In small molecule drug development, it is often said that the low-hanging fruit has already been harvested. Most structures that are chemically accessible, manufacturable, and druggable have already been considered.
Somewhat analogously, the leading edge in biologics development are proteins that for one reason or other present development or manufacturing challenges. Reasons vary, from difficult transfection or poor expression, to low yields or high losses on purification.
Among the speakers at CHI’s recent “PEGS” conference was Anne London, Ph.D., investigator II and lab head at the Novartis Institutes for BioMedical Research, who spoke on evaluating and streamlining platform approaches to preclinical protein production.
Preclinical protein production is a broad discipline, with expectations and goals differing from project to project. Rapid, efficient production are common goals, but challenges arise with differing maturity of programs (e.g., initial antigen production to late-stage in vivo material) and quality specifications. Dr. London presented case studies reflecting her group’s approach to arriving at a platform capable of adapting to fit production requests, with emphasis on cell-line evaluation and impurity clearance.
“The goals for each production vary based on the protein’s intended use,” she said. Live rabbit studies require a large amount of “clean” protein with very low endotoxin levels. By contrast, endotoxins will not affect proteins used in affinity binding assays, where buffer choice and concentration may be more relevant. “Fully understanding the product’s intended use is critical to guide production to the defined endpoint.”
Preclinical production platforms are “much simpler” than a GMP biologics manufacturing platform because they entail fewer polishing steps and less validation. For example, viral clearance is not required at the preclinical stage, so virus removal and inactivation are not explored. “With the goal of simply making enough protein for the intended use, only steps needed to grow, capture, purify, and deliver the protein are considered at the preclinical stage,” Dr. London explained.
A recurring production theme is low endotoxin material. In one case with the production of a His-tagged protein, endotoxin levels were too high (>1.0 EU/mg) for the intended use after standard purification.
“Guaranteeing product quality below 1.0 EU/mg is difficult, if next to impossible, in a non-GMP setting.” The situation resulted in the evaluation of multiple endotoxin-removing membranes and filters, with the final solution being a detergent extraction. Dr. London reports spending “many hours” troubleshooting this process, but eventually the product met quality specifications.
Project scaleup is another issue facing early-stage protein production. Dr. London’s group encountered an antibody that was evaluated at small scale in multiple transient cell lines before scaling into a Wave bioreactor.
“After picking the highest expressing cell line at the small scale, the culture was scaled up in the Wave with poor results; the expression levels in the flask did not scale linearly into the Wave.” This production required further process development at Wave reactor scale to meet the expression levels seen in shake flasks. “Not every project will scale up well, so time must be budgeted to allow for cell culture process development in these rare cases,” Dr. London warned.
An Unusual Phenotype
Haruki Hasegawa, Ph.D., senior scientist at Amgen, presented an investigation on the role of secretory cargo in shaping maximum protein secretory capacity. He began by questioning the nature of a cell’s maximum secretory capacity: Is it simply due to the collective contributions of intrinsic cellular machinery?
Dr. Hasegawa studied an unusual CHO phenotype that, under optimized growth conditions, produced a model human IgG clone as rod-shaped crystals within the endoplasmic reticulum (ER) lumen. Crystal growth was accompanied by cell enlargement and multinucleation, and continued until crystals outgrew cell size and breached the cell membrane.
In this phenotype the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated in the ER lumen until a threshold concentration was reached to nucleate crystals. “Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity,” Dr. Hasegawa said.
Do techniques exist for altering physicochemical properties of secretory cargo generally, for expression systems other than this phenotype? “There are two major ways to alter the physicochemical properties of secretory proteins,” Dr. Hasegawa explained. The first is to alter post-translational modifications, the second is changing the protein’s primary sequence through mutagenesis. “These two approaches are widely employed by protein engineers using various model organisms and any type of protein.”
Commenting on this work on the Faculty of 1000 website, Dr. Jesse Hay at the University of Montana wrote, “What this story tells us is that a CHO cell’s secretory capacity is not inherently limited by its ability to synthesize, oxidatively fold, and assemble functional product—it is limited by its ER export machinery.”
Gene Fusion Method
Morten Nørholm, Ph.D., senior scientist at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark, presented a talk on improved expression of a poorly expressed membrane protein by fusing its gene with that of an N-terminal peptide.
Dr. Nørholm could mimic this effect by re-engineering the 5´ end of the membrane protein with “favorable synonymous mutations.” This effect is surprisingly large compared to substitutions at other locations within the gene.
Gene fusion was conducted in high-throughput fashion using a versatile molecular cloning technology, “uracil-excision”. This PCR-based cloning technology was invented 20 years ago but has been neglected due to incompatibilities with modern PCR techniques.
In 2006, Dr. Nørholm’s group published a paper in Nucleic Acids Research identifying a compatible proof-reading DNA polymerase. “Today the technology is heavily used, open-source, simple, and inexpensive,” Dr. Nørholm explained. “We hypothesize that the peptide simply replaces a suboptimal 5´-end and/or N-terminal peptide sequence of the native construct. It is not rocket science, but a very simple and useful tool indeed.”
How generally applicable is this approach to nonmembrane proteins? “We work almost exclusively with membrane proteins, so it is hard for me to say. But the effect was similar for the few soluble proteins we tested,” Dr. Nørholm explained. “The expressing organisms—bacteria—may be part of the magic due to the strict architectural constraints of the ribosome binding site in relation to the 5´ end of the coding sequence.” However, as Dr. Nørholm pointed out, there have been reports describing optimized species-independent translational leaders for cell-free expression.