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Nov 15, 2007 (Vol. 27, No. 20)

Enhancing Performance in Cell Culture

Hydrolysates from Plants for Medium Supplementation

  • Back in the 1950s, H. Eagle established that a mixture of amino acids, vitamins, cofactors, carbohydrates, and salts are necessary to support in vitro cell growth. Now, however, it’s well known that additional growth factors, hormones, and other biochemical compounds are required to optimally control and regulate cell growth. Traditionally, such supplementation has been achieved by adding animal serum (e.g., fetal bovine serum or FBS) at a concentration of 5–20%.

    Sera do provide a multitude of important growth and regulation factors but are also undefined in terms of absolute composition, can often display batch-to-batch variability, present downstream processing challenges due to its high protein content, and can be extremely expensive, especially if specific countries of origin are required.

    The use of sera for the bioproduction of therapeutic proteins, antibodies, and even viral vaccines has fallen out of favor primarily due to safety concerns. Contending with the possible presence of adventitious agents—be they prions responsible for transmissible spongiform encephalopathy (TSE or BSE in cows), viruses (a recent study found that 20–50% of commercial FBS was virus-positive), or bacteria (and their undesirable endotoxins found upon cell lysis)—has truly spurred significant movement away from the use of sera.

    Consequently, national and international legislatures are increasingly opposed to the use of animal-derived products such as FBS in biopharma production. In January, the FDA announced its intention to limit the materials used in some medical products, “in order to keep them free of the agent thought to cause BSE.”

  • Serum-free Media

    The ideal cell culture medium would allow mammalian cells to grow at the same rate as sera without any loss in protein yield. Accordingly, many serum-free alternatives have been developed since the late 1970s.

    The first step toward serum-free media (SFM) was the use of animal-derived protein hydrolysates (peptones), produced with animal-derived enzymes and/or purified proteins from animal or human sources.

    In the search for a growth medium that most closely replicates the benefits of animal-derived sera without its attendant biosafety risks, there has been growing interest in the use of plant-based (vegetable) hydrolysates such as those derived from soy, wheat, and cottonseed.

    Kerry Bio-Science, with its Sheffield™ Pharma Ingredients (www.sheffield-products.com) brand products, was among the first to market nonanimal-derived protein hydrolysates for both serum replacement and general media/system optimization efforts. Examples of Kerry Bio-Science products include Hy-Soy™ or HyPep™ 1510 (both derived from soy), HyPep™ 7504 (cottonseed), and HyPep™ 4601 (wheat gluten).

    Various test results clearly demonstrate the suitability of vegetable protein hydrolysates to replace animal-derived media components for the cultivation of most cell lines (e.g., hybridoma, BHK, CHO, Vero, MDCK). The first biotherapeutics produced using plant-protein hydrolysates have already reached the market and many more are in various stages of development.

    Nonetheless, plant-based hydrolysates have met with criticism concerning lack of definition and lot-to-lot variability. Although most suggest such variability results from key processing steps employed during hydrolysis and refinement, the raw material itself—be it seasonal effects in plant crops or intrinsic differences among specific enzyme activities—can also contribute.

    One possible solution has been the use of chemically defined growth media (CDM), which attempt to optimize individual biochemical formulation constituents without the need to supplement with a protein hydrolysate. Although CDM has had its successful applications, development and optimization time lines can be long and expensive, process performance may be somewhat limited (yield loss, culture viability concerns), and formulation utility or versatility may well prohibit the development of a platform CDM suitable for both wild-type host cells and their bioengineered progeny.

    Despite advances in SFM and CDM, some particularly fastidious yet industrially important mammalian cell lines seem to still require serum components (e.g., attachment factors) to perform in a viable culture.

    Sheffield Pharma Ingredients has developed a new hydrolysate platform that addresses the need for animal-free cell culture medium supplements while also minimizing variability concerns. The platform is based upon a novel approach to enzymatic digestion and more refined processing.

