The 8th Annual Bioassays and Bioanalytical Method Development conference, organized by the Institute for International Research (IIR) in Sarasota, FL, attracted more than 100 scientists to the classic Claremont Hotel in Berkeley, CA, October 1–3, 2012. Dr. Mark Ma, Director of Preclinical Research at Amgen (Thousand Oaks, CA), started the meeting with a quick overview of the goal of the symposium: 1) incorporating the latest technological advances in analytical biochemistry into the work flow; 2) developing, validating, and managing robust bioassays, particularly for potency and stability; 3) method transfer of complex cell-based assays (CBAs); 4) critical reagent identification and management; and 5) development of biosimilars.
A bioassay (BA) measures the effects of a substance on living matter. Bioassays often appear to be empirical, but for very large materials such as antibodies, our state of knowledge and technology is insufficient to establish a mass/response relationship (mg/pill) as is practiced with small-molecule drugs with a defined molecular formula. BAs are essential in the development of biotherapeutics and especially biosimilars. Further, the FDA insists that assays for potency and stability of a biotherapeutic be similar in Mode of Action (MoA) to the intended biological process.
In the past, BAs involved whole animals as targets, but due to costs, response time, and ethical standards, animal testing has been replaced by live cells or small model organisms such as zebrafish embryos. Cells are fickle, though, and so are cell-based assays.
Interest in biosimilars is high, but there are many high hurdles, especially in the U.S. Dr. Jingyi Xiang (Eureka Therapeutics, Emeryville, CA) compared the pricing of generic small-molecule drugs to biotherapeutics. The models for price competition are quite different. Small-molecule generics see an 80% decline in price during the five years post-introduction. While the data are scarce for biosimilars, he believes the price model shows only a 20% price reduction during the first five years. Part of this is due to differences in treatment plans. Generics may be taken for weeks to years, but the Rx plan for biotherapeutics is usually less than five doses, and is often only one.
Development costs are another factor. Because of the complexity of the drug, the development costs are much higher. A related presentation from Merck KGaA (Darmstadt, Germany) estimated that biotherapeutics cost 20 times more than a comparable small-molecule drug to develop.
Another high hurdle is that there is little harmonization of the regulatory process for biosimilars between the U.S. and Europe. Indeed, the American requirements are complicated and onerous. Thus, the regulatory and business models for generic small-molecule drugs and biotherapeutics are quite different. Forecasts of significant price reductions from biosimilars seem quite optimistic.
Need for better bioassays
Despite this economic analysis, many developers still show interest in biosimilars. The interchangeability requirement mandates that the dose be the same as the original. This means that one needs accurate potency assays, preferably in vitro (i.e., a bioassay). This effort will probably continue, according to Dr. Zuben E. Sauna of the Division of Hematology, CBER, FDA, who reports that the FDA anticipates that improved assays will be developed and refined during the lifetime of the product.
Merck reports that “[t]here is a critical need for increasingly accurate and precise nonclinical, in vitro assays for measuring drug potency, as these are the cornerstone of quality control of manufactured therapeutics. A recent survey showed that 32% of drugmakers declared that innovations in assay technology were required to meet the demands of proving biosimilarity. Such assays can better determine lot-to-lot variability in the manufactured product, assess the impact of process changes on drug quality, assess drug stability, and more. Therefore, increasing the precision of an assay improves the assay’s statistical power, facilitating the comparison between biosimilars and innovators.”1
More than half of the new biotherapeutics in the regulatory pipeline are based on antibodies, which are potentially immunogenic. Immune responses are varied and impossible to predict. A complete lack of immune response across a population is not likely or expected, however.
Since some immune response is expected, the FDA advises that: “A true comparison of immunogenicity across different products in the same class can best be obtained by conducting head-to-head patient trials using a standardized assay that has equivalent sensitivity and specificity for both products.”2 In 2012, the following was added: “The proposed product and reference product should be assessed in the same assay with the same patient sera whenever possible.”3
Immunogenicity of biosimilars
Avoiding expensive clinical trials was the pivotal assumption of early business models on biosimilars. The passages above show that this is probably not the case, especially for antibody candidates. The cost and time required for immune testing are probably less than are required for a new immunotherapeutic, but are still significant.
In the technical part of his lecture, Dr. Jingyi Xiang described Eureka’s approach to developing a biosimilar (called ET801) to alemtuzumab. The reference drug is indicated for specific leukemias. It was withdrawn from the market in September 2012 to prevent its off-label use in treating multiple sclerosis.
