Abstracts (Alphabetical by Speaker/Presenter)

Secure and Traceable Acoustic Liquid Handling — An Integrated System for Regulated Laboratory Environments

• Presenter: Russell Burge, Labcyte Inc.
• Abstract: The Labcyte Key to bioassay success is implementation of multifactorial optimization approaches such as Design of Experiment (DOE) and sample randomization to control for inherent experimental biases. Both methodologies benefit greatly from automated liquid handling solutions that are reproducible, accurate and enable any-well to any-well liquid transfers.
  Acoustic liquid handlers utilize sound energy for rapid, non-contact any-well to any-well liquid transfer thus making them ideally suited to the needs of bioassay development and deployment in pharmaceutical, biotech and CRO organizations. Data quality improvements over manual or automated tip based liquid handling systems are readily measurable at significantly lower transfer volumes. Lower transfer volumes lead to reagent savings and enable direct dilution for dose-response testing eliminating dilution error propagation. Compared to automated tip-based systems, the complex transfer protocols required for DOE and randomization approaches are readily accommodated on acoustic liquid handlers and non-contact dispensing removes any sample/tip interactions eliminating sample loss due to adhesion to or contamination from the tip plastic itself.
  Within the listed organizations, bioassays are often run in FDA regulated environments and, as such, benefit from instrumentation that supports user access control, protocol traceability and versioning, protocol approval enforcement and controlled reporting. A barrier to adoption of acoustic liquid handling for bioassay work has been the lack of such controls. Now, Echo® Liquid Handlers can meet FDA 21CFR Part 11 requirements. Newly available software is tightly integrated with Echo® systems and software applications for a seamless experience. We will provide details on how such acoustic liquid handlers can be deployed in regulated labs.
• Contributing Authors: Iain Russell, Russell Burge and Harry Vlahos
• (Poster)

Converting an Enzymatic Potency Assay to a Relative Potency Assay

• Speaker: Yeeling Chong, aTyr Pharma Inc.
• Abstract: (Pending)
• (Day 2: Bridging Assays Session)

Reproducible, MoA-reflecting Reporter Based Bioassays to Enable Drug Development of Biosimilars and Biobetters

• Speaker: Mei Cong, Promega
• Abstract: The human immune system is comprised of a complex network of immune checkpoint receptors that are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune-mediated disorders. Co-inhibitory immune checkpoint receptors play a critical role in maintaining immune homeostasis and are genetically and functionally associated with autoimmune disease, cancer and viral infections. Blockade of co-inhibitory receptors has emerged as a promising new approach to enhance anti-tumor responses. While immunotherapies directed against PD-1 and CTLA-4 are showing unprecedented efficacy in the treatment of cancer, some patients and tumor types remain refractory to these therapies. This has resulted in a broadening of immunotherapy research and development to include additional co-inhibitory receptors (e.g. LAG-3, TIGIT, Tim-3) targeted individually or in combination with other immune checkpoint receptors.
  A major challenge in the development of biologics is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable and fail to yield data quality required for drug development in a quality-controlled environment. To address this need, we have developed a suite of immune cell line-based bioluminescent reporter bioassays for individual and combination immune checkpoint immunotherapy targets including PD-1 (PD-L1 or PD-L2), CTLA-4, LAG-3, TIGIT, PD-1+TIGIT and more. These assays consist of stable cell lines that express luciferase reporters driven by specific response elements under the precise control of intracellular signals mediated by the TCR and co-inhibitory receptor target(s). These MOA-based bioassays are available in “thaw-and-use” format and demonstrate high specificity, sensitivity and reproducibility. The bioassays are pre-qualified according to ICH guidelines and demonstrate the performance required for use in antibody screening, potency testing and stability studies.
• Contributing Authors: Mei Cong, Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan and Zhi-jie Jey Cheng
• (Day 1: Bioassays for BioSimilars Session)

Does it or Doesn’t it? Only the Assay Control Samples Know for Sure

• Speakers: Stan Deming, Statistical Designs and Mike Sadick, Catalent
• Abstract: (Pending)
• (Day 1: Bioassays for Product Characterization Session)

Improved T Cell Activation Bioassays to Advance the Development of Bispecific Antibodies and Engineered T Cell Immunotherapies

• Presenter: Steven Edenson, Promega
• Abstract: T cells play a central role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings.
  To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.
• Contributing Authors: Steven Edenson, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong and Zhi-jie Jey Cheng
• (Poster)

Reference Materials, A Moving Target?