    At the heart of the product is a rationally designed nonanimal enzyme cocktail, including both proteases and nonproteolytic enzymes, whose activities can also liberate primary components of the polymerized nonprotein portion of the raw material. This enzyme blend is added during a highly optimized process step that targets specific enzyme-substrate reactions to expand the range of beneficial nutritional factors made available to cells in culture.

    Such factors are fundamental to improving the bioperformance of the culture system, as they provide not merely growth-promoting peptides and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that improve both rate and quality of protein expression and serve to improve culture life due to osmoprotectant and antiapoptotic properties.

    Also of significant note is that, compared to typical hydrolysates, the production process is greatly reduced and requires fewer steps, intrinsically yielding a better-controlled and therefore more reproducible product.

    Finally, the more sophisticated approach to enzymatic digestion renders hydrolysates more amenable to sterile filtration, allowing hydrolysate end users to experience streamlined media preparation and bioreactor supplementation activities.

    UltraPep™ Soy, the first commercial product manufactured using this process, was released earlier this year.

  • Experimental Results

    Click Image To Enlarge +
    Figure 1

    Experimental data shows four significant benefits of UltraPep Soy hydrolysates: elevated cell densities, prolonged cell viability, enhanced target protein production when compared with nonsupplemented media controls and media supplemented with traditionally manufactured hydrolysates, and greater consistency among lots.

    The experiments employed a transfected CHO-K1 line, engineered to constitutively express secreted embryonic alkaline phosphatase (SEAP). Monolayer cultures were grown in six-well microplates containing a final medium volume of 3 mL/well. The basal medium consisted of 50% CDM and 50% Ham’s F12-K, 1 mg/mL G-418, supplemented with 5% FBS. Cultures were seeded at 2x105 cells/well and incubated at 37°C in 5% CO2. Data was collected after five days in Sheffield Products’ standard assay.

    Four separate lots of UltraPep Soy were used for the experiment. These were manufactured at pilot-plant scale on equipment closely resembling the full-scale manufacturing line. To investigate consistency, soy raw materials were dual sourced and two different lots from each supplier were employed, as well as three different lots of enzymes.

    The dose response (Figure 1) shows that UltraPep Soy leads to increased cell densities and sustained cell viability. After seven days, SEAP in the UltraPep Soy-supplemented medium surpassed the control medium. Furthermore in contrast to control soy hydrolysate, UltraPep Soy in elevated doses exhibited significant increases in SEAP irrespective of cell density as supplementation enables sustained cell viability over time.

    The control soy hydrolysate supplemented cultures exhibit a rapid loss of viability (to 40% or less) after five days at 10 g/L dosage (Figure 2A). Whereas the same dosage of UltraPep Soy maintains cell viability at 75% or above for at least 12 days. Results also suggest that at elevated dosages (20 g/L), traditionally manufactured hydrolysates can also improve cell viability (Figure 2B).

    In Figure 3, the performance of all four pilot lots are compared as a percentage of a control soy hydrolysate supplemented control and clearly demonstrate improvements in both the functionality of cultures supplemented with UltraPep Soy and the lot-to-lot consistency of the four pilot lots.

    Finally, tests showed a high level of compositional consistency among the four pilot lots of UltraPep Soy (Figures 4A and B), which is especially notable since the production process drew upon such a diverse range of ingredient sources. It’s also important to note that UltraPep Soy is ultrafiltered, ensuring endotoxin content and, since most peptides are <10 kD, it is considered by most to meet the protein-free claim.

    In conclusion, UltraPep Soy and future products manufactured using this approach clearly represent a new generation of hydrolysates for the cell culture market.

  • Click Image To Enlarge +
    Figure 2a
  • Click Image To Enlarge +
    Figure 2b
  • Click Image To Enlarge +
    Figure 3
  • Click Image To Enlarge +
    Figure 4a
  • Click Image To Enlarge +
    Figure 4b


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