Riding the WAVE to the STAR; To Infinity and Beyond

• Speaker: Nicole Abello, Seagen
• Abstract: Bioassays are an important component of CMC regulatory compliance and require cell banks with low lot-to-lot variability to deliver high-quality potency data. Frozen ready-to-use (FRTU) cells offer many benefits including reduced assay variability; however manual banking of FRTU cells is time consuming, labor intensive, prone to variation, and places constraints on bank sizes. Additionally, accelerated CMC timelines, including multiple bioassays for release as well as stability and characterization testing, are driving the need to improve operational efficiency and turnaround times without compromising on data quality.To overcome the limitations of manual cell banking, we have implemented a streamlined semi-automated suspension/adherent cell banking workflow that uses WAVE bioreactors (suspension)/dissolvable microcarriers (DMC) (adherent), a Hamilton STAR liquid handler, an automated tube labeler with barcoding capabilities, and a controlled-rate freezer (CRF).The business impact of this workflow is significant. The workflow increases the throughput of the cell banking process, generating >1,000-vial high-quality, homogenous cell banks. These banks provide reproducible assay performance and are used across both development and QC labs. The added benefits of this workflow are avoiding the need to continually requalify small banks for purity and function, reduced cost, labor and ergonomic burden.
• (Day 3: Automating Bioassays Session)

Pseudoparticle Expression and Characterization of SARS-CoV-2 Wuhan Strain and Its Variants

• Speaker: Martine Boyer, Nexelis
• Abstract: Reliable assays to measure neutralizing antibodies induction following vaccination is required to address the humoral response of SARS-CoV-2 coronavirus and its variants. Given the highly infectious and pathogenic nature of this coronavirus, the use of replication-restricted pseudoviruses bearing the viral spike glycoprotein (S) represents a safe and useful surrogate.
  In this talk, we describe our efforts to optimize and characterize the production of SARS-CoV-2 Wuhan Spike-bearing pseudotyped virus. We highlight our ability to generate large scale SARS-COV-2 pseudotyped virus and associated variants for pseudotyped neutralization assays (PNA) through all clinical trial phases. Our team honed the pseudotyped generation process and established a rigorous bridging plan (biophysical and immunogenicity) to ensure critical lot-to-lot consistency while avoiding any potential drift/shift with our PNA assay. In addition, we implemented a long-term stability plan to ensure reliability in the PNA results.
• (Day 2: Bioassays for COVID-19 Therapeutics Session)

Quantitative Cell-Based Assays for Anti-SARS-CoV-2 Therapeutic and Vaccine Development

• Speaker: Jey Cheng, Promega
• Abstract: SARS-CoV-2 is a newly emerged coronavirus responsible for current COVID-19 pandemic. Existing in vitro methods used for antiviral drug testing rely on live viruses or pseudotyped viruses which have high biosafety requirements, high assay variation and take days to complete. Here we report the development of two rapid, quantitative cell-based SARS-CoV-2 assays to quantitatively measure neutralizing activity and Fc effector function for SARS-CoV-2 antibody. The SARS-CoV-2 HiBiT-PsVLP assay is sensitive, fast, can be conducted under low biosafety level (BSL1). It can measure differential neutralizing activities of therapeutic antibodies to SARS-CoV-2 HiBiT-PsVLPs carrying different spike variants (e.g., Alpha, Beta, Delta). Secondly, we developed ADCC and ADCP bioassays using engineered target cells stably expressing spike protein and showed positive Fc function for anti-SARS-CoV-2 S antibody in both reporter-based ADCC/ADCP assays and PBMC ADCC assay. These bioluminescent cell-based SARS-CoV-2 assays can be valuable tools for measuring the antiviral activities for therapeutic antibodies and also potentially for the antibodies derived from patient’s samples after vaccine administration.
• (Day 2: Bioassays for COVID-19 Therapeutics Session)