• Speaker: Cindy Grant, Janssen R&D
• Abstract: (Pending)
• (Day 1: Reference Materials Session)

Bioassay Basics – What We (Should) Do in the Lab and Why

• Instructors: Bassam Hallis, Public Health England and Jane Robinson, Consultant
• Abstract: (Bioassays differ in important ways from other analytical techniques, presenting challenges for those new to the field. All too often, experienced bioassayists forget to explain the reasons behind the “obvious” practices they take for granted. An additional problem for many novice bioassayists is that initially they are involved in only one step of the process of assay development, validation and routine usage: understanding the full process can help in recognizing issues that may impact subsequent steps or suggest where to look for previous evidence of a problem.This workshop will cover the basic principles of bioassays, showing how appropriate assay design and analysis are essential to obtain meaningful results. We will discuss assay variability, the concept of relative potency and its practical implications, use and development of reference standards, setting assay acceptance criteria, monitoring, trending, and validation, all the time highlighting common problems and ways of avoiding them. This is the opportunity for workshop participants to bring questions and their own bioassay problems to the discussion, and hopefully leave with solutions or useful suggestions)
• (Workshop1-AM)

The Role and Requirement of Bioassays in Assessing Analytical Similarity of Biosimilar Products to Reference Products

• Speaker: Xu-Rong Jiang, AstraZeneca BioVentures
• Abstract: (Pending)
• (Day 1: Bioassays for BioSimilars Session)

Bioassay for Characterization of Peptides

• Presenter: Sandhya Kashi, Merck
• Abstract: As therapeutic products, peptides occupy a niche between typical small molecule drugs and larger proteins. They feature a modular structure with amino acids linked by peptide bonds as base units. Cell-based Potency assay for peptide therapeutics is central to its characterization and provides the platform for product specification, analysis of product comparability, and stability. Determination of potency of the peptides is also required for regulatory submissions.
  This poster will focus on comparison of cell-based potency assays for measuring 3′-5′-Cyclic adenosine monophosphate (cAMP, cyclic AMP) using two different platforms, advantages and disadvantages of each assay. In vitro potencies of peptides are determined in Chinese hamster ovary (CHO) cells stably expressing target receptor A or target receptor B using either Meso Scale Discovery (MSD) platform which uses electro-chemiluminescence or Cisbio cAMP Dynamic 2 assay, which uses time-resolved fluorescence energy transfer technology.
  The MSD cyclic-AMP assay is a competitive immunoassay based on the displacement of a cAMP molecule carrying an electro-chemiluminescent label. In the absence of cAMP, a large proportion of the labeled cAMP is bound by an antibody on the surface of a disposable carbon electrode. Elevated concentrations of cAMP proportionally displace the labeled analog. When a potential is applied to the electrode, bound label produces light and a quantitative measure of cAMP is recorded.
  Cisbio’s HTRF® (homogeneous time-resolved fluorescence) is based on a fluorescence resonance energy transfer between a Europium cryptate (donor) and a second fluorescent label (acceptor). This is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the dye d2. The tracer binding is visualized by a Mab anti-cAMP labeled with Cryptate. The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
  Cisbio’s HTRF no-wash assay was preferred over MSD platform since it was less variable, robust reproducible. In addition, the no-wash assay approach of Cisbio’s HTRF assay saves time and is more readily implemented in a quality control laboratory.
• Contributing Authors: Sandhya Kashi and Kevin Gurney
  Merck, Bioprocess and Formulation Dept., Kenilworth. NJ 07033
• (Poster)

