June 15, 2008 (Vol. 28, No. 12)

Getting the Most from Cells Is Part Nature and Part Nurture

Cell-line optimization has become a critical step for generating the robust, highly productive cells used in biomanufacturing, cell-based assays, and reagent production. While numerous techniques exist for incorporating desirable traits into mammalian cell lines as well as into yeast, bacteria, and insect cells, one can make the case for viewing optimization as a holistic exercise whose components include transfection and expression methods, media development, and process optimization.

Optimization has become routine wherever cells are used, from drug discovery to manufacturing, for small-scale reagent production to super-sized mammalian cell cultures—everywhere that success depends on expressing desirable traits in cells.

GenWay Biotech (www.genwaybio.com), which sells proteins and antibodies, optimizes its production cells for purity and protein function. All cell-line optimization work, whether ultimately for biomanufacturing or reagent production, begins with these two considerations, says Sergey Sikora, Ph.D., vp, business development. The company began in drug discovery, but today focuses on producing catalog antibodies, proteins, and reagents for plasma proteomics based on chicken IgY antibodies. Based on this expertise, GenWay also optimizes production cells for customers, many of whom are virtual companies.

Production-Worthy Cells

The top concerns that Invitrogen (www.invitrogen.com) hears from its pharma customers are related to project cost, protein titer, and speed—how fast a concept can be turned into a robust cell line. The company estimates that it takes six to nine months for it to create a robust cell line, depending on the complexity. Related to cost is the freedom to generate lines unburdened by royalties or licensing fees.

Product quality is another area of interest, particularly how it relates to post-translational modifications. Assuring that cells produce what they were meant to requires having strong analytics behind the cell-optimization process.

Increasingly, service companies recognize the interplay between cell genetics and media—nature and nurture. Invitrogen’s cell science group, therefore, works closely with the firm’s GIBCO® media and process groups to provide what it calls a “total solution.” This way, by the time cells are optimized for genotype, the customer also knows which medium will accentuate their best characteristics.

Invitrogen’s high-throughput cloning service utilizes the ClonePix FL cell-analysis system from Genetix (www.genetix.com). ClonePix FL performs high-throughput, fluorescence-based detection, isolation, and selection of single clones of hybridomas, adherent and suspension mammalian cells, and stem cells. Invitrogen scientists have modified some of the instrument’s workflows and protocols for faster, more efficient selection of high-producing lines.

Similarly, SAFC Biosciences’ (www.safcbiosciences.com) Cell Express performs high-throughput clone screening combined with media optimization.

Cell Express allows screening cells at an very early stage and measures both productivity and growth rates for individual cells in a media-optimized manner,” says Bruce M. Lehr, director of marketing.

For Lehr, the question of nature vs. nurture is not entirely relevant because they are not independent variables. “The parental cell plus the gene you insert into it demand certain nutritional requirements, but the nutritional environment also provides a selection pressure, so it is possible to influence the cells you select based only on media.”

SAFC takes about six months to deliver a dependable clone to customers who supply transfected, development-stage cells, which SAFC scientists screen and test under various media conditions. More commonly, customers do some of the cloning work and present SAFC with a first-approximation line that may, for one reason or other, not meet performance criteria. SAFC can optimize media for lines such as this and perhaps reselect clones for higher productivity or more robust growth.

“There’s always an opportunity to sub-clone out more desirable cells,” notes Lehr, “but matching new cells with optimized media improves the line even more.”

Altogen Biosystems (www.altogen.com) specializes in polymeric, lipid- and nanotech-based, and also traditional methods for delivering genes into mammalian and bacterial cells. The company performs custom cell-line development and designs kits for permanent and transient transfection.

Cell-development services will always be in demand because most desirable cell lines—particularly suspension cells and primary cells—transfect only with great difficulty, at less than 1% efficiency. Moreover, clone-picking for growth is complicated by the need of many cells to “communicate” chemically with surrounding cells before they multiply. Cells plated singly in microwells are therefore unlikely to proliferate unless they are spiked with supernatant from growing cell lines or prepared solutions of growth factors.

While biotech companies can purchase transfection kits and try them in succession, they can often save time and effort, and perhaps cost, by outsourcing this operation, says senior scientist Alex Voltz, Ph.D. “Altogen is equipped to assess the cell and try multiple technologies,” he notes, and can supply various clone-picking techniques that may not be available to biotech customers.