Resetting Expectations for CAR-T Potency Assessment

• Speaker: Avinash Chinthala, Precision BioSciences
• Abstract: Potency assays are an essential aspect of analyses of biotherapeutics, including CAR-T therapeutics. Further, the potency assay format that will eventually go on to be used as a release assay for CTM (in a QC environment) will need to be as accurate, precise, and robust as possible. Here, we present an improved approach to a CAR-T potency measurement while providing rationale to, and data for, the transition from currently used interpolation-based cytokine induction assays, with minimal number of dilutions (one), to full-curve analyses based upon MOA-reflective cellular responses. This method uses engineered reporter cell lines that express the target antigen (for PBI’s Allogeneic CAR-T products) on the surface and co-express an enzyme complementation fusion protein (called HiBiT) intracellularly. The HiBiT fusion protein, on binding with its complementary half (LgBiT) present in the detection reagent, forms a functioning nano-luciferase. Co-culture of the CAR-T cells with their respective engineered target cell lines activates the CAR-T effector cells in a TCR independent manner to induce membrane perforation and granzyme mediated apoptosis of the engaged target cells. The lysis of the target cells releases the HiBiT fusion protein into the well, which on addition of the LgBiT and a detection substrate, emits a luminescent signal, directly proportional to the number of cells lysed in the well. Collectively, we demonstrate the successful development and pre-validation of an assay that directly quantifies the cell killing ability of our CAR-T cells. The analysis based on complete dose-response curves results in a greater degree of confidence in the measurement of the cell-killing ability of the CAR-T products, and the relative potency approach with respect to a reference standard better aligns with regulatory guidelines regarding potency assays capable of supporting GMP QC release testing for late stage clinical and commercial products.
• (Day 2: Bioassays for Novel Cell-Based Therapeutics Session)

Switch from an In-Vivo to an In-Vitro Potency Test for QC Market Batch Release

• Speaker: Aurora Dadone, Merck
• Abstract: Saizen/Serostim Merck drugs currently require in vivo bioassays for batch release. Since more than 20 years animal tests are routinely applied to ensure the release on the market of such drugs
  Currently, the bio-identity test is performed for finished products according to the in vivo method described in the “Somatropin bioidentity tests” USP<126>. The method is based on body weight gain in rats, and it foresees 2 weeks for animal acclimatation and 10 days of sample administration. The high consumption of animals coupled with the mission to improve the analytical testing panel with state-of-the art methods has motivated the Company Team to initiate the project to replace the in vivo method by the in vitro one recently included as an alternative in the General Chapter USP<126>. An in-vitro method based on the stimulation of a rat cell line with hGH and measuring cell proliferation as a direct indicator of cell growth.
  Merck Scientists started working on this assay to evaluate the feasibility and the applicability of this test in our labs. However, some rooms for improvement were identified in the compendial procedure, and an evaluation started on the possibility of improving the method. Direct application of an assay already published in a pharmacopeia requires less work in terms of method validation and potentially an easier approach to the Health Authorities for the approval. However. the benefits of the improved method were estimated to have a final overall better chance of obtaining the final replacement of the in-vivo test and this route was followed.
  Consequently a few but relevant modifications were introduced to improve the performance of the method together with an assessment of its robustness. The improved in vitro assay was discussed in the Type C meeting with FDA in December 2017.
  Upon endorsement, the method validation strategy has been identified, associated to data collection and comparability. a method. The validation package is strictly in line with the most recent Health Authority requirements and includes Specificity, Linearity, Accuracy and Precision. The following comparability study was executed, testing release and stability batches in parallel with existing in vivo bio-identity test.
  The outcome of the study demonstrated the comparability among the in vivo and in vitro approaches, providing the green light for the submission of the method variation to FDA and Canadian Health Authority that was performed in January 2021 which was finally approved in June 2021.
  The outcome of submission is a small but relevant brick of the sustainability path leading to animal-free scientific environment during drug development, at least at some steps. Such ethical approach combines the animal welfare to the relevant quality standard mandatory to guarantee the efficacy and safety of the marketed drugs.
• (Day 1: Stages in the Life-Cycle of Bioassays Session)

Statistical Analysis of Laboratory Data. Or: Bioassayists, Statisticians, Working Together