An MOA Reflective Bioassay for a Bi-Specific Immuno-Oncology Product

• Speaker: Hyun Jun Kim, AstraZeneca BioVentures
• Abstract: (Pending)
• (Day 2: Product Specific Bioassays Session)

Strategies for Functional Characterization of an IgG with Effector Function

• Speaker: Veerendra Koppolu, MedImmune
• Abstract: Biological potency assays are widely used in biopharmaceutical drug development and commercialization as indicators of biological activity, stability, and quality of biological products. Precision and accuracy in determining the potency are very important in biopharmaceutical drug testing and lot release. Bioassays, in general, are tedious and involve many time consuming steps leading to less sample throughput and more possibility of manual errors. During the past several years, the United States Pharmacopeia Convention has focused on public standards to modernize and expand the bioassay requirements in the United States Pharmacopeia (USP).
  In order to improve the throughput and eliminate the tedium of bioassays, automation is highly desired. Use of robotic liquid handling systems facilitates the automation of multiple steps in biological and analytical methods while maintaining great precision and accuracy. We have developed an automation module in Hamilton robotic system that is adaptable to several bioassays. The automation module is validated for cell-based reporter gene assays, cell based binding assays, and non-cell based binding assays. Our results demonstrate that both manual and automation methods meet the acceptable qualification criteria for repeatability, accuracy, and linearity, while automation shows equal or less overall variability. Automation has increased the throughput of sample testing and reduced the sample preparation time by 3 to 4-fold. The presentation will cover the need for bioassay automation, our automation approach, and advantages and challenges of bioassay automation.
• Contributing Authors: Veerendra Koppolu, Craig Lamison and Jon Borman
  MedImmune, Bioassay Development, Analytical Sciences, Gaithersburg, MD, USA, 20878
• (Day 2: New and Improved Technologies Session)

Hitting the Gas: Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Co-Stimulatory Immune Checkpoint Receptors

• Presenter: Gopal Krishnan, Promega
• Abstract: The human immune system is comprised of a complex network of immune checkpoint receptors that are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune-mediated disorders. Immunotherapies designed to block co-inhibitory receptors (e.g. PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of cancer. However, not all patients and tumor types respond to this approach. This has resulted in broadening of immunotherapy research programs to target additional co-inhibitory (e.g. LAG-3, TIGIT, Tim-3) and co-stimulatory (e.g. GITR, 4-1BB, OX40, CD40, CD27, HVEM/LIGHT) receptors individually and in combination.
  A major challenge in the development of biologics is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable and fail to yield data quality required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell line-based bioluminescent reporter bioassays for co-stimulatory immune checkpoint targets including GITR, 4-1BB, OX40, CD40, HVEM/LIGHT, CD27 and more. These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of intracellular signals mediated by each immune co-stimulatory receptor. These bioassays reflect mechanisms of action for the drug candidates designed for each co-stimulatory immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. In summary, these reporter-based bioassays can serve as powerful tools in immunotherapy drug development for antibody screening, potency testing and stability studies.
• Contributing Authors: Gopal Krishnan, Jun Wang, Michael Beck, Jamison Grailer, Jim Hartnett, Julia Gilden, Frank Fan, Mei Cong and Zhi-jie Jey Cheng
• (Poster)

Seamless Implementation of Qualified Bioassays from Development to QC Lot Release

• Speaker: Jane Lamerdin, Eurofins US
• Abstract: A quantitative and robust bioassay that is reflective of the MOA of the drug is a critical component of any development program. We have expanded our PathHunter® cell-based assay platform to provide simple bioassays for potency determination and stability testing of biological drugs. These quantitative and robust assays rely on the native biology of the relevant receptor, allowing developers to choose a readout that is truly reflective of the MOA of their drug. Importantly, these are homogeneous assays that use “thaw-and-use” cryopreserved cells and simple protocols to minimize assay variability compared to traditional assays, and are highly scalable and suitable for automation. We will share case studies from our large portfolio of qualified bioassays for several different biosimilar and immuno-oncology targets.
• (Day 2: New and Improved Technologies Session)

