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Sep 1, 2014 (Vol. 34, No. 15)

Therapeutic Proteins, Made Better

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    Protein Sciences’ Flublok represents a new class of influenza vaccine, called RIV3 (recombinant hemagglutinin influenza vaccine, trivalent formulation). It is produced in insect cell culture, not in chicken eggs.

    To boost productivity in the manufacture of therapeutic proteins, one may try improving specific production elements—cell lines, processes, or any of the ancillary tools related to transfection. One may even try improving purification, which not only impacts quality but also contributes to overall productivity.

    Producers of non-monoclonal antibody therapeutics are beginning to appreciate the benefits of platform processes that obviate the need to re-invent the wheel constantly. A group at Protein Sciences led by Nikolai Khramtsov, Ph.D., has reported on a universal process for expressing and purifying influenza recombinant hemagglutinins (rHAs), components of the company’s commercial Flublok® vaccine, at different scales without the need to redevelop the process for new rHAs. Apparently the company’s manufacturing approach, which occurs in insect cell expression systems, works for all seasonal and pandemic rHAs.

    Access to a platform production method is significant, since the rHAs change every year. By expressing these antigens in insect cells, Protein Sciences avoids the legacy egg-based production method and streamlines manufacturing.

    “rHA sequences change all the time, but our process of expression and purification remains the same,” Dr. Khramtsov tells GEN.

    Flublok is only the fourth FDA-approved product manufactured in insect cells. The cells are transiently transfected using a proprietary baculovirus expression vector.

    At present, Protein Sciences employs a simple batch strategy; however, the company plans to implement its recently patented fed-batch system, which Dr. Khramtsov says will be similar to plasmid-based mammalian fed-batch processes. Scales are typically in the 600–2,500 L range, with titers at mg/L levels. One Protein Sciences customer has scaled the baculovirus process to 20,000 L.

  • Getting Genes Where They Need to Be

    MaxCyte has been successful at using flow electroporation to streamline development of biotherapeutics. Electroporation involves subjecting cells to an electric field that temporarily permeabilizes cell membranes, allowing external materials such as genes to enter cells. The company has used electroporation to express genes transiently in insect, CHO, and approximately 70 other cell lines, providing high yields of antibodies, antibody-like molecules, and vaccines.

    The technique easily transfects 10 billion cells—a quantity sufficient to produce up to low gram quantities of recombinant protein. “Traditionally, stable pools or stable clones must be generated to produce comparable quantities,” says James Brady, Ph.D., director of technical applications. “By eliminating the need to generate stable pools or clones, our process significantly shortens development timelines.”

    MaxCyte produces and sells three instruments for various cell types and quantities.
    Flow electroporation yields much higher levels of transfection efficiency and post-transfection viability relative to other transient transfection methods. The differences are especially pronounced with CHO cells, which are challenging to transfect with conventional technology. Due to the efficiency of transient expression, stable or targeted integration is not required to generate high protein titers with the MaxCyte process.

    “We and our customers have obtained titers exceeding 1 g/L following transfection of CHO cells with human IgG1 expression plasmids,” Dr. Brady continues. A single-flow electroporation with MaxCyte’s STX instrument provides enough cells for up to 4 L of high-density cell culture, enabling production of up to 4 g of protein from a single transfection. Tenfold higher levels of protein can be generated from a single electroporation with the company’s VLX transfection system.

  • Design of Experiment

    Hanuman Mallubhotla, Ph.D., research director for biopharmaceutical development at Syngene International, has achieved excellent results in work to improve antibody protein production through a statistical design-of-experiment (DoE) approach in mammalian cell fed-batch processes.

    Dr. Mallubhotla applied this strategy using JMP, a statistical software package available from SAS Institute, to evaluate 15 basal and 7 feed media while controlling for feed rate and temperature. He optimized media and feed based on statistically observed interactions and amino acid/metabolite profiles. The result was a titer increase from 0.5 g/L in shake flasks to more than 3.0 g/L in bioreactors. Investigators collected samples and analyzed for glucose, lactate, titer, and amino acid content.

    “Optimization of the cell culture processes usually happens as an afterthought,” Dr. Mallubhotla observes. “Most companies are under severe pressure to produce the material under aggressive deadlines. We accomplished a step-by-step DOE methodology to optimize the cell culture process before going into manufacturing.”

    Organizations that build this methodology into its standard development plans will be capable of developing a cell culture processes for antibodies and perhaps for other molecules, quickly and predictably, Dr. Mallubhotla adds. “It will be like an assembly line.”

    Companies have attempted to reach platform development processes, but each requires revision and rework to accommodate molecular differences. Through this process—perhaps system is a better term—Syngene provides its customers with clinical and commercial material while shortening time-to-market. “We are talking about a principle that can applied to many situations, as opposed to an application platform itself,” Dr. Mallubhotla says.

    A good deal has been discussed regarding scaledown methods involving small, parallel bioreactors. In April 2014, contract manufacturer Gallus Pharmaceuticals officially adopted the ambr15™ microbioreactor from TAP Biosystems, a Sartorius Stedim Biotech business unit.

    “We use the ambr15 to perform many types of screening and response surface designs,” says Matthew Zustiak, Ph.D., principal scientist, cell culture development, Gallus. Optimizations include media and feed screening, feed strategy optimization, feed timing, feed quantity, and process parameters such as pH and temperature.

    According to Dr. Zustiak, ambr’s main strength is its ability to run a 48-vessel, fed-batch study that closely mimics conditions inside a larger-scale bioreactor, and to perform large studies examining multiple process variables in a single device. “The ambr excels at individual control of the feed supplementation or pH control strategy since each vessel has independent control.”


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