Complex Protein Development: Assay Early, Assay Often

By Matthew Pillar, Editor, Bioprocess Online

Just days before our August Bioprocess Online Live event Early Workflow Considerations For Novel Protein Therapeutics, I was thrilled to learn that Lena Tholen, Ph.D. would be joining my panel, which included SOTIO Head of CMC Meinhard Hasslacher, Ph.D. and Vera Therapeutics SVP, Product Development and Manufacturing Neeraj Pakala, Ph.D. Dr. Tholen is Director of Cell Line and Bioprocess Development at FyoniBio, and her contributions to the discussion were clutch, given her deep and broad experience in cell line development. That experience juxtaposed quite nicely with that of Drs. Hasslacher and Pakala, offering our audience unique perspectives on how process decisions made very early on can influence – beneficially or detrimentally – the efficiency of upstream and even downstream operations in the development of novel protein therapeutics. Those novel therapeutics include the fusion proteins Vera is developing and the even more complex ADCs (antibody-drug conjugates) SOTIO is working on.

Selecting cell lines for those novel therapeutics, says Tholen, is not as simple as choosing a CHO and running with it. She says it’s best to evaluate multiple human and CHO cell lines, as early as the pool stage, for the attributes that best contribute to your desired protein. “This early decision can have such a high impact on your final GMP production process, so it's better to spend a little bit more money in this early stage than it is to get into a clinical stage or reproduction process and then need to go back because your critical parameters are not fitting well,” she says.  

Dr. Hasslacher offered a specific example of Tholen’s point from the ADC world. His experience with post-translational modifications of the antibody element in ADCs, he says, indicates that they require stricter specifications than those required for simple mAbs. “In ADCs, you want to strictly avoid the uptake of such molecules by healthy cells,” he says. As such, he suggests that early assessment of the content of afucosylated glycans, for instance, should be a critical quality attribute considered at the cell line selection stage. “In ADCs, the conjugation step is unique unit operation not seen in other biologics. You have to concentrate on this high-cost step to reach high yields and high DAR (drug-antibody ratio) values. Typically, DOE approaches are applied to get there early on,” he says.

Regulatory Scrutiny Awaits

Dr. Pakala is currently feeling the impact of early process decisions made in years gone by. Vera acquired atacicept, a fusion protein developed some 15 years ago, long after its initial quality attributes were determined. Now in a phase III study in IgAN (immunoglobulin A nephropathy ) on the heels of successful data in phase IIb, he says the molecule’s productivity is not optimal, and he recognizes opportunities for further optimization in its downstream process related to the complexity of the molecule. “If you have a really nice DOE and you run through an extensive product quality analysis, even during the clone selection process, you can gain a lot of understanding of how robust later-stage processes can be,” says Pakala. “Seek to understand the failure modes, whether it's cell culture or understanding how impurities need to be addressed, so that downstream processes and even the control strategy are set up in a robust way. The way we understand the CQAs now, our control strategy could be slightly different. Some of the decisions made 10 years ago are really affecting us now.”

They say hindsight’s 20/20, but those days-gone-by decisions are putting a bit of pressure on Pakala and his team as it enters the later clinical phases and becomes subject to increased regulatory scrutiny. “My recommendation to any company that’s trying to make later-stage changes is to get in alignment with the regulatory bodies, whether FDA or EMA, to make sure you’re in good shape. We’re now going to the regulators to get the alignment and develop the approaches to bridge pre- and post-change processes.”

Specific Attributes To Monitor Early

At the request of an audience member, Dr. Tholen outlined some of the key attributes to monitor during the complex protein therapeutic cell line development process. While acknowledging specifics depend on the particular product being developed, she first stressed the importance of aggregation. “I would definitely screen for aggregation at the pool level, as well as glycosylation, particularly in ADCs,” she says. “This will give you a direct look at the conjugation efficiency, which is important because post-translational modification can definitely influence conjugation efficiency.”

She also suggests that complex proteins should be subject to extensive binding assays at the pool level, and that a panel of aggregation, glycosylation, and binding assays should again be conducted at the single-cell and clone levels. “With novel technologies, you will see differences at the clone level,” she says. “With transferase systems integrated into single-cell cloning, you will get more homogenous pools, but there will be differences.” As such, she suggests screening a lot of clones. “Don’t just screen 24 clones, broaden that approach to find the one that best fits your requirements.”

Making Early Go/No-Go Decisions

Asked what criteria is used to make the Go/No Go decision for novel and difficult-to-produce complex proteins, all three of my panelists brought enthusiastic responses. Dr. Hasslacher stressed the importance of conducting a late-stage developability study with SOTIO’s CDMO prior to getting into IND development. “We produce the molecule in a stable pool of clones, and then typically apply thermal, sheer, Ph, and light stresses to see how the molecule behaves and whether it’s robust or extremely sensitive,” he says. That data determines whether to move into clinical process development or to continue discovery work to improve the molecule’s design. “If there’s a strong propensity to protease degradation, we recognize that this would be hard to fix without adding a lot of protease inhibitors during the entire manufacturing process,” he says. “See the risk, evaluate the risk, and if necessary, go back to the molecule design.”

The discussion on shelf life-limiting factors led Dr. Tholen back to her commitment to early formulation and product stability studies. “You can do these at the pool production level and begin to determine formulation and product stability,” she says. Dr. Pakala agrees, adding that understanding shelf life-limiting factors like light sensitivity, aggregation, and degradation is key to the decision to move forward. “If you determine that you’re not going to get even a year of shelf life, that’s a huge problem,” he says. Pakala also leans back into productivity. “If your productivity is so low that you’ll have to go into a 40,000-liter to get an amount of drug, I think you need to pause and consider what you’re looking at from a cost of goods standpoint.”

On that point, Tholen shared an anecdote from her unique perch overseeing the development of novel protein candidates from several sponsor companies. “In the past year, more often than not, I’ve seen companies that already have a product in phase II clinical studies coming back to us when they’re ready to jump into phase III, and they’re realizing their productivity is still too low for commercial viability,” she says. She suggests spending more time and money on USP development early, which will not only increase productivity, but improve product quality as well.

Throughout our 60-minute-plus discussion, Drs. Tholen, Hasslacher, and Pakala entertained these and many more early process development consideration questions from their peer attendees. While it’s too late for you to ask a question of this particular panel of experts, the conversation in its entirety is available here to watch on-demand. And, if analytical methods are in your wheelhouse, be sure to keep an eye out for a Bioprocess Online Live event coming this December 3, focused on analytical method development and transfer in the cell therapy space.