Combining Run Results to Calculate Reportable Values

• Speaker: Laureen Little, BEBPA
• Abstract: (Pending)
• (Day 2: Interactive Survey)

From Continuous to Thaw & Go….What’s in YOUR Bioassay

• Instructors: Mike Merges and Mike Sadick, Catalent
• Abstract: (Establishment of dependable master and working analytical cell banks (MACB and WACB, respectively) is an essential foundation of a strong and dependable bioassay program. The fact that there are so many different types of cell and cell lines, each one often with quite particular growth needs and characteristics (e.g., adherent vs. non-adherent), presents a number challenges. Additionally, some bioassays utilize cells immediately from thaw (Thaw&Go), while other bioassays use cells maintained in continuous culture, necessitating different approaches to cell bank generation. This workshop will address the requirements for developing and optimizing the generation, establishment and testing of analytical cell banks. Combining interactive discussions and case study presentations, we will address the strategies and equipment/instrumentation to generate consistent MACBs and WACBs. These include strategies for both Thaw&Go cell banks as well as continuous culture cell banks, for both adherent and non-adherent cells and cell lines.)
• (Workshop 1-PM)

A PLATFORM TECHNOLOGY FOR THE RAPID GENERATION OF ROBUST ANTI-IDIOTYPIC BINDERS FOR CLINICAL PK ASSAYS

• Presenter: Chris Miller, Avacta Life Sciences
• Abstract: Affimer® proteins are next-generation affinity scaffolds with great potential for the generation of both novel biotherapeutics and renewable research and diagnostics tools. Based on the Type 1 cystatin protein fold, the Affimer® scaffold has been engineered to accept peptide loops such that target-specific Affimer® proteins can be selected from highly diverse libraries of random loop sequences. Using phage display, it is possible to generate highly-specific anti-idiotypic Affimer binders using a 12 week development process. The resulting Affimer® binders can be used as reagents in typical pharmacokinetic and immune response assays, such as bridging assays or Fc detection assays, for characterisation of monoclonal biotherapeutics. We have exemplified this process with the development and characterisation of specific Affimer binders for Trastuzumab, anti-CTLA4, anti-CD20 and anti-TNFa antibodies, and analysed their performance via Fc detection sandwich assay. We have demonstrated that the scaffold has properties (including generation of fusion proteins, ease of production, speed to discovery, specificity and stability and low batch-to-batch variation) supportive for the development of critical reagents to underpin clinical monoclonal drug development programs.
• (Poster)

Development, Validation, and Implementation of an ELISA for Determining Potency of an Influenza Vaccine

• Speaker: Michael Murphy, Medicago
• Abstract: Medicago has developed a biomanufacturing platform that utilizes expression of recombinant proteins in the plant Nicotiana benthamiana. Our lead clinical product, quadrivalent seasonal influenza vaccine, consists of virus like particles (VLP) containing the influenza antigen hemagglutinin anchored in a plant lipid bilayer. Historically, the potency of seasonal influenza vaccine has been determined by the Single Radial Immunodiffusion (SRID) assay; however, this assay does not accurately predict the potency of the plant based seasonal influenza vaccine. Therefore, Medicago sought an alternative potency assay that was more predictive of immunogenicity. This objective led to the development of a strain specific Competitive ELISA potency assay that is stability indicating as shown by accelerated stability studies performed with Medicago’s vaccine and from an egg-based commercial vaccine. Upon transfer of the assay to the Quality Control organization, the assay was validated for potency determination of monovalent VLP Drug Substance and quadrivalent VLP Drug Product vaccine. Subsequently, the Competitive ELISA was successfully implemented for release, formulation, and stability testing of Medicago’s seasonal influenza vaccine.
• Contributing Authors: Dan Hastings and Michael Murphy
• (Day 2: Product Specific BioAssays Session)

