Facing Up to Recombinant Polyclonals
Therapeutic recombinant antibody technology is largely based on building better monoclonal antibodies, largely because of their unrivaled specificity. Polyclonal antibodies, however, work synergistically and exhibit high affinities, a property that has spurred investigators to explore their therapeutic potential as well.
At the meeting, Andrea Porchia, Ph.D., senior scientist at Symphogen, discussed the particular challenges involved in purifying a coterie of recombinant polyclonal antibodies. The company’s technology is founded on the creation of a recombinant mixture of antibodies that bind to different regions of the same antigen or multiple antigens, mimicking the natural immune response.
Symphogen scientists start with antibody-producing B lymphocytes, isolated by cell sorting from humans that have been exposed to desired target antigens. From these cells, variable light and variable heavy regions are isolated using PCR primers that preserve the original pairing of the light and heavy chains. The product consists of the variable heavy chain gene joined to a variable light chain and constant light chain gene through a linker section.
These recombinant antibody genes are then cloned into a Fab expression vector, and the clones screened without phage display. The resulting pools of recombinant antibodies mimic the behavior of naturally occurring antisera, attaining high levels of specificity, diversity, and affinity. “The resulting antibodies capture the advantages of antibody polyclonality, while eliminating the safety risk associated with the sourcing of human material,” Dr. Porchia explained.
Dr. Porchia and her colleagues are working with two different Symphogen products—an anti-Rhesus antibody for treatment of hemolytic disease in newborns and an anti-Vaccinia virus antibody for counteracting the adverse effects of smallpox vaccination.
On the upstream side of the process, the cells were grown in 10–500 L bioreactors and subsequently characterized and processed. Dr. Porchia emphasized that the purification process must retain diversity and also perform in a simple, robust fashion with good quality product and satisfactory yields.
The establishment of diversity within the antibody population is critical and quite different from the situation with monoclonal recombinant antibodies in which uniformity is the goal. This characterization is carried out by ion-exchange chromatography fingerprint profiles. Identification of unique marker peptides by liquid chromatography is followed by mass spectrometry.
This process allows certification of batch-to-batch consistency and maintenance of diversity during downstream processing. The Symphogen team was able to identify all 25 antibodies in four different batches using this approach, thus authenticating the performance of the purification protocol.
The purification process, which employs MEP hypercel chromatography at pH 4.5, maintains diversity, but it must be carefully optimized, Dr. Porchia reported. The industrial-scale purification of the recombinant polyclonals is a critical step in the development of a satisfactory commercial product.