Genetic Engineering & Biotechnology News

NOV1 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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16 | NOVEMBER 1, 2018 | Genetic Engineering & Biotechnology News | GENengnews.com In Balance It comes as no surprise that as biologicals become more complex, their manufacture becomes correspondingly—per- haps 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 modifica- tions (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 qual- ity of protein is produced from the cell lines," says Gregory Bleck, Ph.D., global head of R&D, biologics, Catalent Phar- ma Solutions. Similarly, a "normal" antibody can be tagged for down- stream conjugation by engineering in a five-amino-acid-long consensus sequence, and allowing the endogenous formyl- glycine-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 en- zyme 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 sulf- oxide (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, explor- ing 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' mem- branes 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 tre- halose without having to do anything complex to it," Dr. Brockbank explains. "It essentially equilibrates with the ex- tracellular fluid on the inside of the cell." The third method is to add the sugar to the media of actively phagocytic cells, Bioprocessing At a biologics manufacturing facility maintained by Catalent Pharma Solutions in Madison, WI, the capabilities on offer include mammalian cell line engineering. For example, Catalent uses its GPEx technology to create stable, high-yielding mammalian cell lines that can express complex proteins and expedite the generation of Master Cell Bank (MCB) candidates. Biomanufacturers Take the Heat off Their Cell Lines Josh P. Roberts 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's not just the way cells are frozen down. The way cells are thawed and treated post- thaw can lead to high rates of cell loss as well as functional deficits among surviving cells.

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