Thaw-and-Use Target Cells Pre-Labeled with Calcein AM for Antibody Dependent Cell-Mediated Cytotoxicity Assays

• Speaker: Van Nguyen, Genentech
• Abstract: (Pending)
• (Day 2: New & Improved Technologies Session)

Introduction to Statistics for Potency Bioassays

• Instructors: Nancy Niemuth, Battelle and Ann Yellowlees, Quantics BioStatistics
• Abstract: (Pending)
• (Workshop 2)

Overcoming variability issues in cell-based neutralizing antibody assays by using modified data normalization procedures

• Presenter: Alena Nikolskaya, Intertek
• Abstract: High fluctuations in raw signals are a common problem for cell-based Neutralizing Antibody (NAb) assays. Even the slightest variations in cell culture conditions may result in considerable well-to-well and run-to-run variability. Additionally, the assay responses from individual test subjects are intrinsically variable due to the patient-specific matrix interferences. These variabilities considerably affect NAb assay cut point values leading to compromised assay sensitivity and affecting the detection of neutralizing antibodies.
  Traditional approach to decreasing run-to-run variability for cut point calculations involves raw signal normalization procedures relying on the assay responses from antibody-negative control samples and applying ether multiplicative or additive normalization factors. The compensation for patient-specific matrix interferences usually based on the so called Inhibition Ratio (IR) defined as the assay response from individual sample tested with the addition of the assay drug divided by the response from the same sample tested without the drug. As each sample is considered being normalized to itself, IR is used directly for the confirmatory cut point analysis.
  We propose a more thorough data modeling approach to define raw assay signal transformations that would provide the best possible correlation between the assay responses from the controls and individual patient samples across different assay runs. Additionally, we utilize modified subject-specific normalization procedures aimed to further decrease variability at the individual sample level. This is achieved by taking into account the dynamic ranges of each assay run to additionally compensate for inter-run differences.
  The feasibility of modified normalization approach was demonstrated by strong compensation for both inter-run and subject specific differences of individual sample responses leading to better separation of positive and negative assay responses and increased NAb assay sensitivity.
• (Poster)

Performance Characteristics of iLite® Cell-based Reporter Gene Assays: Case Study of iLite® IL-23 Assay Ready Cells

• Presenter: Frida Pauly, Euro Diagnostica
• Abstract: Biologic activity is an essential quality attribute for the assessment of the potency and stability of biological drugs, and the successful development of such biologics is dependent upon the establishment of validated and standardized assays that allow direct comparisons of the relative potency and stability of different batches. We have developed a high throughput assay platform, iLite®, based on the use of engineered reporter-gene cell lines that is applicable to most biologics and that allows the direct comparison of drug potency and stability in the same assay. Furthermore, since biological activity is measured, the same assay system can be used to determine the presence of neutralizing antibodies. Here, using our iLite® IL-23 Assay Ready Cells as an example, we show the robustness of our assay system, describing parameters such as accuracy, precision, and plate homogeneity, as well as assay characteristics such as EC50 and LLOQ.
• Contributing Authors: Elsa Grenmyr, Lone Frier-Bovin, Karin Blume, Anna Pramhed
  Euro Diagnostica, Lundavägen 151, 212 24 Malmö, Sweden
• (Poster)

Development of a Cell-Based Relative Potency Assay for Botulinum Neurotoxin-Based Drug Products