Dr. Xiang explained that biosimilars are “highly similar to the reference product, notwithstanding minor differences in clinically inactive components.” Further, “there are no clinically meaningful differences between the biological products and the reference product in terms of the safety, purity, and the potency of the product.” Others might add that the dosing, administration, and package insert need to be the same.
Since ET801 is an antibody product, similarity of the Fc and Fab binding needs to be demonstrated. The first step was to compare in vitro tests for binding to the target antigen. Small differences are expected. These were observed between different batches of the reference product and also between ET801 and commercial lots of alemtazumab. Many tests were run, including surface plasmon resonance (SPR) studies with various ligand probes to compare receptor binding, bioassays for antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Differences in glycosylation were found with high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and correlated with activity.
ET801 was produced with Eureka’s proprietary human antibody expression vector. The antibody was characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, peptide mapping, glyco-profile, endotoxin content, etc. Potency was measured in vitro with a novel whole blood killing assay that provides direct visualization of apoptosis. The MoA is similar to in vivo MoA of the reference drugs.
Interlaboratory method transfer appears to be the highest hurdle for laboratories. It is a difficult process that cannot be avoided, since the complexity of registration and production of biotherapeutics involve many functions spread over several locations. Until an organization has experience with method transfer, the difficulty is always underestimated. However, the case studies sound like a compelling mystery that only lab rats would appreciate.
Several lectures provided anecdotal reports on the basics:
- Water quality, which can vary from lab to lab
- Pipetting protocols and individual technique
- Biobanking of samples, including history of access and return
- Protocols for freezing and thawing samples, cells, and critical reagents
- Managing critical reagents (vendor selection to disposal)
- Other tribal knowledge and practices
- Maintaining proficiency.
Method transfer case study
Method transfer is often the most problem-prone laboratory activity. This is especially true for cell-based assays, since they usually have several critical reagents coupled with ill-defined manual techniques. Dr. Laura Geagan, Bioanalytical Development at Genzyme (Cambridge, MA), presented a case history of method transfer of a cell-based potency assay for a therapeutic protein to three new sites: two abroad and one domestic.
First, the assay was validated in accordance with ICH Q2B guidelines. This included assay design, which mimicked the expected mechanism of action of the drug product on the cells. The cell line was evaluated for stability and growth. Master and working cell banks were created, stored, and documented. Assay performance was evaluated for accuracy, specificity, dynamic range reproducibility, robustness, and purpose (potency, stability, identity, etc.). Acceptance criteria were established and performance verified.
The method transfer exercise started with a detailed plan that described the purpose, basis of assay design, and transfer plan guidelines. Other topics included training, surveillance, reporting, critical reagents including master and working cell bank, instruments, and data analysis and acceptance criteria for the receiving labs.
Staff from the receiving labs were trained in the originating laboratory. Multiple analysts were involved in each receiving laboratory. Post-training, all labs experienced failing assays that appeared to be operator-dependent. A thorough investigation found and solved problems with cell culture technique, cell thawing technique, unstable detection reagents, and instrument response. The debugging process was very interesting because the focus on detail was essential to discovering unanticipated contributing factors.
Credits and comments
The symposium organizers deserve special thanks for choosing lecturers that could talk from experience. The Claremont provided warm creature comforts that promoted networking.
However, many lecturers did not release copies of their slides. This is apparently a consequence of the American process on biosimilars that requires demonstration of equivalency by in vitro methods. Thus, analytical methods might be useful to the follow-on firm in demonstrating equivalency, since they could cite similar results using the innovator’s method. In the absence of such a reference point, the comparison is harder to make and defend since the innovator can claim the method and the results supplied by the follow-on firm are different than theirs, and irrelevant. My Philosophy of Science course taught that failure to see a difference between two different samples might be due to using a method that is blind to the differences, or the samples are indeed identical. This is just one example of how the American process on biosimilars has been stacked in favor of the innovator.
- Effective In Vitro Bioanalytical Assays for Comparing Pharmacodynamics of Biosimilar Monoclonal Antibodies; http://www.millipore.com/techpublications/tech1/an4475en00.
- FDA Draft Guidance on Biosimilars, 2009; http://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/TherapeuticBiologicApplications/Biosimilars/default.htm.
- FDA Draft Guidance on Biosimilars, 2012; http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm291232.htm?source=govdelivery.
Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.