• Co-Speakers: Stan Deming, Statistical Designs; David LeBlond, CMCStats; Tim Schofield, CMC Sciences
• Abstract: Co-Authors Stan Deming, Tim Schofield & David LeBlond tell the true story of an analytical chemist (Stan, a devotee of statistics to better understand his analytical results) who for many years had wanted to do something with statistical confidence intervals applied to bioassay practitioners: relative potencies that he had never seen done before. He was frustrated that he could never get a statistician to talk with him about this approach. Finally, a biochemist (David, who became formally trained in statistics) looked at it and said, “No problem. We Bayesians do this sort of thing all the time, but from a different perspective than the frequentist approach you’ve been using. If we make a couple of assumptions, you’re pretty much good to go.” Another experienced statistician (Tim, always a trouble-maker), watching from a distance, said, “Well, I’m not surprised. Just one more example of the marvelous things that can be done when researchers better understand statistics and statisticians better understand the research objective.”
• (Day 4: Ins and Outs of Calculating Reportable Values Session)

R We Nearly Square Yet?

• Speaker: Brooke Franklin, Quantics Biostatistics
• Abstract: At BEBPA Europe 2021 the survey asked: Do you use R2 as a suitability criterion in your 4PL analyses?
  57% responded “No” – we agree and this year we will explain why! Hopefully helping the 43% explore the issues too.
  At Quantics we always encourage the use of the most appropriate statistical methods. R2 can tell us about the strength of the relationship between doses and responses… but does it tell us about the shape of our data?
  Join us to discover why R2 is not the best suitability criterion for goodness of model fit as we explore what R2 does and doesn’t tell us about different dose response models.
• (Day 4: Ins and Outs of Calculating Reportable Values Session)

Assays for CAR-T Cells

• Speaker: Ulrike Herbrand, CRL
• Abstract: T cells engineered with chimeric antigen receptors (CARs) have revolutionized the field of cell therapy. Allogeneic CAR-T cells are being explored for off-the-shelf therapy.
  A bioactivity test shall be used to determine whether a CAR-T cell product has the expected therapeutic ability, which is closely related to the efficacy of the product. The mechanism of CAR-T cell therapy is manifold and complex.
  However, a typical approach to monitor the function of antigen-specific T cells is the evaluation of the killing ability of CAR-T cell products to target antigen-positive tumor cells and the release of specific cytokines like IFN in vitro. In addition, specific activation markers like CD25 and CD95 can be evaluated by flow cytometry.
  In a case study the bioactivity determination for a CAR-T model targeting a solid tumor surface antigen will be presented.
• (Day 2: Bioassays for Novel Cell-Based Therapeutics Session)

A Modular Approach to Automating Relative Potency Assays (and Everything Else!)

• Speaker: Sean Johnston, Biogen
• Abstract: The development of monoclonal antibody (mAb) therapeutics requires the fast turnaround of an array of analytical readouts that drive decision-making during process development. While automated assays have been established to reduce turnaround time and standardize results generation, each successfully deployed script incurs development time and increases the burden of support. Despite the high complexity of these methods, most assay workflows can be broken down into a series of simple tasks. Through the development of a non-linear script and the modularization of assay steps, these simple tasks can be accomplished using generic input-driven blocks of code, which can be linked together to form complex procedures. The result is a single automated script that promises to address a vast majority of analytical preparations, providing a flexible and efficient answer to some major challenges associated with results generation for process development.
• (Day 3: Automating Bioassays Session)

Steps Towards Strategic Control of Potency Bias Caused by Allowed Non-Similarity

• Speaker: David Lansky, Precision Bioassay Inc.
• Abstract: Small amounts of some types of non-similarity cause appreciable potency bias. Precision, sample size, and power considerations (or more simply the typical width of similarity confidence intervals) lead to a strategic approach: equivalence bounds based on bias limits with assay size based on precision and power needs for similarity. This approach has important implications for assay design and analysis. This talk will illustrate these issues with examples.
• (Day 4: Ins and Outs of Calculating Reportable Values Session)

The Interchangeability of Various Software Used for the Bioassay Analysis and Potency Result Calculation