• Presenter: Timothy Piazza, BioSentinel, Inc.
• Abstract: Purpose: Potency testing for quantification of botulinum neurotoxin (BoNT)-containing drugs and substances for clinical and pharmaceutical purposes has historically been performed using the mouse LD50 bioassay. While the mouse bioassay offers excellent sensitivity, it is mired by low throughput, low precision, high failure rates, and animal welfare concerns. Cell-based assays (CBAs) can eliminate the use of animal-based methods for BoNT-detection while increasing throughput, accuracy, and precision. BioSentinel Inc. has developed a CBA for the detection of BoNTs. The BoCell® CBA is a valid replacement for mouse LD50 testing due to its ability to capture all cellular mechanisms required for BoNT-intoxication: cell receptor binding, light-chain membrane translocation, and target SNARE protein proteolysis.
  Methods: We developed the BoCell® CBA by stably expressing fluorescently-tagged SNAP-25, the natural substrate for BoNT serotypes A and E, into an immortalized neuronal cell-line. Next, we demonstrated dose-dependent BoCell reporter cleavage with BoNT holotoxin. The BoCell CBA was insensitive to recombinant BoNT light-chain. Further, competition assays using BoNT receptor-binding domain preparations rendered the CBA insensitive to BoNT holotoxin, indicating cell receptor binding, endocytosis, and translocation of the light chain were required for proteolysis of the BoCell reporter. We tested both Active Pharmaceutical Ingredient (API) and drug product (DP) in a parallel-line relative potency assay format using the BoCell CBA. We used PLA software to analyze data using both linear-regression and 4-parameter logistical fitting. System suitability testing was performed using F-test assessments of non-parallelism, significance of slope, and non-linearity with an = 0.95. Method linearity was tested using DP test samples with a nominal concentration range of 70-130% and method precision and accuracy was assessed over repeated method events.
  Conclusions: The BoCell CBA provided excellent precision and reproducibility with an overall study mean of 102.4% relative accuracy and a 7.0% coefficient of variation. We found that the BoCell CBA was extremely accurate over the range of potencies tested (70 -130%). Lastly, because the BoCell assay is performed in a 96-well, high-throughput format and does not require the use of animals, we believe it to be a suitable and preferred replacement for mouse LD50 testing during manufacturing and commercial release of BoNT-based drug products.
• Contributing Authors: Timothy M. Piazza, Theresa L. Geurs, Sara F. Hendricksen, Füsûn N. Zeytin and Ward C. Tucker
  BioSentinel, Inc. 505 South Rosa Rd, Suite 105, Madison, WI 53719; (608) 441-8172; info@biosentinelpharma.com
• (Poster)

Road Map from Mechanism of Action (MOA) to Reportable Results

• Presenter: Emma Reyes, Pfizer
• Abstract: (Pending)
• (Poster)

Bioassays: The Past, the Present and the Future

• Speaker: Jane Robinson, Consultant
• Abstract: In the nearly 20 years since the adoption of ICH Q6B, we have witnessed an increasing rate of developments in the field of bioassays for potency measurement, driven or facilitated by developments in biological medicinal products, understanding of product MOA, novel analytical methods and more powerful statistical tools for data analysis. Reviewing some of the experiences from the last few years can help in devising strategies for meeting current and emerging challenges.
• (Day 1: Opening Keynote Talk)

AR BioAssay© – Clinically-Enabled Live Cell Assay for Pan-Androgen Measurement

• Speaker: Fred Schaufele, Xcellassay
• Abstract: Living cells are exquisitely sensitive and accurate devises that constantly access their internal and external environment. However, they are rarely considered as tools for highly sensitive and reproducible measurements. Here we discuss a case study of a live cell assay, AR BioAssay that exhibits exquisite sensitivity, repeatability, accuracy, reproducibility, as well as inherent specificity, in measurement of androgenic compounds in human urine. We validated AR BioAssay against industry’s golden standard, mass-spectrometry, by conducting a limited clinical trial using urine from hypoandrogenic testosterone-supplemented men. This trial showed outstanding concordance between mass-spectrometry and AR BioAssay results, with correlation coefficient R2=0.98. Evaluation of the potency of AR BioAssay against a panel of 51 steroids showed outstanding specificity of AR BioAssay for intact androgens, either natural or synthetic. Unlike mass-spectrometry that can only measure a known compound in question, such as testosterone, AR BioAssay reports on the cumulative action of androgens that is of particular interest when assessing pan-androgenic status in children, women (in particular women diagnosed with polycystic ovary syndrome, PCOS), and prostate cancer patients. To our knowledge, this is the first example of highly accurate and reproducible clinically enabled live cell assay for measurement of biologically active compounds.
• (Day 2: New & Improved Technologies Session)

