Downstream Processing

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The “Recovery and Purification” track began many years ago as a conference of its own. In October 2004, IBC Life Sciences brought it together in Boston, MA, with three other events (“Cell Culture and Upstream Processing,” “Production and Economics,” and “Scaling Up from Bench to Clinic”) to create the program of the first BPI Conference and Exhibition.

Just like BPI magazine’s editors, the event producers have depended on industry advisors since the beginning. Our editorial advisory board (EAB) members give generously of their time and expertise to help us keep the magazine content relevant, trustworthy, and forward-looking. Similarly, each events’ group of scientific advisors assist the producers in bringing together the best presenters and covering the most timely topics each year.

Some of our EAB members have been with us from the beginning. And some experts have come back to help IBC Life Sciences for two or more yearly events. For the “Recovery and Purification” track, those have included

  • Joanne Beck (Shire HGT)

  • David Kahn (GlaxoSmithKline)

  • Duncan Low (Amgen)

  • Jill Myers (BioPro Consulting)

  • Uwe Gottschalk (Sartorius Stedim Biotech)

  • Charles Schmelzer (Genentech/Roche)

  • Peter Wojciechowski (Baxter).

People such as these — as well as loyal readers and attendees, authors and presenters, advertisers and exhibitors — have been key to our success. We couldn’t do what we do without you!

What’s Different: By 2006, downstream process engineers were already facing the challenge that has characterized this past decade in recovery and purification. “Recent improvements in mammalian cell culture processes and process development have led to significant increases in product titers,” wrote David Kahn in that year’s conference guide. “These large increases in product mass delivered from the upstream process pose a large challenge for downstream processing, at both the harvest and subsequent purification steps.”

The so-called downstream bottleneck has yet to be fully ameliorated. Companies have considered many possible solutions: new chromatography resins and methods; precipitation, crystallization, extraction, and other less familiar techniques; monoliths and membranes and other novel approaches to the chromatography concept. At the same time, everyone wanted to lower the costs of production and processing — especially with biosimilar competition looming on the horizon. Early BPI Conference programs included vendors of single-use technology demonstrating the potential benefits of using disposables in downstream processing. Later on, early adopters presented their results. And now, users are sharing challenges and solutions in the implementation of single-use technology.

What Remains the Same: Although many alternatives have been considered, none has yet displaced chromatography and filtration as our main downstream workhorses. But as Peter Wojciechowski told us back in 2008’s conference guide, “Good science is the mechanism by which the conflicts between business and compliance can be reconciled.” This is true in every high-technology industry — no less so in bioprocessing, where good science is the basis of good process engineering.

Quality by design (QbD) grows out of that “good-science” approach. As more companies set out on the road of QbD implementation, they are finding ways to model their downstream processes — whether in silico, at small scale with or without laboratory automation, or all of these — to create a design space and determine the sources of potential problems as early as possible. The more information you can gather about your product and process, the better you can define that space. For example, proteins are stable within certain temperatures and pH ranges, and chromatographic chemistries depend on salt and buffer concentrations as well as time and other parameters. Taken together, all those specifications help create the “space” within which a process can best perform.

Process analytical technology (PAT) was already in discussion at the first BPI Conference. But it was the subject of more questions than answers. Now, 10 years later, presenters and attendees are offering real, practical ways to achieve process monitoring and control.

Where Do We Go from Here? In addition to downstream process modeling, monitoring, and control, discussions of potential alternatives to chromatography (and protein A affinity, in particular) continue. Some companies are even investigating the idea of continuous processing, knocking down the walls between upstream and downstream, and making “batches” a thing of the past. High-density cell cultures may complicate matters, but good scientists and engineers are hard at work on solving those problems and more. The BPI Conference is where they can come together and compare notes.

As always, this event is all about the people involved in it and the work that they do. IBC’s Jennifer Pereira spoke with several presenters this past summer about their topics as well as experiences with the BPI Conference over the years. Here, in Q&A format, is what they had to say.

Lam Raga Anggara Markely (Biogen Idec)

Lam Raga Anggara Markely, PhD (a scientist in cell culture development for the high-throughput analytical group at Biogen Idec) will be joining us for the “Advances in Analytics and Models to Aid Downstream Process Development” session on Thursday afternoon, 19 September 2013. Lam’s case study is titled “High-Throughput, Small-Scale, Ion-Exchange Protein Purification,” and it contains unpublished data.

