The exponential increases in yields of recombinant proteins by various expression systems continue to place demands on the downstream end of the process. Proteins generated at high densities can take on undesirable properties as the molecules crowd together.
These changes must be carefully described and monitored if the investigator has any hope of tracking down and eliminating the problem. Moreover, alternative platforms are beginning to compete with mammalian cells, long the mainstay of the industry. As their properties are different from one another, their expressed proteins require serious analysis and description. A number of companies are currently considering these challenges.
Jesús Zurdo, Ph.D., head of advanced protein technologies at Lonza, is knowledgeable about protein engineering approaches to obviate aggregation of recombinant products. According to Dr. Zurdo, “proteins by and large are unstable molecules, subject to rapid degradation, of which aggregation is one of the most intractable. By following a Quality by Design strategy we can make modifications in the molecule as we develop the process, increasing our chances of success.”
Aggregation is a significant problem in protein expression and purification, causing reduced half-life, altered activity, and loss of bioavailability and affinity. In addition, aggregated proteins possess increased immunogenicity and toxicity toward the host.
In an intact organism, aggregation is usually not an issue since a number of natural barriers exist, including chaperones and other proteins that act to stabilize the products. But biologics are produced outside the natural environment and likely have been substantially modified through genetic engineering, a practice that usually focuses on performance rather than durability. After the process is complete, a further layer of difficulty is added, since the proteins may be stored under highly non-physiological conditions. Because it is difficult to define, aggregation may require sophisticated methods of characterization in order to monitor and resolve the problem.
“In developing the parameters that reflect aggregation propensity, it is necessary to have the physical descriptors of the proteins,” Dr. Zurdo says. “These include the physico-chemical properties of the amino acid sequences comprising the protein, the sequence patterns that could prevent self-assembly, as well as the solvent accessibility.”
This analysis is aided by Aggresolve™, a software program that yields in silico predictions of the behavior of the protein. These aggregation predictors allow the investigator to discard sequences that appear risky and to design modifications in the protein that can be confirmed by in vitro validation. Dr. Zurdo and his colleagues analyzed a number of Fab antibody fragments with different sequences in silico, and then confirmed their behavior in the real world.
As predicted, those sequences that were at high risk for aggregation in the in silico model had a much higher propensity for aggregation, as shown by their migration patterns on acrylamide gels. When whole antibodies were subjected to in silico and in vitro analysis, it was possible to achieve substantial improvements in their aggregation potential by eliminating aggregation hot spots.
“We are moving toward an in silico formulation design,” says Dr. Zurdo. “The sequence will dictate the properties and behavior of the protein and enable us to specify purification requirements.”
These productivity predictors can allow the modification of the target protein in a fashion that optimizes performance and will allow better and safer pharmaceutical products.