Development and Validation of Droplet Digital PCR assay for Viral Vectors Used in Cell and Gene Therapy

• Presenter: Andrew Schneider, MilliporeSigma
• Abstract: Droplet Digital PCR (ddPCR) is a powerful technology which combines the principles of standard PCR and QPCR to perform absolute quantitation of a test sample. Advantages of using Droplet Digital over conventional QPCR include greater accuracy and precision of quantitation, end-point instead of real-time analysis, higher signal to noise ratio for decreased inhibition, and the ability to quantitate without the use of a standard curve.
  Accurate quantitation of vector genome copies is critical for viral gene therapy products. Several variations of the method have been developed for accurate titration of the most commonly used Viral and Gene Therapy Vectors: Adenovirus, Adeno-associated virus (AAV), Retrovirus, and Lentivirus. To accommodate the variety of products existing in every viral vector category, primers/probe sets have been designed to target common regions present in every viral genome type. All assays have been optimized to reduce sample manipulation steps leading to ddPCR and are fully validated to demonstrate specificity, accuracy, intermediate precision, repeatability, linearity, range and limit of quantitation. The methods may be customized for specific vector applications. Combined with an automated platform, results may be obtained within 1 day allowing for important process development, manufacturing, and patient dosing decisions to be made with the most accurate data that the industry has to offer.
  As applications continue to grow with this emerging technology, additional offerings for copy number variation and to monitor viral integration for Lentivirus and Retrovirus applications are in development.
• Contributing Authors: Andrew Schneider, Ahae Woo, Marian McKee, and Bradley Hasson
  MilliporeSigma BioReliance® Biosafety Testing Services, Rockville, MD
• (Poster)

The Use of Surface Plasmon Resonance for Extended Biological Characterization

• Speaker: Jeongsup Shim, Genentech
• Abstract: (Pending)
• (Day 1: BioAssays for Product Characterization Session)

USP Bioassay Chapters: Changes Afoot

• Speaker: Robert Singer, R. Singer Consulting
• Abstract: The suite of USP bioassay chapters has now seasoned for a few years. The chapters have been generally well-received and broadly implemented. Some of the bloom is off the rose now, though, and attention is being directed to some small thorns along the stem. Work on the chapters has been initiated at the USP, and this presentation will address that.
• (Day 1: Pharmacopeia and the BioAssay Session)

Review of the New USP <1210>: Challenge and Opportunities

• Speaker: Perceval Sondag, Pharmalex
• Abstract: (Pending)
• (Day 1: Pharmacopeia and the BioAssay Session)

Potency Assays for Immuno-Oncology Products

• Speaker: Tara Stauffer, Bristol-Myers Squibb
• Abstract: Bioassays are a critical component of immuno-oncology product development. Whether used for release and stability, biological characterization or to inform CMC development, the bioassay must reflect inherently complex systems and serve as a surrogate measure of the biological effect of the product. A high degree of bioassay accuracy and precision is required for product control strategy, in addition to the ability to discern molecular changes that influence structure-function relationships. The complexity of immuno-oncology mechanisms of action presents additional analytical challenges. Case studies addressing these challenges associated with the integral role of bioassay in immuno-oncology product development will be presented.
• (Day 2: Product Specific Session)

Visualization and Assay Simulation Tools for the Verification of Equivalence Test Systems

• Speaker: Ralf Stegmann, Stegmann Systems
• Abstract: (Pending)
• (Day 1: BioAssays for Product Characterization Session)

Strategies for Bridging Late Phase Cell-Based Potency Assays

• Speaker: Amy Teale, Regeneron Pharmaceuticals
• Abstract: (Pending)
• (Day 2: Bridging Assays Session)

Development of a Guinea Pig Potency Assay for a Next-Generation Anthrax Vaccine Candidate: Generation of a Stabilized Vaccine Reference Standard

• Speaker: Daniel Tishkof, Emergent Biosolutions
• (Day 1: Reference Materials Session)

