Valita®Titer – a novel plug-and-play IgG quantification tool for Bi-specific antibody screening

Bi-specific antibodies (bsAbs) have shown great promise with respect to the enhanced target specificity and novel mechanisms of action these formats allow. Until now however, difficulty in producing bsAbs has limited their advancement and wider clinical application. Here we describe a novel plug-and-play IgG quantification screening tool, Valita®Titer, that is easily integrated into an automated workflow to optimise the bsAB clone selection process in a high-throughput format. 

Background

Bi-specific antibodies are a diverse class of next generation biologics, characterised by their ability to bind two separate epitopes or antigens. This feature allows bsAbs to maximise potency and specificity, and accesses new mechanisms for the treatment of disease – where for example a single bsAB may recruit cytotoxic T-cells to tumour cells by binding to separate antigens on the respective cells [1]. It is not surprising therefore that cancer therapies represent a majority of the over 80+ bsAb therapies currently in development (clinicaltrials.gov); although bsAbs are also increasingly investigated for use in other clinical areas, from autoimmune and infectious diseases, to haemophilia and even Alzheimer’s Disease [2,3]. Even though only two bsAb therapeutics are commercially available to date, the establishment of over 60 different technological platforms currently for bsAb development [4] reflects the extensive market opportunities forecast for bsAb applications.

Production of bi-specific antibodies

Capitalising on the modular nature of antibodies, over 100 distinct bsAb formats have been developed by combining heavy chain (hc) and light chain (Lc) fragments of separate variable domain regions (Fv)  to form a variety of architectures. Across these formats bsAbs may vary in molecular weight, number and spatial organisation of binding sites, valency for each antigen, pharmacokinetic profile, and ability to elicit secondary immune functions [5]. Thus, while bsAbs can be highly tailored to the intended clinical application, the unique and complex nature of each bsAb poses significant demands to large-scale manufacturing and process development.

Most modern bsAbs undergoing preclinical and clinical investigation fall into 3 formats: homodimeric knob-in-hole Abs, Bi-specific T-cell engager (BiTEs) and dual-affinity retargeting Abs (DARTs) [6]. These formats are generally produced using either chemical conjugation of constituent IgG fragments, or by genetic engineering, with each method adding specific requirements to the clone selection process. When combining separate IgG fragments, automated microscale high throughput protein production processes are indispensable to screen a high number of hits for each target, before subsequent assembly of separate Fv fragments into the correct bsAb format. Therefore, in addition to the conventional challenges of mAb production – including expression, titre, variable quality, stability of product – bsAb production must identify optimum clones early on for each target before assembly of bsAb formats. Crucially, costly custom purification steps for bispecific formats that remove incorrectly formed Ab fragments, or differentiate between homodimer and heterodimer, are often required in bsAb production [7], and so efficient clone selection analytics at the pre-purification stage is required to reduce cost and decrease cycle time.

High throughput and automated solutions for bsAb production

Successful bsAb production – that produces more complex molecules at high throughput (HTP) scale – must therefore consider format, expression, platform and manufacturability as early in the process as possible. And so by its nature, the diversification of antibody formats has necessitated the employment of innovative HTP strategies that reduce cost and time, and identify failed leads early on [4,6]. 

Figure 1. Overview of an automated, microscale high—throughput bsAb production process which integrates Valita®Titer for clone ranking, based on expression. Selected clones are brought forward for downstream functional analysis, reducing the time and cost associated with bsAb production and screening.

The Valita®Titer assay is one such HTP screening tool that utilises fluorescence polarisation (FP) technology to achieve rapid and precise IgG detection and quantification in conditioned media – in either 96- and 384-well format (Figure 1). As such, the assay can be readily applied in bsAb production workflows to provide expression information at HTP scale during clone ranking, productivity and stability assessments – with no need for additional reagents and minimal hardware requirements (plate reader with fluorescence detection capabilities; Figure 2). 

Critical for HTP bsAb applications, Valita®Titer can be employed with low [5-60mL] sample volumes (suitable for microscale production process) and relies on an FP signal that is robust to interference from cells. These features of the assay make it ideal for quick determination of expression level in conditioned media (<15 min for plate prep and analysis of 96 samples, based on manual preparation) across a high number of leads at pre-purification stages. Particularly relevant for bsAb process development is the automation-friendly nature of Valita®Titer. The plug-and-play format is easily integrated into automated workflows and supports the associated benefits of such workflows by facilitating enhanced reliability and reproducibility, decreased cycle time and significant cost reduction [Figure 2].

Figure 2. Each well of the plate is pre-coated with a fluorescently labelled Fc-specific probe (1). An IgG sample binds to the probe (2). Binding is measured via fluorescence polarization and rotational diffusion (3). 

[1] Lejeune, Köse, Duray, Einsele, Beguin, Caers (2020). Bispecific, T-Cell-Recruiting Antibodies in B-Cell Malignancies. Front. Immunol.

[2] Labrijn A, Janmaat M, Reichert J, Parren P (2019). Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18:585–608.

[3] Brinkmann U, Kontermann R (2017). The making of bispecific antibodies. MAbs 2017 Feb/Mar; 9(2):182-212.

[4] Hofmann T, Krah S, Sellmann C, Zielonka S, Doerner A (2020). Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies Int. J. Mol. Sci. 2020, 21, 6551.

[5] Spiess C, Zhai Q, Carter G (2015). Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology. 67-2, Part A, Pages 95-106

[6] Sedykh S, Prinz V, Buneva V, Nevinsky G. (2018). Bispecific antibodies: design, therapy, perspectives. Drug Des Devel Ther. 12:195-208. 

[7] Zwolak A, Leettola C, Tam S, (2017). Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding. Sci Rep 7, 15521.

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