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Methods Improving HIV Protein Expression: Cell Substrate and Protein Purification

Description:

Background
Despite the widespread use of GMP-established pharma cell substrates (e.g., CHOs, 293 etc.) in development of recombinant HIV Env protein antigens, critical bottlenecks still exist in their use for large-scale, high-yield GMP manufacturing; yields often are on the order of mg/L compared to mAbs at gm/L. Some of the limitations relate to their intrinsic incapacities to metabolically produce high levels of stable properly folded, properly glycosylated recombinant HIV Env protein, often times requiring extensive clonal screening to identify the rare high-level producer clone. These constraints have a cascading effect in increasing the overall cost and time for production of HIV vaccine antigens from millions of dollars and years of upstream and downstream process development. As such, there is an urgency to evaluate alternative strategies/technologies capable for developing highly productive cellular substrates suitable for high yield GMP manufacturing of HIV antigens and reduced product development lead times. Traditional downstream purification processes for HIV Env purification are equally plagued with similar inefficiencies either requiring expensive lectins or multi-step purification cycles resulting in low yields. Alternative approaches to HIV Env purification are needed to improve yields and expedite the overall purification process and costs.
Project Goals
The objective is to evaluate and modulate the molecular pathways involved in regulating and enhancing HIV envelope/antigen expression in mammalian cell lines and to accelerate development of purification platforms in a CGMP manufacturing setting. Key areas of support will include, but are not limited to, the following:
Phase I activities may include the following non-CGMP activities:
 Exploration of methodologies to improve HIV Envelope protein expression in mammalian cell substrates. The following approaches may include: alteration of codon usage, improvements in expression cassettes including the use of novel selection markers or other selection approaches, evaluation of Env mRNA sequence
 Exploration of methodologies to improve of existing cell substrates by removal of deleterious proteases, targeting of genes involved in glycosylation, improved secretion or other post-translational modifications that enhance yield and/or stability, or removal of endogenous retroviruses.
 Methodologies to improve HIV Env expression or cell substrates can include traditional gene modification approaches as well as novel technologies such as siRNA and/or CRISPR/Cas9 gene targeting.  Development of strategies to accelerate phase appropriate manufacturing including transient transfection or stable cell pool approaches for HIV Env GMP manufacture  Improvement of HIV envelope downstream protein purification methodologies including affinity purification approaches or other strategies.
Phase II activities may include the following CGMP activities:

 CGMP development of the improved Cell Substrates explored in Phase I, including additional IND-enabling
characterization studies, development of technical reports, generation of MCB, etc  CGMP Process Development of the improved Downstream purification methodologies developed in Phase 1, including
technical reports, development of scale-up approaches, etc.

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