Scalable transient protein expression.

Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. This review will summarize developments in this field, properties of the components of a transient expression system that enable maximal protein production, and detailed protocols for this application.

Scalable purification of Bacillus anthracis protective antigen from Escherichia coli.

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.

A phase 1 study of PAmAb, a fully human monoclonal antibody against Bacillus anthracis protective antigen, in healthy volunteers.

BACKGROUND: Inhibition of the binding of Bacillus anthracis protective antigen (PA) to its cellular receptor can abrogate the downstream toxin-mediated deleterious effects of the anthrax toxin. A fully human monoclonal antibody against B. anthracis PA, PAmAb, was previously shown to provide a survival advantage in rabbit and monkey models of inhalational anthrax. METHODS: A randomized, single-blind, placebo-controlled, dose-escalation study with 105 healthy volunteers was conducted to evaluate the safety, pharmacokinetics, and biological activity of PAmAb. Subjects received PAmAb or placebo as a single intramuscular injection (11 subjects/cohort) or intravenous infusion (10 subjects/cohort). Three intramuscular dose levels (0.3, 1.0, and 3.0 mg/kg) and 5 intravenous dose levels (1.0, 3.0, 10, 20, and 40 mg/kg) were studied. Two separate intramuscular injection sites (gluteus maximus and vastus lateralis) were evaluated in the cohorts (hereafter, the "IM-GM" and "IM-VL" cohorts, respectively). RESULTS: PAmAb was well tolerated, with no dose-limiting adverse events. All adverse events were transient and mild to moderate in incidence and/or severity. The pharmacokinetics of PAmAb were linear within each route and site of administration but were significantly different between the IM-GM and IM-VL cohorts. The mean terminal elimination half-life ranged from 15 to 19 days. The bioavailability of PAmAb is approximately 50% for IM-GM injection and 71%-85% for IM-VL injection. The biological activity of PAmAb in serum, assessed using a cyclic adenosine monophosphate assay, correlated with serum concentrations. CONCLUSIONS: PAmAb is safe, well tolerated, and bioavailable after a single intramuscular or intravenous dose, which supports further clinical development of PAmAb as a novel therapeutic agent for inhalational anthrax.

Development of an edema factor-mediated cAMP-induction bioassay for detecting antibody-mediated neutralization of anthrax protective antigen.

Intoxication of mammalian cells by Bacillus anthracis requires the coordinate activity of three distinct bacterial proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Among these proteins, PA has become the major focus of work on monoclonal antibodies and vaccines designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. Here, we present the development of a sensitive, precise, and biologically relevant bioassay platform capable of quantifying antibody-mediated PA neutralization. This bioassay is based on the ability of PA to bind and shuttle EF, a bacterial adenylate cyclase, into mammalian cells leading to an increase in cAMP that can be quantified using a sensitive chemiluminescent ELISA. The results of this study indicate that the cAMP-induction assay possesses the necessary performance characteristics for use as both a potency-indicating release assay in a quality control setting and as a surrogate pharmacodynamic marker for ensuring the continued bioactivity of therapeutic antibodies against PA during clinical trials.