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Methods Improving HIV Protein Expression: Cell Substrate and Protein Purification



Methods Improving HIV Protein Expression: Cell Substrate and Protein Purification

Fast Track proposals will be accepted Number of anticipated awards: 3-4 Budget (total costs, per award): Phase I: up to $300,000 for up to 2 years; Phase II: up to $1,000,000 per year up to 3 years



There is an urgent need to have multiple HIV envelope immunogen components for use in HIV vaccine clinical studies. Results of the RV144 vaccine clinical trial indicated that antibody responses to the gp120 Env proteins were the major components of the vaccine that contributed to the efficacy signal. In addition, the discovery of both broad and potent neutralizing antibodies against the HIV envelope in HIV infected individuals, provides additional evidence that the human immune system can respond effectively to the HIV envelope and so envelope could be the major component of an effective HIV vaccine.

Results of contract manufacturing organizations (CMOs) efforts to produce envelope proteins using pre-existing platforms developed for Chinese hamster ovary (CHO)-based monoclonal antibody production have been disappointing. Despite the widespread use of GMP-established pharma cell substrates (e.g., CHOs, 293 etc.) in development of monoclonal antibodies and recombinant protein antigens, critical bottlenecks still exist in their use for large-scale, high-yield GMP manufacturing of HIV protein antigens. For example, the use of CHO-based protein expressions systems previously used for the generation of monoclonal antibodies (mAbs) have resulted in significantly lower yields of HIV envelope proteins (100-1000x fold lower) compared to mAb expression (mgs/l of envelope product compared to the typical g/l yield of mAb). Additional limitations relate to their intrinsic incapacities to metabolically express high levels of stable, properly folded and glycosylated recombinant HIV Env protein; often times requiring extensive clonal screening to identify the rare high-level producer clone.

Beyond issues of primary yield of the expression system, traditional downstream purification processes for HIV Env purification are equally plagued with inefficiencies due to multi-step purification cycles resulting in low yields. As a result, subsequent purification schemes for mAbs are not readily transferrable to HIV envelope purification and often result in 80-90% losses of envelope material. Moreover, purification schemes developed for one HIV envelope are not necessarily suitable for another HIV envelope due to the potentially large differences in post-translational modifications. While, monoclonal antibodies may be able to withstand harsh viral clearance procedures, whereas HIV envelopes which are more sensitive glycoproteins may not be able to sustain the harsh viral clearance procedures.

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. These problems demonstrate the need for new approaches to enhance and expedite the screening, production and purification of HIV envelope protein candidates. As such, there is an urgency to evaluate alternative strategies and technologies capable for developing highly productive cellular substrates suitable for high yield GMP manufacturing of HIV antigens and reduced product development lead times.

Project Goals

The goal of this contract solicitation is to support research to improve the expression yield in a specific cell culture system (i.e. CHO), and the purification yield using specific purification regimens designed for HIV envelope protein suitable for use as clinical immunogens. Projects may focus on any step of envelope expression and yield, improvement of substrates (i.e. CRISPR/Cas9 editing, siRNA delivery and gene silencing) by evaluating and modulating the molecular pathways involved in regulating and enhancing HIV envelope/antigen expression in mammalian cell lines. The projects may also focus on development of purification platforms.

Phase 1 activities may include:

Improving HIV Env protein expression in existing cell substrates or development of novel cell substrates should be explored through the following approaches: improving existing cell substrates o altering codon usage

o targeting host cells genes that increase expression

o improved expression cassettes for the recombinant protein and novel selection marker genes

o identifying auxiliary proteins essential for protein production, modifying components of secretory and processing pathways, enhancement of cellular processes (e.g. chaperonins)

o functional phenotypic screening

o enhancement of transcription or of evaluating mRNA sequence and structure

o alteration of epigenetic targets

o methodologies to alter post-translational modifications including glycosylation or disulfide composition, and secreted protein

o the production of intracellular, membrane-associated, or secreted protein.


• Improving existing cell substrates o by removal of deleterious proteases,

o addition of enzymes involved in glycosylation

o removal of deleterious proteases

o alteration in epigenetic targets


• Using of functional genomics to identify gene function and editing. Focusing on siRNA technologies and delivery methods into cell substrates coupled with high throughput screening and analytics, modulation of gene function, expression, regulation and mutation of target cell. Evaluating newer technologies such as CRISPR/Cas9 targeted gene editing stimulating genetic modifications to prepare productive stable cell lines will also be evaluated.

• Evaluating transient transfection/gene expression technologies to support and accelerate phase appropriate manufacturing

• Developing and improving HIV envelope protein purification methodologies including (but not limited to), affinity purification approaches and/or other highly novel strategies;

• Developing analytical assays and testing methods for characterization, identification, quantitation of expressed product during cell line development

• Establishing conditions for removal of host cell protein clearance, endogenous retroviruses (viral inactivation and clearance).

• Developing research cell bank

• Testing and characterizing cell lines and evaluating quality attributes/metrics prior to advancing the cell substrate and processes to GMP scale

• Establishing conditions for removal of host cell protein clearance, viral inactivation and clearance.

Phase 2 activities may include:

GMP development and manufacturing lead cell substrates (e.g., Master Cell bank generation)

• Translating and scaling-up of process development activities including downstream purification unit operations to GMP setting

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