August 1, 2008 (Vol. 28, No. 14)

Development and Evaluation of New Downstream Methodologies Is Leveling the Playing Field

Upstream processing may be attracting a lot of attention lately due to breakthroughs in protein production levels, but there are plenty of downstream advances to report as well. New approaches to antibody processing were front and center at the recent “European Downstream Forum” hosted by Sartorius Stedim Biotech. Several participants discussed their experiences with enhanced purification of antibodies and other recombinant proteins, and the merits and limitations of new purification devices were covered in detail.

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.

Squeezing Out Contaminants

“We have fine-tuned a generic approach for contaminant removal in the purification of recombinant proteins,” stated Thierry Ziegler, Ph.D., process development manager at Merck Serono’s France Etablissement de Martillac. Dr. Ziegler discussed his group’s activities in contaminant removal strategies, including charged membrane technology, ultrafiltration/diafiltration (UF/DF), and depth filtration.

According to Dr. Ziegler, the Martillac facility has adopted a generic strategy for process development, and minimizing production time from six to nine months. In order to adhere to this tight timeline, their protocol takes advantage of alternative contaminant removal procedures. The charged membrane technology was used in three out of five of the company’s recent development projects.

The Serono group used Sartorius Stedim Biotech’s Sartobind Q step to remove DNA, followed by a protein A capture step, and then an AEX membrane-polishing step to clear the host cell proteins. Ramping of amounts to production scale levels did not alter the outcome.

Dr. Ziegler also discussed the use of UF/DF technology for concentration and buffer exchange. He showed that host cell protein contamination can be substantially reduced through the use of a 100 kD cutoff unit, trapping the target Fc-fusion protein of 210 kD molecular weight.

Finally, Dr. Ziegler covered the application of depth-filtration technology to recombinant protein purification for harvest clarification and viral filtration. The procedure is notable for its ability to remove cell debris, endotoxins, and a motley collection of other malefactors. Involving the use of a matrix medium, it combines high surface area and charge interaction for greatly augmented absorptive performance.

In order to develop a robust filtration process, Dr. Ziegler argued that an understanding of the mechanisms behind contaminant removal is essential. “We find that the use of alternative approaches not aimed at contaminant removal has helped improve the final purity of our product,” he said.

Options for the Polishing Step

Klaus Tarrach, Ph.D., senior product manager for purification technologies at Sartorius Stedim Biotech, discussed viral removal and the final polishing steps in recombinant protein purification. According to Dr. Tarrach, retrovirus-like particles are ubiquitous in CHO cells, the most commonly employed cell line for antibody production.

Dr. Tarrach’s group examined the challenges of potential contamination with small nonenveloped viruses such as MVM, which calls for an orthogonal viral safety concept. The orthogonal concept of purification is based on complementary protocols for viral removal.

Nanofiltration using Sartorius Stedim Biotech’s Virosart CPV, a polyethersulfone-based 20 nm virus filter, allows for robust removal of small nonenveloped as well as larger enveloped viruses, Dr. Tarrach reported. However, contaminants such as aggregates and host cell DNA can clog the 20 nm filters, which requires appropriate polishing steps prior to the nanofiltration step. According to Dr. Tarrach, the preferred positioning of a 20 nm virus filter is within the polishing phase of the biopharmaceutical product, ideally after the anion exchange step.

“Our design will target the removal of the host cell-derived contaminants within the initial recovery process,” Dr. Tarrach explained. “These dedicated depth filtration steps will have the capability to significantly reduce the overall load of DNA and host cell contaminating protein.”


The implications of these new bioprocessing technologies with respect to antibody technology are significant. Hermann Allgaier, Ph.D., managing director for Merckle Biotec, discussed the outlook in a keynote address. “Molecular biology and upstream development have been the major innovation drivers in biopharmaceutical manufacturing, forcing upstream yields from milligrams to hundreds of milligrams to grams per liter,” he stated. “So now we hear yelling at every conference that we should change this or that to bring the up- and downstream processes in line.”

In order to address the problem on an appropriate scale, he cited the Framework for Downstream Initiative, a collaborative program established by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung—BMBF; www.bmbf. and the German industrial and academic sectors.

The BMBF plan of action will target development and evaluation of new methods to widen the existing field of separation techniques to achieve higher yields, purity, and more favorable economics for biotech products. It will also establish cooperative networks of the various players to optimize technology transfer.

Finally, the program proposes the establishment of centers of excellence to nurture focused interdisciplinary working groups. The hierarchical framework will consist of young but experienced research scientists, selected from within Germany and abroad, as leaders. The proposals are under evaluation at this time, and funding is scheduled to commence in October. “I believe we can really do something to transform the downstream world,” Dr. Allgaier concluded.

K. John Morrow Jr., Ph.D., is president of Newport Biotechnology Consultants. Email: [email protected].

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