Editor’s Note: At the time of this recording. Dr. Ultee was with Gallus Biopharmaceuticals; however, he has since started his own consulting company, Ulteemit Bioconsulting, where he is employed full time.
Advances in protein production, including biomaterials and cell culture, have resulted in new methods for bioprocessing that can increase both yield and quality as well as decrease cost and time. Trisha Gladd, editor of Pharmaceutical Online and Bioprocess Online, spoke with Dr. Mike Ultee, Chief Scientific Officer at Gallus Biopharmaceuticals, about not just these new options but also the future of bioprocessing.
Trisha Gladd: Hello, this is Trisha Gladd, editor of BioProcess Online and I want to welcome you to our podcast today with Dr. Mike Ultee, Chief Scientific Officer at Gallus Biopharmaceuticals. Welcome Dr. Ultee, and thank you for being with us today.
Dr. Mike Ultee: Thank you for inviting me. Happy to be here.
TG: Today we’re going to talk about flexibility in bioprocessing. Can you explain to me what that means?
MU: Sure, Trisha. Flexibility means choices; options that one can take to produce any particular therapeutic protein. Traditionally, these types of proteins were made in large, stainless-steel tanks by production cells grown in cell culture media. Following harvest of the bioreactor mixture and removal of the cells, the protein of interest is purified using a series of glass and steel chromatography columns containing resins suitable for protein purification. In the last decade, however, thanks to advances in many areas, including biomaterials and cell culture, many new options have opened up to these traditional methods. These provide choices for the bioprocessor in developing a robust process to increase both yields and quality, and at the same time, do so with less cost and time.
TG: Why is this important?
MU: Biopharmaceuticals are complex, large protein molecules that require sophisticated methods to produce them from living cells. Typically, they are 100 to 1000 times larger than a small-molecule drug like aspirin or Lipitor. There are typically many routes that one can take to produce a protein therapeutic, including type of production cell line, bioreactor, and purification process. Allowing options at each stage enhances the chances for success.
TG:OK, let’s start with the cell line. What options are available there?
MU: Well, that first depends on whether or not your protein has what are called “Post-Translational Modifications,” or sometimes abbreviated “PTMs”. What this means is whether or not the protein composed only of chains of amino acids, or are some of these modified to include other structures, such as carbohydrates. Simpler proteins, such as Interleukin-2 or IL-2, have no PTMs. These can be made most efficiently in bacteria production cells, such as those from E. Coli. Bacterial cells grow quickly and produce a lot of protein. But they cannot do any PTMs – their cells are incapable of this.
For that, one needs to go to more complex systems, such as mammalian production cells. These allow full addition of complex carbohydrate, and these are the system of choice for most glycoproteins, such as monoclonal antibodies. This class of proteins, often abbreviated as “mAbs”, represented the dominant and largest group of biopharmaceutical therapeutics. HerceptinÒ, RituxanÒ, and HumiraÒ are well-known examples of these drugs, and are all block-buster drugs. Looking then at mammalian-cell systems, there are several types, the best known being CHO cells. These originated long ago from a Chinese hamster ovary. These cells are robust production cells that have been extensively used to produce mAbs and other therapeutic proteins.
Now there's an alternative type of mammalian cell line that has been growing in popularity and that's the human cell line called PER.C6. It has the advantage producing purely human PTMs, while CHO cells can produce a slightly different version that hamsters have. At Gallus, we have had extensive experience with both CHO and PER.C6 cell lines. Finally, there are also production cells in between bacterial and mammalian systems, such as yeast and green plants. These have advantages and disadvantages of their own.
TG: What about flexibility in bioreactors?
MU: Here, flexibility can take many forms. While the steel-tank bioreactors are commonplace in the biopharmaceutical industry, their high capital costs and fixed sizes make them less flexible than the newer, single-use models. Furthermore, the steel tanks required extensive and validated cleaning between runs using ample quantities of cleaning agents. These would include water for injection, which is expensive to produce. Single-use technology avoids this concern by beginning with a clean, sterile bag for each run. The changeover time is thereby also minimized.
Even stainless steel, however, can be made more flexible, through the use of quick connect/disconnect fittings using polymeric lines, and by making the steel tanks movable on wheels. Finally, there is a wide range of options for the types of mixing that bioreactors employ, from the traditional impeller stirring in a cylindrical tank, to rocker or Wave-type of mixing, to paddle and rotating drum designs. Each of these bioreactor designs offer different advantages and disadvantages, giving the bioprocessor more flexibility.
Additionally, there is flexibility as to whether the bioreactor is run in fed-batch mode, or perfusion mode. In fed-batch, cells are grown in a final large tank and they're fed until they begin to loose viability, which is typically after about two weeks. Then the bioreactor is harvested. Perfusion, on the other hand, has the bioreactor culture media, containing the product, removed either continuously or daily and replaced with fresh media. This goes on for much longer periods than fed batch – typically 30-90 days for a perfusion process. Each technique has its advantages and disadvantages. Fed-batch is the industry standard for large-scale production of stable proteins, such as monoclonal antibodies, but some companies choose to make these proteins via perfusion mode. Perfusion is better for less stable proteins, such as enzymes, since the protein is not retained for a long time in the bioreactor. Also, perfusion is amenable to continuous processing, which can be an advantage.
TG: How about downstream or purification flexibility?
MU: It used to be that one dealt with a mix of column chromatography and sizing filtration to purify protein therapeutics. The columns, typically packed in place with a limited number of resins, were made from some combination of glass, acrylic, and stainless steel. Thus, flexibility was limited. Today, thanks to advances in downstream processing, there are a host of different types of chromatographic resins to choose from. In the standard types of affinity, ion-exchange, and hydrophobic interaction modes, there are now multiple choices for each. The newest types allow faster processing, more bioavailability for very large proteins, greater capacity, and varied selectivity.
There are also new modes combining more than one modality. For example, by combining ion-exchange and hydrophobic interaction into one resin, producing what is called a mixed-mode support, allows one to exploit the power of both modes in one operation.
Beyond columns, membranes have come a long way in developing as high-throughput choices for protein purification. Now we have available multiple types of ion-exchange membranes for protein purification, including newer salt-tolerant types and hybrid membrane-gel types that have increased capacity. Such membranes allow high-flow rates, speeding processes, and are ready to use after simple flushing and equilibration. Columns are now also available in pre-packed, or “plug and play,” formats that save time and labor. All of these downstream advances provide much needed flexibility in designing efficient processes.
TG: Where do you see the future in bioprocessing?
MU: Flexibility is the name of the game as bioprocessing continues to tackle new and more challenging proteins. Eventually, I foresee smaller scale, fully-integrated upstream and downstream operations relying more and more on single-use technologies to speed the processes. We will be looking to achieve even higher productivity from our production cells, allowing for smaller production lines. On the downstream side, higher capacities, more powerful separation steps, and vertically-integrated downstream processes, again relying on single-use technologies, will purify proteins efficiently in a single pass from clarified bioreactor harvest through to final bulk drug substance.
Such a “factory of the future” is moving from concept to reality with systems, such as the flex-factory, developed by Xcellerex and now provided by GE Healthcare. If continuous purification process were also coupled to perfusion bioreactors, such processes would allow continuous production of the therapeutic protein from cell culture through to finished drug substance.
TG: That’s very interesting, Dr. Ultee. Thank you so much. Unfortunately, that’s all the time we have for today. I’d like to thank Dr. Ultee for being with us. I’m Trisha Gladd. Thank you for listening to this BioProcess Online podcast.