- Oral presentation
- Open Access
BEAT® the bispecific challenge: a novel and efficient platform for the expression of bispecific IgGs
© Moretti et al; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
The binding of two biological targets with a single IgG-based molecule is thought to be beneficial for clinical efficacy. However the technological challenges for the development of a bispecific platform are numerous. While correct pairing of heterologous heavy and light chains (Hc and Lc) can be achieved by engineering native IgG scaffolds, crucial properties such as thermostability, effector function and low immunogenicity should be maintained . The molecule has to be expressed at industrially relevant levels with a minimum fraction of contaminants and a scalable purification approach is needed to isolate the product from potentially complex mixtures. This article introduces a novel bispecific platform based on the proprietary BEAT® technology (Bispecific Engagement by Antibodies based on the T cell receptor) developed by Glenmark.
Materials and methods
Stable cell lines were generated by co-transfection of three proprietary expression vectors pGLEX41_GA/GB coding for the Hc, Lc and Fc-scFv under optimized stoichiometric conditions in CHO-S cells. Cell lines were selected according to expression and heterodimerization during small scale fed-batch cultures performed in TubeSpin bioreactors (TPP, Trasadingen, Switzerland). For high throughput (HT) screening, the fraction of BEAT® molecule was evaluated using the Caliper LabChip GXII Protein Assay (PerkinElmer, Waltham, Ma, USA). Titers were measured by HPLC-PA after 14 days of culture. The fraction of heterodimer in CHO supernatants was measured by CE-CGE on Protein A (ProtA) purified supernatants harvested on day 14. The actual BEAT® titer was obtained by multiplying the concentration measured by HPLC-PA by the fraction of heterodimer measured by CE-CGE in ProtA purified supernatants. The BEAT® was produced in 3 L STR bioreactors (Mobius CellReady Bioreactor, Millipore) in fed-batch. Supernatants were typically harvested on day 14 by centrifugation and dead-end filtration. A single Protein A step was performed for purification, where two isocratic steps allowed the selective elution of the bispecific product. The thermostability of the BEAT® molecule was measured by differential scanning calorimetry (DSC) in PBS.
At 3 L bioreactor scale, titers of 3 g/L with 90 % of secreted heterodimer could be obtained in fed-batch with minimal feeding optimization. After harvest the molecule is purified by Protein A (ProtA). For purification purposes the BEAT® was designed with a missing ProtA binding site on the Hc of the molecule. Consequently, residual monospecific IgG contaminants (harboring 2 Hc) do not bind to the ProtA column and are thus easily separated from the products of interest. In addition, the BEAT® molecule and the homodimeric Fc-scFv contaminant exhibit a different affinity for Protein A as the molecules harbor one and two binding sites for ProtA, respectively. Thus, the BEAT® molecule can be separated by ProtA via a two-step isocratic elution as illustrated in Figure 1 D. Applying this purification strategy for harvested bioreactor material, a level of purity of 97 % could be obtained post ProtA.
This work introduces a new bispecific IgG format called the BEAT ® . Glenmark's BEAT® platform allows the generation of stable clones with volumetric productivity of several g/L and a high heterodimerization level (> 90 % secreted BEAT® in CHO supernatants). Generated clones harbor stable product quality profiles, e.g. level of heterodimerization, over at least 75 PDL. The developed purification strategy allows a purity reaching 97 % post ProtA. The BEAT® platform combines a unique CH3 interface for heterodimerization, an efficient cell line selection strategy and an industrial relevant purification process for the production of pure bispecific antibody at several g/L.
The authors would like to thank Emilie Vaxelaire and Farid Mosbaoui for their contribution to this work.
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