Process development for manufacturing eCD4-Ig

ABSTRACT eCD4-Ig is an antibody-like therapeutic for HIV that was constructed by fusing CD4 domains 1 and 2, and an HIV coreceptor-mimetic sulfopeptide, to the N- and C-termini of the constant Fc region of an antibody. eCD4-Ig will join an emerging class of long-acting therapeutics for treating and preventing HIV infection. As a consequence of being built from parts of HIV’s receptors, eCD4-Ig neutralizes 100% of HIV isolates, and even neutralizes simian immunodeficiency virus (SIV). Thus, eCD4-Ig presents fundamental barriers to escape. This grant will fund the work that is needed in between cell line development and IND-enabling studies to bring eCD4-Ig to the clinic. In Phase I, we will validate assays for critical quality attributes (CQAs) including the sulfotyrosine content of the coreceptor-mimetic sulfopeptide, and then show that the eCD4-Ig protein made by new cell lines meets pre-defined CQAs. In Phase II, we will optimize our upstream process, downstream process, formulation buffers, and generate a GLP lot of eCD4-Ig protein for IND-enabling studies. Features of the upstream process that will be optimized include media and feed selection, a temperature shift, and fed-batch versus perfusion culture. Our downstream process is based on convective chromatography, including for Protein A affinity chromatography, anion exchange (AEX) chromatography, and hydrophobic interaction (HIC) chromatography. These processes efficiently remove poorly sulfated eCD4-Ig, aggregates, and host cell protein (HCP). We will develop a formulation for intravenous injection, and a high-concentration formulation with acceptable viscosity for subcutaneous injection. Production of a GLP lot of protein for IND-enabling studies will be outsourced to a CMO after a technology transfer process. These are the essential next steps towards bringing eCD4-Ig to the clinic.