• Speaker: Natasha Leshinsky, Omeros
• Abstract: In the current global economic environment, it is often required to perform testing for a commercial product in multiple countries utilizing various contract testing organizations (CTO). Achieving alignment of the results from these different testing sites requires attention to establishing similar approaches for test performance and result analyses across these organizations. One element that often differs between CTOs is the use of different software for the calculation of the dose-response curves of the bioassay runs needed for obtaining the final reportable value.
  This talk offers an overview of the challenges associated with the harmonization of data analysis for a potency assay across various CTOs. We will discuss the alignment of the data analysis strategy for a potency assay (4PL parameters’ ratios, % RP calculation, and outlier tests), the differences and limitations to the data analysis associated with the use of the various analysis software or different versions of a software (PLA, SMP, Gen5), and will offer for your consideration what we see as the simplest way to harmonize the data analysis for a potency test around the globe.
• (Day 4: Ins and Outs of Calculating Reportable Values Session)

Strategy for the Potency Assessment of a Monoclonal Antibody: Development, Harmonization, and Comparison of Three Binding and One Cell-Based Potency Assays

• Speaker: Adriana Rimesso, Biogen
• Abstract: Summary: Our drug MOA is complex, and its physiological target is known to have variants. In order to achieve thorough drug characterization and strengthen our regulatory filing, we developed three binding assays. In one binding assay the drug binds to the canonical protein form, and in the two other assays it binds to known pathogenic variants of the target protein. We also developed a technically challenging cell-based assay. We harmonized, qualified, and correlated all methods, using our correlation study to select a release assay.Methods:
  DELFIA-based competitive immunoassays evaluating drug binding to full-length, truncated and fibrillized target protein. One cell-based assay evaluating drug-mediated inhibition of cellular pathogenic fibril seeding.Results:
  Qualification yielded 102% accuracy and 8% RSD for full-length target protein binding, 99% accuracy and 7% RSD for truncated target protein binding, 99% accuracy and 5% RSD for fibrillized target protein binding and 103% accuracy and 9 %RSD for fibril seeding inhibition assay. A cross-comparison of stressed and unstressed sample results from the three binding methods showed the assays were equally sensitive. Stability and chemically stressed sample evaluation by the seeding assay showed inferior sensitivity to potency changes, although the seeding and binding methods trended together.Conclusions:
  We show that drug binding to full-length target protein is representative of binding to truncated and fibrillized protein and is more sensitive and accurate than the cellular fibril seeding assay. Based on the results, full-length protein binding was chosen as the release activity assay.
• (Day 1: Stages in the Life-Cycle of Bioassays Session)

Development of an iLite® Reporter Cell Platform for the Quantification of Anti-AAV Neutralizing Antibodies

• Speaker: Jordi Rodó, Svar Life Science AB
• Abstract: The efficacy of gene therapies using recombinant adeno-associated viral (AAV) vectors is limited by the presence of pre-existing humoral immunity against AAV as a consequence of prior exposure to the virus, often in early childhood, to one or more AAV serotypes. The existence of anti-AAV antibodies, which can be observed in up to 70% of the population for some serotypes, negatively impact clinical efficacy as it can both drastically reduce AAV transduction and enhance adverse events. Thus, the development of sensitive and precise cell-based in vitro assays for the quantification of anti-AAV neutralizing antibodies (NAbs) is necessary for a clinical environment to achieve early clinical proof-of-concept and to ensure safe and successful results.
  Here we present a novel two-component system for the detection and quantification of NAbs directed against recombinant AAV vectors, the iLite® AAV Platform. The platform comprises a packing cell line for the production of the relevant AAV serotype and a reporter-gene cell line incorporating a reporter gene-promoter construct that responds specifically to the AAV transduction. This platform can be used for detecting and optimally quantitating anti-AAV NAbs directed against the capsid in a test sample. Furthermore, the availability of reporter cells in a frozen, thaw & use format, obliviates the need for cell culture or specialized equipment and provides a means for obtaining highly reproducible results superior to those obtained using the same cells maintained in culture. Moreover, the time necessary to run the assay is half of the time required in the current alternatives.
  These features combined allow the advantages of a cell-based assay to quantify neutralizing anti-AAV antibodies, as recommended by regulatory agencies, to be combined with the versatility and ease of an immune-detection assay for use in AAV-based gene therapy trials.
• Contributing Authors: Jordi Rodó, Lone Frier Bovin, Benoit Vallette, Christophe Lallemand, Michael Tovey, Katarina Håkansson
• (Day 3: Potency Assay Method Development: Case Studies Session)

