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European Biopharmaceutical Review

Analytical Methods

Testing requirements for novel anticancer and anti-inflammatory therapeutic antibodies and antibody fragments differ from requirements for former therapeutic drugs. For the latter, the determination of dose-dependent proliferation, or suppression of proliferation, is sufficient for bioactivity testing in most cases. However, the disadvantage of proliferation assays is that they do not reflect mode of action (MOA), which is often required by authorities for approval of such therapeutics.

The setup of in vitro assays that reflect the MOA assays of therapeutic antibodies is highly challenging, due to the fact that most of these assays are based on primary effector cells or complement derived from primary sources. Nevertheless, the assays must be robust, reproducible and reliable in order to be useable for preclinical and clinical testing.

This article will discuss the setup of an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and present complement-dependent cytotoxicity (CDC) and validation data for a supporting flow cytometry-based binding assay for the therapeutic antibody Rituximab.

Rituximab is an already approved chimeric monoclonal antibody directed against CD20, which is primarily found on the surface of B cells. Rituximab is used in the treatment of many lymphomas, leukaemias and some autoimmune disorders, as well as in transplant rejection. Rituximab induces the death of target cells via ADCC as well as CDC.

During development, preclinical and clinical testing, therapeutic antibodies must be tested with highly specific assays reflecting the MOA, producing robust, reproducible and precise data.

Most therapeutic antibodies can induce the death of their target on one or more of the following pathways: antibody-dependent cellular cytotoxicity, complement-dependent cellular cytotoxicity and programmed cell death (PCD) (see Figure 1) (1).

In comparison to classical proliferation assay approaches for bioactivity testing, several additional challenges must be addressed:

  • A suitable target cell line with constant cell surface receptor expression is a prerequisite for these types of assays. The range needs to be determined precisely to achieve reproducible data
  • The donor-dependent variability of primary natural killer (NK) cells or the human complement must be accounted for to avoid inaccurate results due to donorto- donor variability
  • Appropriate choice of the source of primary material as well as careful handling with precise procedures, titration of all assay components, and testing of the optimal assay steps and conditions, must be performed for each individual pair of therapeutic antibody and target cell line

MOA assays are expensive and therefore it often makes sense to perform bridging experiments. The idea of a bridging experiment is to show that other less expensive and time-consuming methods (for example, flow cytometry-based binding assays) are suitable to achieve results comparable to MOA assays. They can, for example, reflect changes in the bioactivity of the drug due to improper storage conditions comparable to MOA assays. Bridging experiments are accepted by authorities for stability studies as supportive methods.

In the following section, a complete package for testing of the therapeutic antibody Rituximab, reflecting the possibilities and borders of MOA assays, are presented using the human Burkitt's lymphoma cell line Ramos as a target.

Rituximab Binding Assay

A flow cytometry-based assay for the measurement of the binding efficacy of Rituximab to CD20 expressing Ramos cells was set up and validated. Rituximab was titrated on the cells and a fluorescently labelled anti-human secondary antibody was used to detect the dose-dependent binding of Rituximab. The binding efficacy was determined using PLA analyses. The assay was validated following ICH guideline Q2 (R1).

The Rituximab binding assay was validated for its ability to determine the relative activity/binding efficacy of Rituximab to CD20 on the surface of Ramos cells. The parameters of intermediate precision, repeatability, linearity, accuracy, range, robustness and specificity were evaluated following ICH guideline Q2 (R1). The results are summarised in Table 1. Rituximab ADCC Assay ADCC refers to a cell-mediated reaction. Lysis of antibody-coated target cells is induced by effector cells with cytolyticactivity and specifi c immunoglobulin receptors called Fc receptors. Within this test, NK cells, derived from periphery blood mononuclear cells (PBMC) of healthy donors by negative selection were used as effector cells. The quality of the NK cell purifi cation was tested by fl ow cytometry using commercially available antibodies directed against the NK cell markers CD16 and CD56. Typically, more than 80 per cent of the purifi ed cells are positive for both markers (see Figure 2).

NK cells recognise their target cells via the receptors for the Fc fragment of IgG (FcgRIIIA, CD16) that bind to antibodies bound to the surface of the target cells. Binding of NK cells to target cells induces the release of preformed cytotoxic mediators by granule exocytosis.