First, why did you choose to present at the BPI Conference? My supervisor, Shashi Prajapati, went to the same conference last year. She had a very good experience with the presentations and discussions with other scientists.

What general challenges do pharmaceutical companies face in modern downstream process development? To my knowledge, since I was in graduate school at the Massachusetts Institute of Technology (MIT) working with Professor Daniel I.C. Wang, there has been an unsolved issue on how to purify highly concentrated cell culture samples.

Does quality by design (QbD) help, or does it complicate matters? If the root cause of the problem is a limitation in the binding capacity of a chromatography resin, then quality by design may not help much.

Do you use design of experiments in your work and, if so, how do you handle the associated biostatistics? Does Biogen Idec have statisticians on staff? And do you see this as an increasingly vital part of process development? I don’t use DoE for my work, but I have to do a lot of statistical analysis. I work in the high-throughput analytical group, and we develop a lot of high-throughput analytical assays. When we develop an assay, we need to analyze its limit of quantitation (LoQ), limit of detection (LoD), accuracy, precision, day-to-day variability, person-to-person variability, and well-to-well variability. So we do analytical analysis for those studies. We don’t have biostatisticians in process development. My colleagues who use DoE attend some training to learn how to use it.

Recovery and Purifcation Sessions

Tuesday, 17 September 2013

8:00–9:45 AM  New Paradigm: Continuous Processing in Biomanufacturing

10:15–11:45 AM  Nonchromatographic Methods: Revisiting the Past?

11:45 AM–1:30 PM  Concurrent Technology Workshops

1:45–3:15 PM  Point–Counterpoint Discussion: Capture Options in Antibody Purification — Current Limitations, Emerging Options, and Obstacles to Change

Wednesday, 18 September 2013

8:00 AM&#8
211;12:00 PM  Purification Advances Using Mixed-Mode Chromatography (hosted by Bio-Rad Laboratories)

12:00–12:30 PM  Concurrent Technology Workshops

1:45–3:30 PM  Purification Strategies for Novel Modalities and High-Density Cell Cultures

Thursday, 19 September 2013

8:00–10:15 AM  Innovation at the Interface of Upstream and Downstream Processing

10:15 AM–3:15 PM  Expanding the Tool Box: Novel Purification Methods and Single-Use Applications (sponsored by Life Technologies)

12:00–12:30 PM  Concurrent Technology Workshops

3:45–5:00 PM  Advances in Analytics and Models to Aid Downstream Process Development

What are some challenges in ion-exchange (IEX) chromatography? What parameters are best to consider in optimization? The main challenge comes when the samples contain impurities that strongly interact with the IEX resin through ionic interaction. If impurities bind to the resin through the same ionic interaction as the product, then it will be difficult to separate the product from those impurities. There also may be some impurities that bind strongly to the product through ionic interaction, thereby disrupting interactions between the product and resin. As a result, the purification yield will be low.

Based on our experience in this study, salt concentration is one of the key parameters for optimization. At low salt concentrations, charged molecules will have strong interactions with the resin; at high salt concentrations, those interactions will be weak. By adjusting salt concentrations, you can optimize the process to maximize yield and minimize impurities in your purified samples.

Can you describe why it is important to develop a high-throughput, small-scale protein (HT-SSP) purification? It is important for product quality analysis during cell line and process development. In cell line development, we need to screen hundreds of clones to select certain ones that can produce our product at a certain level with certain product quality attributes. Similarly, in process development, you need to optimize your process to produce the product at a certain level with certain product quality attributes. To support these product quality analyses, we need to purify many samples in parallel in a short time. So we develop high-throughput protein purification for this purpose.

Can you briefly describe your development work and the new process itself? The SSP aims to purify a basic protein in the presence of polyanionic compound supplemented to the cell culture to improve its performance. This is a two-step purification in a 96-well plate format. The first step removes the polyanionic compound using anion-exchange Q Sepharose Fast Flow resin in flow-through mode. The polyanionic compound binds to the QFF resin, and the product is collected in the flow-through fraction. The second step purifies the product from host-cell proteins and other impurities in the QFF flow-through. This is achieved using a cation-exchange SP Sepharose XL resin.

