November 1, 2018 (Vol. 38, No. 19)
The creation and preservation of unique cellular samples are cornerstones of modern biomedical research and its translation. The Waterside Conference, to be held in Norfolk, VA, November 12–14, will feature topics ranging from the manufacture of biopharmaceuticals to the freezing down and thawing of patient samples. Below, four of the event’s presenters discuss highlights from their upcoming talks.
It comes as no surprise that as biologicals become more complex, their manufacture becomes correspondingly—perhaps exponentially—more demanding as well. It’s no longer just a question of how much protein can be expressed, but sometimes it’s a question of a protein’s relative amount, for example, or whether the correct post-translational modifications (PTMs) can be effected.
Products such as bi-and trispecific antibodies may require the expression levels of “three, four, or five different genes to come together in specific ratios to ensure that the best quality of protein is produced from the cell lines,” says Gregory Bleck, Ph.D., global head of R&D, biologics, Catalent Pharma Solutions.
Similarly, a “normal” antibody can be tagged for downstream conjugation by engineering in a five-amino-acid-long consensus sequence, and allowing the endogenous formylglycine-generating enzyme (FGE) to convert the sequence’s cysteine residue to formylglycine. But the conversion may not be able to keep up with overexpressed heavy and light chains, so Catalent created conversion-capable lines by transfecting in FGE. However, they didn’t know how much would be needed. “We didn’t have a good assay for the enzyme at the time, and so we created a range of cell lines with varying copy number,” Dr. Bleck explains. “Luckily, even at the higher copy number/expression level, it didn’t seem to affect cell growth and we were able to utilize the ones expressing the most enzyme as our base cell line to add our antibody genes to.”
Sometimes a cell line, or in this case, its species, just isn’t up to the task of producing the desired protein. There is a push in the vaccine world to make more virus-like particle (VLP) vaccines, where you might need 10 copies of a protein, 5 of a second, and 1 of a third to approximate the normal viral coat structure of a particular virus while creating the VLP, Dr. Bleck notes. He gives an example in which Catalent needed to engineer the VLP in a human cell line, rather than the more traditional Chinese hamster ovary (CHO) cells, “in order to get full function, and have the function that the partner wanted.”
Sweet Frozen Cells
For more than 60 years, the organic solvent dimethyl sulfoxide (DMSO) has been used to freeze down cells, “first as a research product, and then clinically when they first started to do bone marrow transplants,” says Kelvin Brockbank, Ph.D., CEO of Tissue Testing Technologies (TTT). His talk focused on TTT’s efforts to find new ways of preserving cells that remove some of the cons of current practice. Among these, he says, is “the potential toxicity of DMSO in patients if you deliver it intravenously.” Cells frozen down in DMSO need to be washed and cleaned prior to infusion.
TTT has been looking instead into the sugar trehalose (and to a lesser extent sucrose) as a cryopreservative, exploring three methods to introduce it into the cells. In the first, trehalose-containing liposome-like structures are incubated with the cells, allowing them to merge with the cells’ membranes and release the cells’ contents. The second method uses the p2x7 purinergic cell surface receptor, found on various cell types (especially those of hematopoietic origin). “Any cell type that has that, you can actually deliver trehalose without having to do anything complex to it,” Dr. Brockbank explains. “It essentially equilibrates with the extracellular fluid on the inside of the cell.” The third method is to add the sugar to the media of actively phagocytic cells, “and they will take it up by pinocytosis.”
Dr. Brockbank says that scientists in his subfield within cryobiology prefer, and preferentially tend to use, trehalose over other sugars. For one thing, trehalose is not metabolized by mammalian cells, “so if you can get it into a cell, that concentration is stable until the cell divides.” Sucrose, on the other hand, “is more difficult because cells tend to metabolize it, so accumulation is not always easy.” Yet while trehalose is a common excipient for pharmaceutical products, and thus there would be “very little concern” about using it in a patient, there “are no questions about residuals” at all with sucrose.
The results of using sugars to preserve cells vary with cell type, and for the most part are “still not quite as good as DMSO,” Dr. Brockbank admits. “But I think it’s more a matter of time and effort.”
It’s not just the way that cells are frozen down. The way that cells are thawed and treated post-thaw can lead to high rates of cell loss as well as functional deficits among surviving cells. “In an area like cell therapy, clearly getting back a high number of what you put in, and having the cells function the same way as they did prior to the preservation process, is really important,” stresses John Baust, Ph.D., president, founder, and lead scientist at CPSI Biotech.
Dr. Baust discussed his group’s research into characterization of approaches to efficiently and effectively thaw cells. To this end, they have developed “a dry thawing system that gives reproducible, controllable, hands-free thawing of cell products,” akin to controlled freezing devices currently on the market.
Once thawed, though, the cells are still not out of danger. “Over the next 24–48 hours is when this whole stress response process manifests,” he notes. “If we can intervene in that post-preservation period—what would be basically the recovery or culture period—and help the cell repair itself and have a lower level of stress response, then it would be a better functioning cell.”
Dr. Baust’s group is working on identifying different molecular, proteomic, and genomic targets involved with the activation of cell death pathways, and with protein folding and recovery processes. From there, “we can identify compounds that could be additives to the culture media after the thawing process is over.” Among the leading candidates are molecules such as those in the N-acetylcysteine class to control oxidative stress, as well as caspase inhibitors.
There are literally billions of already-frozen samples that can’t benefit from any improvements in freezing technologies, points out Dr. Baust, but “we still want to be able to get back the best product we can.”
Holistic approaches to systems biology mean that every data point—from haplotype to age of disease onset to smoker status—is important. Parsing out that data and subsetting it into meaningful groupings—the foundation of precision medicine initiatives—can contribute to a better understanding of health and disease as well as help guide individual treatments. But this all means that the exponentially growing collections of samples, along with their associated metadata digests, need to be stored in ways that they are easily searchable and readily retrievable.
Part of the answer may be automated biobanking. Here, a robotic system receives, moves, and warehouses barcoded biological materials through a cold space under database control. Allocated space can range from the size of a typical –80 °C lab freezer up to an entire building, depending on the needs and budget of the research group. “They don’t have to worry about where the sample is or deciphering or remembering its location,” explains Steve Broach, global sales and marketing manager for LiCONiC Instruments. “Nor do they have to remember anything about the data associated with that. All that’s done for them automatically.”
Broach says he may discuss the concomitant return on investment (ROI), how an automated biobank favorably compares to a traditional manual cold storage setup in terms of energy costs, space usage, labor, and the like. But “the data and the integration with the LIMS (laboratory information management system) is where most people are seeing the value.” Having at hand millions of samples associated with their searchable metadata enables questions to be answered that could not even be asked otherwise.