Cell-line engineering holds particular promise in the design of relatively uncommon expression systems, i.e., yeast for mAb production. Yeast are attractive as an economic alternative to CHO cells, but the prevailing belief is that yeast present problems with post-translational modification, specifically glycosylation. ApoLife (www.apolife.com) has demonstrated, through its “twin cassette” gene-insertion technology, that this need not be so.

According to Nalini Motwani, Ph.D., president of Apolife, there is no experimental evidence for immunogenicity in immunoglobulins produced in yeast, nor is there any a priori reason to believe it might occur. Yeast may be programmed to produce homogeneous, human-like glycosylation patterns. Moreover, Dr. Motwani believes that antigen-dependent cellular cytotoxicity—a measure of an antibody’s biological activity—may be higher in yeast-manufactured proteins.

To be effective, cell-engineering efforts should begin early in development. Selexis (www.selexis.com) focuses on cell optimization at the protein drug discovery stage, with the expectation that the cells will eventually be used in GMP manufacturing. Retaining the same cells, says vp of business development Andrew Sandford, saves considerable time and effort, although there is an upfront investment since stable transfection takes longer than transient transfection. One notable benefit, according to Sandford, is greater assurance or product quality and consistency throughout drug development.

Selexis claims that through its Sure Cell Line DevelopmentSM and Genetic Elements™ technologies, customers can obtain high-expressing, high-performing cells in as few as five weeks.

Transient transfection is, of course, faster if the only goal is producing small quantities of protein. “But if someone had the choice between obtaining either stable or transiently transfected cells, time being equal, they would choice the former,” says Sandford.

Selexis licenses its technologies and performs optimization work on a contract basis for companies that lack this expertise. Part of their custom work involves designing and synthesizing genetic elements to improve gene expression.

In contrast to cell-line optimization at the genome or media-development stage, organizations like the Massachusetts Biomanufacturing Center (MBMC; www. uml.edu/centers/biomanufacturing) specialize in optimizing processes at the bioreactor level. MBMC typically receives cells that have already been tweaked for productivity but may have quality or consistency issues such as folding, aggregation, and inappropriate post-translational modifications.

According to MBMC director Carl Lawton, Ph.D., biotech companies have been slow to pick up on optimization strategies that include chaperonins and heavy/light chain stoichiometry in antibody production. Chaperonins assist in proper protein folding and their misfunction may result in aggregation. Heavy/light chain stoichiometry can affect the optimal assembly of mAbs. Both factors could, of course, be programmed into cells, but optimizing them during production is a possibility as well.

“These factors are particularly important as processes are scaled up,” says Dr. Lawton. “If the built-in cell machinery does not keep up with the scale-up, productivity will fall.”

In theory, cell-line developers should insert more of the light-chain gene into cells to assure that light-chain folding, the first step in antibody assembly, occurs correctly. The question is, how much? An overabundance of light chain means the cell spends inordinate energy making and assembling it; if there is not enough light chain available, then antibody production falls.

Creating Custom Cell Lines

Stem cell optimization will become more important as these cells gain wider acceptance in R&D. “Many of the things we do, before the customer gets our cells, are similar to what occurs in any cell-line optimization,” says John Hambor, Ph.D., CEO of Cell Design. The company utilizes stem cell lines to generate physiologically relevant human and animal cell lines used in drug discovery.

Cell Design offers numerous human cell types: neuronal, hepatic, mesenchymal, and others derived from the appropriate stem cells. Further differentiation into subtypes of somatic cells is also possible. Finally, the company can optimize cells for various phenotypes, for example, expression of GPCRs, transporters, ion channels, or any other trait useful in drug discovery. These traits are produced by changing culture conditions—growth factors, cell densities, the extracellular matrix, and time to differentiation to name a few.

No recipe exists for programming most traits into stem cells. “It’s an empirical science,” notes Dr. Hambor. Researchers obtain cues from the literature, but progress is generally made through trial and error. Once a method is established for a cell line and a trait, it may serve as the springboard for further optimization.

Predictable proliferation, from research through industrial scale, is key to the stem cell business, explains Dr. Hambor. “If you can’t expand these cells they are not of much use. It takes a great deal of expertise to control the process, to repeat it reproducibly.”

One of the Cell Design’s current projects is to create stem cell collections for various cell and tissue types that reflect the genetic makeup of large populations. This project could transform how companies perform cell-based drug discovery assays.

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