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February 19, 2015

Smart Process Development Strategies

Making the Challenge of Manufacturing Proteins a Commercial Reality

Smart Process Development Strategies

CellVessel single-use customizable benchtop bioreactor from CerCell.

  • Since a large proportion of the cost of goods (COGs) of a biologic is determined during the drug design phase, it is critically important to design the molecule from a manufacturing point of view and compensate for heterogeneity right from the start.

    That’s the view of Emma Harding, Ph.D., head of molecular chemistry, manufacturing, and controls (process research) at Glaxo-Smith Kline, who spoke at Knowledge Transfer Network’s recent Process Development for the Manufacturing of Challenging Proteins meeting.

    However eliminating variance is not a simple task, as Andrew Kaja, senior scientist at GSK, pointed out.

    “When producing biologics, cells are not predictable and don’t always generate product and express it in the supernatant at a specific time,” he explained.

    Even when the protein is being expressed there can still be problems, which can occur at any stage of upstream and downstream bioprocessing.

    “Proteins can ‘break bad’ by forming aggregates, precipitating out or co-purifying with undesirable proteins, all of which can affect the yields and efficiency of your biomanufacturing process,” added Richard Tran, Ph.D., principal scientist at GSK.

    These issues can make manufacturing protein-based therapeutics within a commercially viable timeframe challenging but much can be overcome with considered approaches.

  • Finishing School for Biopharms

    According to Dr Harding, GSK process research scientists perform a mini Quality by Design (QbD) assessment, known as a Molecular Design Intent (MDI) process on promising leads before molecules are taken through bioprocess scaleup. They use an in silico analysis to check for hot spots, including glycosylation and deamidation and to identify potential solubility and aggregation issues and predict how this might affect the pharmacodynamics, pharmacokinetics, and potency of the molecule.

    Using this information, they rank their lead biological candidates as low, medium, or high risk for difficulty of manufacturing. When the candidates have been ranked, the scientists discuss if there are upstream or downstream process or genetic modification methods of eliminating those potential solubility and aggregation issues identified with the problem molecules.

    “Our MDI 1 process is like a finishing school for biopharms, they have to pass this before we can consider investing time in moving them on into bioprocess development,” said Dr. Harding.

    When molecules are being considered for scale-up, bioprocess scientists are faced with a number of options to optimize clone selection and process parameters to try to ensure that proteins will not present titer and formulation challenges. They can use the traditional shake flask and reusable benchtop bioreactors approach or rely on disposable versions of these to speed up the process. Increasingly, many are now utilizing fully automated micro bioreactor mimic systems.

    “The scale-down equipment you use to predict process conditions will have an impact on the type, quality, and quantity of data you can produce, so you have to choose according to the complexity of information you need,” cautioned Kaja.

    He then described how GSK is using a range of different technologies to solve its bioprocess scale-up questions. For reasons of simplicity, cost, and throughput, many initial screening projects use shake flasks and microtiter plates.

    “When we want to further characterize clone performance in these uncontrolled environments we use the RAMOS automated shake flask system,” he noted. “For example, when we wanted to find out the effects of different maximum oxygen transfer rates and of changing concentration of IPTG for induction had on one set of clones, we used this system with cultures induced with either 10 µM IPTG or 500 µM IPTG and with fill volumes of 10 mL and 40 mL, respectively.”

    From the automated independent gas analysis Kaja and his team were able to see that at the lower IPTG amount and in combination with a higher maximum oxygen transfer rate, the carbon source was depleted at 12 hours but at the higher amount of IPTG and with less oxygen available, the fermentation was maintained for longer and it was 25 hours before the carbon source was depleted.

    “Using the shake flasks allows us to look at a couple of parameters and pick out strain specific differences. Then we can move promising clones to an HTS system, and this gives us a better and more robust screening technique,” he said.

  • Zeroing in on Costs and Time

    For studies that require a larger design space than a shake flask, GSK is using the Pall Micro-24 MicroReactor system.

    “It is too expensive and time consuming to run 24 cultures in benchtop bioreactors,” explains Kaja. “Using this system we can control and monitor pH, DO, and temperature in each well, and we’ve found using this system that titers are akin to those we achieve in fermenters, whereas titers are lower in the shake flask system so the Micro-24 is better at mimicking our fermenters in an HTS manner.”

    Now the team uses this plate-based model for strain selection to rank host/vector combinations before fermentation development work takes place.

    For the next stage in process development, GSK is replacing its stainless steel benchtop capabilities with 250 mL single-use benchtop bioreactors. This allows more focus to be placed on expanding and exploring a repertoire of process parameters and quality attributes.

    “With the disposable bioreactor we can generate 60 models, which we layer and analyze to predict conditions for maximum titer and minimum aggregation during fermentation runs,” Kaja pointed out. “This helps us to determine the optimum time point to harvest the proteins so it is good for efficient process development.”

    It seems that single-use benchtop bioreactors are becoming a popular choice for process development as, according to Adeline Fanni, upstream process development scientist at Actavis, they reduce set up, preparation, cleaning, and sterilization time from six to ten hours down to two hours.

    “We’re looking at switching to single-use benchtop bioreactor because they come pre-assembled, there is less chance of the run failing due to incorrect set-up and since it is disposed of at the end of the experiment, this reduces the chance of cross contamination with material from a previous run,” Fanni explained.

    However, one issue she cited with many single-use bioreactors is that they are closed and not versatile systems.

    “There is not a wide choice of impellor or spargers and because of this many of these bioreactors will not perform well in perfusion high cell density fermentation where the oxygen transfer demand is high,” Fanni said.

    For their process requirements, Actavis scientists evaluated the specifications of three 1 L single-use bioreactors and chose the Cercell bioreactor to run comparative tests against a standard glass benchtop bioreactor.

    “We are using the Cercell CellVessel as it is customizable so we can have different impellors, spargers and sensors,” explained Fanni. “Our comparative results show that in a fed-batch culture of a mammalian cell line cultured in either the glass or single-use fermenters, the titer and cell density are comparable. We will continue to evaluate this single-use bioreactor for perfusion high cell density fermentation and will look to use this in process development to develop robust scale-up.”

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