End-to-end approach for the characterization and control of product-related impurities in T cell bispecific antibody preparations

Abstract

Antibody-based T cell-activating biologics are promising therapeutic medicines being developed for a number of indications, mainly in the oncology field. Among those, T cell bispecific antibodies are designed to bind one tumor-specific antigen and the T cell receptor at the same time, leading to a robust T cell response against the tumor. Although their unique format and the versatility of the CrossMab technology allows for the generation of safer molecules in an efficient manner, product-related variants cannot be completely avoided. Therefore, it is of extreme importance that both a manufacturing process that limits or depletes product-related impurities, as well as a thorough analytical characterization are in place, starting from the development of the manufacturing cell line until the assessment of potential toxicities. Here, we describe such an end-to-end approach to minimize, quantify and control impurities and -upon their functional characterization- derive specifications that allow for the release of clinical material.

Keywords: Antibody manufacturing process; CE-SDS, capillary electrophoresis‑sodium dodecyl sulfate; CRS, cytokine release syndrome; ELISA, enzyme-linked immunosorbent assay; End-to-end approach; Fc, fragment crystallizable; Functional characterization; GMP, good manufacturing process; HIC, hydrophobic interaction chromatography; HMW, high molecular weight (species); IEX, ion exchange chromatography; PBS, phosphate buffer saline; Product-related impurities control; SEC, size-exclusion chromatography; SPR, surface plasmon resonance; TAA, tumor-associated antigen; TCB, T cell bispecific; TCR, T cell receptor.

Graphical abstract

Fig. 1

Formats of TCB monomer and side-products. (a) [2+1] TCB molecular format. The binding to TAA occurs in a bivalent mode to achieve high-avidity on target cells, whereas the binding to CD3ɛ is monovalent. These molecules have an approximate molecular weight of 200 kDa and comprise two different heavy chains and two different light chains. (b) Possible side-products occurring during manufacturing or storage of [2+1] TCB.

Fig. 2

Side-product profile of selected TCB expressing clones upon Protein A affinity chromatography elution. (a) Non-reducing CE-SDS analysis. The identity of the components was deduced from their theoretical MW under non-reducing and reducing conditions. (b) SEC analysis. Intensities were normalized to the main peak for clarity. (c) Purity and side-product content analysis of 114 clones, as determined by non-reducing CE-SDS. The size of the corresponding dots is proportional to the product titer. The selected manufacturing cell line is indicated in dark blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Evaluation of different methods for the quantification of HMW species. (a-b) Elution profile of the TAA1-TCB main product and the corresponding isolated dimer and knob-knob after (a) analytical HIC, and (b) analytical IEX. (c-d) Electrophoretic and chromatographic elution profile of purified TAA1-TCB spiked with various amounts of purified knob-knob side-products after (c) CE-SDS and (d) analytical SEC. For the analytical SEC, a Tosoh Bioscience TSKgel SuperSW mAb HR column was used.

Fig. 4

Biological activity of TCB samples analyzed in a cell-based reporter gene assay. TCB monomer and HMW side-products were incubated with Jurkat NFAT-luc reporter cells in (a, c, f) the presence and (b, d, e, g, h) the absence of TAA-expressing tumor cells. The upper panels (c-e) show the data corresponding to the normalized dose-dependent activity of TAA1-TCB, while the lower panels (d-e) show similar data generated with a second TCB (TAA2-TCB). (Note: e and f are magnifications of d and g, respectively).

Fig. 5

Evaluation of TAA1-TCB safety profile in a whole blood assay. The effects of TAA1-TCB monomer alone and in the presence of HMW species (knob-knob and dimers) on T cell activation and cytokine release were analyzed in a spiking assay in vitro. Upon treatment, (a) the percentage of CD4+ T cells positive for the activation marker CD69, as well as (b) cytokine release (exemplary shown here, IL-6 secretion) were investigated. Data represents mean and standard deviation of 6 donors). PBS, cetuximab (anti-epidermal growth factor receptor antibody), muromonab-CD3 (OKT3, anti-CD3 antibody) and alemtuzumab (anti-CD52 antibody) were included as negative controls.