Octet-based Fc receptor panel can accelerate biopharmaceutical drug development.

With the rising interest in biopharmaceuticals, drug developers need to quickly understand and evaluate the binding of  therapeutic antibodies to Fc receptors. In this interview, Michael Sadick, Ph.D., Manager, Biologics Analytical Services, and Dan Papa, Ph.D., Group Leader, Biologics Analytical Services at Catalent Biologics, talk about a new analytical testing panel that can be used of this purpose. Sadick and Papa explain how using an Octet-based  Fc receptor panel may help developers achieve a faster assessment of monoclonal antibody functionality — well before the final molecule is selected.

What is an Fc receptor and why is it important?

Sadick: Fc receptors are widely distributed cell-surface proteins that are essentially communication points between  effector antibodies and their biological implements.

There are several classes of Fc receptors. For instance, Fc-gamma receptor I (i.e., CD64) is responsible for things like phagocytosis and the activation of monocytic cell lines like macrophages, neutrophils, and eosinophils. The Fc-gamma receptor II family (i.e., CD32) is mainly responsible for antibody-dependent cellular phagocytosis (ADCP), while Fc-gamma receptor III (i.e., CD16) is responsible for antibodydependent cellular cytotoxicity (ADCC). These receptors appear on a subset of lymphocytes called natural killer  (NK) cells. There’s also the neonatal Fc receptor (i.e., FcRN), a cellular receptor that’s widely distributed across epithelial and endothelial cells. FcRN has a major impact on the pharmacokinetics of antibodies.

All these receptors bind to the back end — or Fc portion — of antibodies, either IgG1, IgG2, or IgG4.

Why is it advantageous to assess a monoclonal antibody (or mAb) via reactivity with an FcR panel?

Sadick: Different receptors convey different activities to an effector antibody. It’s very important to know how a given mAb interacts with the various Fc receptors.

With purely neutralizing antibodies like an IgG4, you may not have as much binding activity to immunological Fc receptors like CD16, CD32, or CD64. While neutralizing antibodies may have minimal interactions with the “immunological” Fc receptor panel (but they will interact), they should still have strong interactions with FcRN, especially at low pH, which is the high-affinity condition for this receptor.

Meanwhile, with effector antibodies such as ADCC antibodies (which induce lysing of a target cell) or ADCP antibodies (ones that target a cell to be phagocytosed by a macrophage or other scavenger cell), you want to show strong, reproducible interactions wi th the immunological Fc receptors.

For what kinds of product strategies can these analyses be used?

Sadick: You can use these analyses for originator-type molecules/new biological entities. FcR panel testing can be used for release and stability studies because you can potentially change the way the antibodies interact with the effector targets upon degradation of the therapeutic.

Even more importantly, these kinds of assays can be used when establishing biosimilar molecules because you can confirm that the new molecule’s interactions with the Fc receptors are equivalent/similar to that of the originator molecule.

How long does it take to optimize the Octet system for each new mAb?

Papa: Let us provide some background. When you optimize a system, you need to optimize the buffer and background conditions for each new antibody; you can minimize the nonspecific interactions that might happen. Each antibody is quite different from the preceding or subsequent antibodies, and the formulation will be different.

Once you’ve done that, you need to optimize the association and dissociation times for receptors with that antibody.

Generally, it takes about two or three runs for each receptor condition. There are six receptors: CD64, CD32a, CD32b/c, CD16a, CD16b, and FcRN. That’s a panel of about six receptor conditions, and it takes about a week to include all the analyses and documentation required.

What kind of precision can be achieved?

Sadick: Right now, all we have are preliminary numbers. We’ve looked at robustness, which is using different binding conditions and different binding times for the same antibody and receptor. About five different conditions were tried. We had a robustness precision of approximately 18–50%. For the true repeatability precision, we have only done n = 2 thus far (i.e., same run conditions, performed twice, independently), which provides a relative percent difference rather than a relative coefficient of variation. But, we’re seeing relative percentages between 1% and 25%, which given the order of magnitude of binding kinetics — between 10–6 and 10–9 molar — is very good.

How long does it take to run the full FcR panel (once the Octet is optimized)?

Papa: If you don’t have any problems, and everything works like it should, it takes about one to two hours per run. Remember, you’ve got six runs (i.e., six receptors set-ups). That’s about one to two days of on-bench work, and then another one to two days of data analysis, write-ups, and peer reviews. In less than a week, you can run through an entire panel for the receptor under cGMP.

Can the FcR panel testing be done as GMP?

Papa: Absolutely. In fact, we have just finished fully qualifying our Octet RED96 system, so it can be used for all aspects of GMP testing, kinetic testing (which is what we’re talking about here), and quantitative testing. This system is fully CFR Part 11 compliant, and can easily be used for the Fc receptive panel test as a quantitative system, which is like a variant of ELISA.

We have several strategies for how we would validate different assays, and we’re in the process of validating them. For example, if we’re going to do kinetics testing, which is just the kd value, you have to validate around the assay’s precision because you can’t really do linearity or accuracy. If you’re going to do the quantitative version, you would validate around the precision as well as accuracy and linearity, similarly to how you would validate an ELISA.