There are a variety of ways in which the problem of low protein yields in bioprocessing platforms can be addressed. These include vector design, cell-line selection, and media optimization. In addition, the properties of the product must be taken into account, including folding and secondary modifications, especially glycosylation. Oxford Global Conferences upcoming “Proteins Congress” will profile various improvements in performance, demonstrating that the biological limits of productivity have yet to be reached.
Shire Human Genetic Therapies is developing enzyme-replacement therapies for human genetic disorders and currently has four products on the market, according to Paolo Martini, Ph.D., director of protein expression and purification research. The company has targeted lysosomal storage diseases.
Shire has two approved therapies in the U.S. for Gaucher and Hunter disease and four therapies, including ones for Fabry disease and hereditary angioedema, in the non-U.S. market.
“In order to optimize protein production, we carried out an analysis of over 500 plasmids to search out the most productive candidates,” Dr. Martini states. “We narrowed our search down to three or four promoters on seven different plasmids that were strong overproducers.”
“We have a completely natural therapy; the enzymes are produced in human cells,” Dr. Martini continues. “We have found that it is unnecessary to use modifications of the protein molecules in order to obtain optimal performance. The enzymes function in the lysosomes, and modification of their structures appears not to be required.”
Given the plethora of lysosomal storage diseases, it appears that a number of therapies will be forthcoming. “The simplicity of our approach means that this straightforward technology will have broad application for the category of systemic diseases,” Dr. Martini concludes.
Polyclonal Recombinant Antibodies
Symphogen is developing an alternative to monoclonal antibody therapies based on a mixture of polyclonal recombinant antibodies. Such combinations directed against different epitopes on the same antigenic molecule can work together in a synergistic fashion, resulting in an apparent high affinity for the mixture.
“One of our lead projects is Sym001 (rozrolimupab), a polyclonal antibody mixture consisting of 25 different recombinant anti-Rhesus D (RhD) antibodies,” explains Anne Bondgaard Tolstrup, Ph.D., director of antibody expression.
“This is a complex product, one that we are doing in partnership with Swedish Orphan Biovitrum. But in comparison to traditional hyperimmune immunoglobulins (raised in lab animals), it is more consistent and well-defined.”
Sym001 represents an alternative to the conventional anti-RhD hyperimmune immunoglobulins used as therapy for hemolytic disease of the newborn. The development of Sym001 and other products required the preparation of a two-tiered polyclonal master and a working cell bank, by which the chosen antibody clones were mixed, aliquoted, and frozen in a number of ampoules.
For GMP manufacturing, working cell bank ampoules were thawed and expanded for production of the antibody mixture in a single reactor.
Regulatory approval of these complex products has required the development of analytical antibody chemistry procedures to ensure the consistency and quality of the mixtures. Currently Sym001 and Sym004 (an anti-EGFR antibody mixture) are in Phase II and Phase I trials, respectively.
“While some of Symphogen’s early products are highly complex, the more recent efforts tend toward a much simpler antibody mixture,” Dr. Tolstrup explains. “For example, Sym004, designed to inhibit ligand binding, activation, and subsequent receptor signaling, consists of just two antibodies against the epidermal growth factor receptor.”
Sym004 induces rapid and efficient internalization and degradation of the EGF receptor, bringing about the immune-mediated killing of the cancer cells.
Currently, there are two FDA-approved monoclonal antibodies targeting EGFR—cetuximab (Erbitux) and panitumumab (Vectibix). These are used for the treatment of metastatic colorectal cancer, but their costs are extraordinary, running to $10,000 or more per month, and clinical trials have shown only limited extension of life.
“Our preclinical data demonstrate that Sym004 exhibits superior anticancer efficacy in vivo compared to currently marketed anti-EGFR monoclonal antibodies,” Dr. Tolstrup concludes.