Managing Variability from Probabilistic Viral Infection in Bioassays that Detect Viral Replication

• Speaker: Alex Rodriguez, PPD
• Abstract: Assays to measure infectious titer (infectivity) and replication competency (RCA) are common tests for viral drug products. Both assays involve the use of viral products at lower concentrations, where infection becomes a stochastic event. One format of infectivity assay involves infecting cells in replicates with increasingly dilute doses of virus. The number of infected wells at each dilution level is then used to calculate the tissue culture infectious dose where there is a 50% chance of infecting cells (TCID50). Many potency assays have system suitability requirements where a general curve shape is well characterized, and measures of curve fit quality can reject bad curves. For infectivity assays, there is an expected pattern that fewer wells should be infected as the virus becomes more dilute. However, because of the probabilistic nature of infection at lower concentrations, it is occasionally possible for more dilute virus to show more infected wells than a more concentrated dose. Due to the limited number of replicates allowable per dilution in a 96-well assay format, it should be carefully considered whether these unexpected patterns should be accepted as endemic to the assay and within normal variability, or instead rejected as testing failures.
  RCA assays generally follow a scheme where virus is added to cells and then subsequent infections are performed with the supernatant or lysate of the previous infection. If the virus is replicating, then the amount of virus will increase with each subsequent infection. If it is not replicating, then the amount of virus detected will decrease. Detection limit is a key parameter of RCA assays, as it must be clear what amount of replication competent virus can be detected in a sample. A replicating wild-type virus is often used as a positive control to ensure replication can be detected. However, as the positive control virus is diluted further to demonstrate a lower detection limit, unlike most potency assays where the result will also decrease, the result for RCA becomes probabilistic. A positive control diluted too far may infect sometimes and not others on repeated tests. When it infects and replicates, even single infection events could be amplified to show an increase in viral concentration, similar to the mechanism observed in the infectivity assay, leading to an unreliable detection limit that may not be noticed if the positive control is not tested repeatedly. If used at a dilution where infection is probabilistic, failures of the positive control could incorrectly be deemed system suitability failures. Further, this would mean that the claimed detection limit may not be accurate, as replication competent virus present at the same concentration in the sample may not always infect. For example, if the positive control has an 80% chance of infection over repeated testing, this also implies that the detection limit is only true 80% of the time, leaving a chance that replication competent virus is present in a concentration above the detection limit but is not detected. Because of the random nature of infection and detection of replication when using low concentration virus, repeated testing during development is an important step to determine the reliability of infection.
• (Day 3: Potency Assay Method Development: Case Studies Session)

Principles and Practices for Reference Standards

• Speaker: Tim Schofield, CMC Sciences LLC
• Abstract: Reference standards are essential to the development and control of biological products. USP recommends reporting potency of test articles relative to a reference standard, while some laboratories use these also or instead as a control. Considering their importance, however, there is no consensus on the source of a reference standard, the basis and means of reference standard qualification and stability evaluation, or the use of a primary standard. This talk will discuss principles and practices related to reference standards used to report potency of biological products and propose strategies for their acquisition and evaluation. Those proposals will borrow from practices related to quality by design for analytical methods, highlighting fitness-for-use of a references standard as well as reduction of uncertainty and the decision risks associated with their uses during development and quality control.
• (Day 1: Stages in the Life-Cycle of Bioassays Session)

Method Transfer and Validation at Multiple Sites

• Speaker: Celine Vongsouvanh, Allakos
• Abstract: Bioassay method transfer from the Sponsor laboratory to the CMO laboratory has many challenging aspects that need to be considered. Criteria for transfer must be rigorously applied to ensure that method is not inappropriately designed or loosely transferred. The main goal is to demonstrate that the method is appropriately transferred and validated at the receiving laboratory on time, and the method shows acceptable robustness to be ran in many countries by many analysts. In this talk, we will outline key success factors to have a successful validation which resides on the work done before and during the transfer; this includes coordination of critical reagent shipments, understanding of the procedure and the differences between laboratories, flexibility in potential optimization and pre-validation data.
• (Day 1: Stages in the Life-Cycle of Bioassays Session)