Directed Molecular Evolution
In an effort to increase the quantities of useful, functionally diverse molecules, researchers embraced rational design philosophies, building upon established knowledge. Maxygen has taken what Robert Whalen, Ph.D., director of infectious disease, calls “the diametrically opposite approach” and adopted the concept of directed molecular evolution, based upon genes’ natural diversity creation using a method derived from nature’s process of gene recombination.
“Evolution has been very successful, and typically produces improvements in organisms,” Dr. Whalen noted. The “directed” part of the procedure prevents the process from becoming a vast scavenger hunt.
Once a product opportunity has been identified, Maxygen finds related genes and places them in a test tube where the genes are fragmented and those fragments are further pulled apart. Those fragments recombine, when the tube cools, at homologous sites. The fragments are then extended, and the genes are recombined to create a library of full-length genes. Those genes are expressed as proteins, resulting in one thousand to one million variants, which are then screened, based on scientists’ understanding of what the ultimate product will require, according to Dr. Whalen.
The recombination process doesn’t cause dramatic changes, he elaborates, and results in a high proportion of functional, useful, genes. “We’ve had some successes,” Dr. Whalen said, outlining Maxygen’s preclinical work developing vaccines for HIV, hepatitis B, and influenza, as well as one vaccine that induces antibody reactions against all four types of Dengue fever.
BIA Separations has developed a process for purifying supercoiled plasmid DNA at milligram and larger scales. The process is based on its high productivity CIM HiP2 Plasmid Process Pack™. “The process contains a selective precipitation and two chromatographic steps on monolith columns for anion exchange and hydrophobic interaction,” according to Matjaz Peterka, Ph.D., manager, molecular biology laboratory.
“In our process, all the chromatographic steps are done on CIM monoliths,” Dr. Peterka said. That takes advantage of the monolith’s flow—independent dynamic binding capacity and separation, high flow rates, flow independent resolution, and low pressure drop, he explained. The process successfully separates supercoiled plasmid DNA from structurally related impurities, like RNA, host chromosomal DNA, and lipopolysaccharides.
“As a direct consequence of high capacity, buffer consumption is low. Because there is almost no limitation on flow rate, purifications can be done in a short time.” These factors result in a more than 50% lower production price per milligram of plasmid DNA.
“We also developed a new HIC monolith called CIM C4 HLD—high ligand density—to separate plasmid DNA isoforms and genomic DNA,” Dr. Peterka said. “Because the monoliths’ structures are the same across all sizes—1, 8, 80, 800, and 8,000 mL columns—scale-up is easy.” And, they are shipped ready to use. Column packing and handling is unnecessary, he added.