In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Summary

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Abstract

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

Introduction

Therapeutic antibodies are recombinant monoclonal antibodies (mAbs) developed for the treatment of different pathologies, including cancers, autoimmune and chronic diseases, neurologic disorders, and others¹. Currently, the FDA has granted approval to more than 40 therapeutic mAbs, and more are expected to reach the market in the following years.

Rituximab is a high-affinity chimeric monoclonal IgG1 antibody approved for the treatment of CD20⁺ B-cell non-Hodgkin's lymphoma (NHL), CD20⁺ follicular NHL, chronic lymphocytic leukemia, and rheumatoid arthritis^2,....

Protocol

1. Evaluation of Target Binding by Flow Cytometry

1. Preparation of biological materials and reagents
   1. Make 500 mL of RPMI culture medium supplemented with 10% heat-inactivated fetal bovine serum (H-IFBS).
   2. Culture Daudi Burkitt's Lymphoma (Daudi) cells and Daudi GFP⁺ cells using RPMI and 75-cm² culture flasks. Maintain the cultures at 37 °C in a 5% CO₂ humidified atmosphere until they reach 6 - 9 x 10⁵ cells/mL.
   3. Make 50 mL of staining buffer by diluting 1/100 H-IFBS in PBS; this buffer is stable at 2 - 8 °C for at least one month.
   4. Prepare the....

Results

Using the protocols described above, target binding and the CDC induction of reference rituximab were compared in parallel with those of a biosimilar rituximab produced and commercially available in Asia.

In Daudi cells, both mAbs bound CD20 in a concentration-dependent manner (Figure 1D). Non-linear regressions of binding data displayed an r² of 0.978 and 0.848 for reference and biosimilar rituximab,.......

Discussion

The patent expiration of a therapeutic mAb is promoting the development of biosimilars. Thus, there is a need for simple methods that can identify differences in clinically relevant activities of these products. CD20⁺ cultured cells were employed for the evaluation of two key functional characteristics of rituximab: target binding and CDC induction. The former activity requires the recognition of CD20 by the Fab region of the mAb, while the latter depends mainly on the interaction of the Fc region with its com.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-1640 medium	ATCC	30-2001	Modify the culture depending on the cell line
Trypan Blue solution	Sigma	T8154	0.4%, liquid, sterile-filtered, suitable for cell culture
Daudi Burkitt's Lymphoma Cells	ATCC	CCL-213	You can modify the cell line depending on the antibody of interest
Fetal bovine serum (FBS)	GIBCO	16000-044	You can modify the source of serum depending of requirements of the cell line
Normal Human Serum Complement	Quidel	A113	It is therefore appropriate for use in biocompatibility experiments including drug development, biomaterials testing and other applications
7AA-D	BDPharmigen	559925	You can use broad range of color options, compatible with most instrument configurations for to analyze viability.
PECy5 Mouse Anti-human IgG	BDPharmigen	551497	Change fluorochrome depending on the filter and laser of your flow cytometer
Human IgG Isotype Control	ThermoFisher Scientific	07-7102	Change depending to mAb
BDCytofix	BDPharmigen	554655	Flow Cytometry Fixation Buffer (1 - 4% formaldehyde or paraformaldehyde )
PBS pH 7.4 10x (Phosphate buffer saline)	GIBCO	70011-044	Phosphatebuffer without Ca2+/Mg2+ [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4] and endotoxin free.
Cell culture plates 96 well, V-bottom	Corning	29442-068	12 x 75 mm round bottom test tubes or 96-well V- or U-bottom microtiter plates
MabThera (Rituximab)	Roche	—	Reference product
Rituximab	Indian	—	Biosimilar product
15- or 50-mL conical centrifuge tubes	Corning	430290 or 430052	—
Pipette Tips	Eppendorf	—	Multiple volume configurations are necessary
Pipettes	Eppendorf	—	Adjustable-volume pipettes are necessary
Centrifuge 5430/ 5430R model	Eppendorf	—	Refrigerated variable-speed centrifuge (4 to 25 °C) with speeds ranging from 10 to 30,130 × g
Flow cytometer	BD Dickinson	—	BD FACSAria III or other flow cytometer
Olympus optical and light microscope	Olympus	—	To quantify and evaluate cell growth
Incubator	SANYO	—	Incubatorfor temperature and CO2 control to culture cells
Biological Safety Cabinet	CHC BIOLUS	—	Biological safety cabinet that is used to protect the researcher, product and environment.