We started by developing the second step. We optimized the SPXL purification conditions to purify the product from cell culture samples without the polyanionic compound present. Then we developed the first step, for which we tested several resins that can be used to bind the polyanionic compound but not the product. After we optimized the first step, we combined both steps into a two-step HTP-SSP process.

What were the main challenges in your assay development? There were two main challenges. First, the SPXL purification did not perform as well as we expected. From our studies, we found that the washing condition was the important part that we need to optimize. We adjusted the salt concentration to minimize impurities and maximize the yield.

Second, the polyanionic compound supplementation significantly decreases the SPXL purification yield because the compound strongly interacts with the product. That disrupts interactions between the product and resin. There could be several approaches to solve such a problem. The idea that came to my mind was to use a resin that could capture the polyanionic compound, but not the product. So we can perform the SPXL purification without a significant decrease in yield. From our studies, we found that under certain conditions QFF resin can be used for that purpose. That is why two steps were required for purification of this product with the polyanionic compound present. Without it, SPXL itself would have been sufficient.

What other types of resin did you try, and how did they perform? We tested Capto Q resin, and it worked well. However, it is more expensive than QFF. We also tested DEAE FF and Capto DEAE resins. They didn’t work well under the conditions that we tried, probably because the pH 9 that we used was close to the upper limit of their buffering capacity.

What are the benefits and limitations of this approach? First, it’s high-throughput. We can purify about 72 samples/day manually. If you integrate it with robotic automation, you could increase the throughput to about 288 samples/day. Second, this approach doesn’t affect product quality attributes. That is very important because we need to study the differences in product quality attributes for different clones and process conditions. The limitation is that the process needs to be reoptimized if the concentration of the polyanionic compound is increased.

Have you attended or presented at past BPI Conferences? What has been your experience? I’ve never been to a BPI Conference, but I’ve been to some other IBC Conferences, and they have been very informative. I had the chance to learn about the latest technologies and projects that other people are working on.

Finally, what are you most looking forward to at this year’s event? I look forward to getting feedback from others on our work as well as learning more about the latest technologies and challenges in the biotechnology field.

Gregory Zarbis-Papastoitsis (Eleven Biotherapeutics)

Gregory Zarbis-Papastoitsis, PhD (senior director of protein production and analytical development at Eleven Biotherapeutics) will be joining us for the “Purification Strategies for Novel Modalities and High-Density Cell Cultures” session on Wednesday afternoon, 18 September 2013. His presentation is titled, “Downstream Strategies for High-Density Cell Cultures.”

Why did you choose to present at the BPI Conference? This is the premier conference to present cases of industrial process development and manufacturing. It attracts process scientists and engineers as well as regulatory and academic people involved in the biotechnology and pharmaceutical industry.

What makes high-density cell culture worth the trouble and challenges involved? The industry as a whole has been moving toward intensified processes, in which the goal is to maximize product titer and minimize the cost of goods. Efforts have been made to increase cell-specific productivity and maximize cell density during fermentation. Increasing those cell densities created significant challenges for harvest and clarification, whereas high titers also challenge the downstream part of the process — especially in relation to low-capacity conventional chromatography resins.

What enabling technologies help companies use that strategy? The use of flocculation during harvest clarification and improvements in chromatography captures help us expand absorption. High-capacity chromatography resins and membranes result in process improvements.

What are the primary downstream challenges of high-density cell culture process streams? High fouling streams for filters and centrifuges; high-product loss and potential product damage; low-capacity resins that result in either big columns or the need for many cycles; expensive processes that challenge the intent of
the intensified processes, which is to lower the cost of goods.

Lately we hear about companies bringing upstream and downstream groups together for more and better communication. Can you describe some experiences you’ve had with this trend? I can witness the communication and efforts of the two disciplines — upstream and downstream — becoming more effective over the past 10–15 years to ensure desired product quality and process efficiency. Understanding the nature of the challenge in communicating the right approach to deal with these issues is now an established practice.

Have you attended or presented at previous BPI Conferences? What has been your experience over the years? I have previously presented at BPI Conferences myself, as have colleagues from groups that I have directed. I do remember not so long ago — in the early 2000s — finding myself in a debate about the utility of single-use technologies in biotechnology manufacturing processes. And years later now, of course, this is not up for debate anymore.

What are you most looking forward to at this year’s event? To see the new trends and advances in technologies that biologics manufacturing will be using in years to come.

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