There has been a maturation of the bioprocessing industry as companies have moved away from a focus on anticancer biologics toward a more diversified portfolio of pharmacological agents. With this diversity of disease targets comes a need for expression technologies to fit the requirements of proteins that may be required in smaller quantities or that may present exceptional problems to be surmounted for successful production.
CHI’s “PEPtalk” conference to be held later this month will feature presentations from a number of scientists who are examining the intricacies of protein expression, and struggling to squeeze the last microgram of performance from their systems, while at the same time optimizing the quality of the recombinant product.
“We found that conditions can be optimized to grow animal cells in shaking reactors at liter scale with the potential to expand to commercial volumes,” says Chao-Min Liu, Ph.D., distinguished research leader at Roche.
Dr. Liu and his research team have been investigating the problem of adapting small-scale technology for the large-scale culture of animal cells in shaker reactors for a number of years. However, the potential of large-scale cultivation in shaker reactors was not considered until recently.
Focusing on the shaker approach, Dr. Liu and his co-workers assumed that this would provide a simple and efficient means of delivering adequate aeration to both mammalian and insect cells. They found that cylindrical bottles with small volumes up to 1 L provided the optimum growth conditions. How-ever the smaller vessels were scalable to much larger volumes using proportionally larger shaking bioreactors.
"Successful implementation of this system has greatly increased the efficiency of our laboratory and our ability to generate large quantities of recombinant proteins for drug screening and development,” Dr. Liu explains.
Florian Wurm, Ph.D., of the Laboratory of Cellular Biology at the École Polytechnique Fédérale de Lausanne Switzerland, has also worked extensively with shaker devices and he agrees with Dr. Liu. “The best approach is the use of cylindrical bottles, which we use in all of our operations with orbital shaking. Erlenmeyer flasks are a poor choice as they limit gas exchange through the narrow neck; devices with impellers are the worst, only useful with densities of cells of less than 2 million cells/mL.”
Dr. Wurm also agrees with Dr. Liu that the cylindrical system can be scaled up to 1,000 L, with similar performance to controlled and “bubbled” stirred tanks. Another positive feature of the cylindrical shaker flasks, according to Dr. Wurm, is their optimal performance with serum-free and protein-free media, when the cells are more fragile.
“They outperform stirred devices, because shear stress is dramatically reduced, since the bulk of the liquid is moved in a laminar way.”
Chemokine Fusion Proteins
“Osprey’s therapeutic approach to inflammation and autoimmunity is to neutralize specific pathological leukocytes using chemokine fusion proteins,” explains Hongsheng Su, Ph.D., director of process development at Osprey Pharmaceuticals.
Dr. Su’s research group is investigating a class of novel fusion proteins called leukocyte population modulators (LPM). The lead therapeutic candidate, CCL2-LPM, is a recombinant fusion protein composed of a member of the chemokine superfamily, CCL2 (also known as macrophage chemoattractant protein-1, MCP-1) fused to a bacterial and eukaryotic protein synthesis inhibitory enzyme (RIP, ribosomal inactivating protein), in this case a truncated Shiga toxin A1 subunit. CCL2-LPM is currently in clinical trials.
Chemokines, a group of small protein molecules, are powerful chemoattractants that participate in leukocyte trafficking, extravasation (exudation of lymphatic fluid into the tissues), and recruitment to specific sites.
The CCL2 chemokine and its receptor, CCR2, play a decisive role in a number of inflammatory diseases, including those of the kidney and some cancers. By recruiting active pathological leukocytes, a cascade is initiated, which facilitates tissue damage, autoimmunity, fibrosis, and metastasis. For this reason, Dr. Su and his colleagues propose that engineering a fusion protein in which the chemokine CCL2 is fused to a protein-synthesis inhibitor would bring this destructive chain of events to a halt.
However, production of a protein that inhibits protein synthesis presents the investigator with a quandary, since it is not immediately obvious how one might prevent the newly synthesized inhibitor from shutting down its own continuing synthesis.
In order to deal with this challenge, Dr. Su and his collaborators developed a protocol using the compound 4-aminopyrazolo(3,4-d)-pyrimidine (4APP), which blocks the action of the Shiga A1. 4APP binds to the SA1, protecting cells from the toxic actions of the SA1 protein.
By employing this strategy the team was able to grow transformed cells containing the fusion protein gene to high densities, and isolate gram/L quantities of the product. The expressed and highly purified protein was effective in neutralizing cells of the monocyte lineage, which possess the CCL2 chemokine receptor. According to Dr. Su, “early clinical trials in IgA nephropathy patients are currently ongoing.”