NK cell-derived cell lysis is measured via the release of lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. LDH in culture supernatants is measured with a coupled enzymatic assay which results in conversion of a tetrazolium salt into a red formazan product. Based on the results of the LDH measurement, the percentage of specific lysis is calculated. Example data are presented in Figure 3.

The ADCC assay was set up and optimised for the therapeutic antibody Rituximab. The quality of the NK cell purifi cation was tested by fl ow cytometry using commercially available antibodies directed against the NK cell markers CD16 and CD56.

Rituximab CDC Assay

The CDC assay was set up and optimised for the therapeutic antibody Rituximab. CDC is a mechanism of killing cells in which antibody bound to the target cell surface fi xes complement, which results in assembly of the membrane attack complex that perforates the target cell membrane resulting in subsequent cell lyses. Not all therapeutic antibodies can induce CDC (1). Rituximab reference and the test item are diluted in the complement matrix and added to Ramos target cells. The target cells are incubated with dilutions of Rituximab antibody and complement.

A flow cytometer was used to measure the dose-dependent, complement-derived cytotoxicity using 7-amino-actinomycin D (7-AAD). This dye intercalates into double-stranded nucleic acids. It is excluded by viable cells, but can penetrate cell membranes of dying or dead cells.

Based on the results of the 7-AAD measurement, percentage-specific lysis was calculated. A potency was determined in comparison to the reference item using a 4-parameter fit based on the flow cytometric histogram data for 7-AAD positive cells and by using statistical software (PLA). The assay was tested over a range of 70 per cent to 130 per cent estimated potency. Example data are given in Figure 4.

Conclusion

MOA assays as well as fl ow cytometrybased binding assays are great tools for the in vitro testing of therapeutic antibodies to ensure that the bioactivity is determined in an approach that is relevant and representative for the in vivo mechanisms of novel complex biotherapeutics. The rising market of biosimilars and biobetters underlines the need of having such test methods in place. This is the only way to compare the bioactivity of biosimilars to originator drugs without using animal models. Most novel biotherapeutics somehow interfere with the immune system, and therefore most animal models, such as rodents, are not suitable models for bioactivity in humans as they have a different immune system, which is usually not representative of the human system.

ADCC assays are highly sensitive and detect dose-dependent cytotoxicity within the μg/mL range, whereas they are saturated at higher concentrations. Therefore, one of the main challenges of MOA assays is the limit of the concentration range. In consequence, all ADCC assays need to be tested over a range of test item concentrations to ensure that the assay can detect variations appropriately within its limits.

Due to the dependency on primary NK cells, ADCC setups are costly and time and consuming, but deliver reliable data on the bioactivity of an antibody in an in vitro model. Especially within the early phases of drug development, data from the ADCC test method are valuable for characterisation of the therapeutic antibody or comparison to an already existing biosimilar.

For therapeutic antibodies that do mediate death of target cells via the complement pathway, the flow cytometry-based CDC assay is a good alternative to ADCC assays, but is less sensitive. In addition, flow cytometry-based approaches are complex and time-consuming in comparison to a proliferation readout-based setup with microplate readers. Nevertheless, it is robust with a sufficient sensitivity and works well over a range of 70 per cent to 130 per cent estimated potency.

The binding assay presented here for Rituximab was successfully validated according to ICH guideline Q2 (R1) and approved as an option of less expensive testing for the later phases of therapeutic antibody development. As is sometimes requested by authorities, it can serve as a supporting method. Bridging experiments can be performed to switch to this method Ė for example stability testing of the drug. The assays are robust and precise, and comparable setups are possible for a broad range of different therapeutic antibodies.


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Ulrike Herbrand joined Charles River Biopharmaceutical Services in 2007 and works as a Scientific Officer in the bioassay department. She earned a PhD in Biological Sciences during her time at the Max Planck Institute for Molecular Physiology in Dortmund, Germany, and worked for five years in post-doctoral positions at medical research centres in the field of cancer research. Email: ulrike.herbrand@crl.com

Simone Scotti has a PhD in biological sciences and joined Charles River Biopharmaceutical Services in 2001. She is currently the Head of the Bioassay Department. She focuses on bioassay and molecular miology aspects of biopharmaceuticals and has over 10 years of experience in GLP/GMP-compliant analytical contract services for the international biopharmaceutical industry. Email: simone.scotti@crl.com

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