Automated Methods for Determining the Potency of Botulinum Neurotoxin Containing Samples

• Presenter: Ward Tucker, BioSentinel, Inc.
• Abstract: Background: BioSentinel has developed and commercialized several biochemical and cell-based assays for detecting and determining the relative potency of samples containing botulinum neurotoxin (BoNT) including drug products and substances as well as food and environmental samples. While these assays offer excellent specificity, accuracy, and precision, assay performance is often dependent on the abilities, training, and experience of the end user. Transfer of methods to the end-user’s laboratory can be complicated by the experience level of the receiving laboratory especially if the receiving laboratory is unfamiliar with biochemical or cell-based methodologies or experiences a high level of employee turnover. BioSentinel, therefore, has automated the BoTest™ Matrix biochemical and BoCell™ cell-based assays to reduce inter-laboratory and operator variability, reduce assay transfer times, and increase end-user throughput.Methods: BioSentinel tested multiple robotic pipetting platforms and spent more than three years developing methods for automated execution of the BoTest Matrix and BoCell assays. Programs were developed that automate all operator-sensitive steps during assay execution, including sample handling and serial dilution, plate washing, sample application, magnetic bead handling, and incubation steps. The operator is only required to dispense reagents, samples, and plastics into the automated platform deck and then, after completion of the automated steps, place the assay plates at the final incubation temperature. Final data collection is also completed by the operator.Results: Six- to ten-event qualifications of the automated BoTest Matrix and BoCell methods indicate that the overall mean relative accuracy of the methods are between 95 and 105% with overall precisions of < 5 %CV. The automated methods meet or exceed the performance of human-executed qualifications of the same methods at BioSentinel.Conclusions: BioSentinel has successfully automated the BoTest Matrix biochemical and BoCell cell-based assays and demonstrated easy transfer of the assay to a second robotic platform of the same make and model. The automated methods can be customized by BioSentinel to accommodate sample-specific needs. Once customized, the methods can be transferred to the end-user’s laboratory with minimal training and transfer time needs.
• Contributing Authors: Burlingham SR, Dunning FM, Piazza TM, Zeytin FN, and Tucker WC
  BioSentinel Inc., 505 S. Rosa Road, Suite 105, Madison, WI 53719, tel: 608 441 8172
  e-mail: wtucker@biosentinelpharma.com
• (Poster)

Application of Droplet Digital PCR in Gene Therapy – A Case Study on Infectious Titer and Residual Host Cell DNA

• Speaker: Yu Wang, Biogen
• Abstract: (Pending)
• (Day 2: New & Improved Technologies Session)

Development and Qualification of a Cell-Free Time-Resolved Fluorescence Resonance Energy Transfer Potency Assay

• Presenter: Engin Yapici, Pfizer
• Abstract: Fluorescence-based detection methods are commonly employed in bioassays. Use of Fluorescence Resonance Energy Transfer (FRET) makes it possible to detect and quantify the interaction between two or more labeled biomolecules. Even though traditional FRET is a very powerful technique, it also has its drawbacks; the major one being high background signals observed in complex solutions. Time-resolved FRET (TR-FRET) alleviates this problem by utilizing the long luminescence lifetime of special rare earth metal organic complexes as agents to label the biomolecules.
  In this study, we developed and qualified a method to determine the potency of a monoclonal antibody (mAb) using homogeneous time resolved fluorescence (HTRF®) reagents to label proteins. Upon binding of mAb to its target protein, we observe a decrease in signal and by using the resulting dose response curve we determine the potency of the mAb in comparison to a reference standard. Our results demonstrate the robustness and sensitivity of the method in potency determination and in identification of forced degradation pathways.
• Contributing Authors: Engin Yapici and Julie A. TerWee
• (Poster)

Statistical Approaches for Bridging Reference Lots

• Speaker: Ann Yellowlees, Quantics Biostatistics
• Abstract: (Pending)
• (Day 1: Reference Materials Session)

Comparison of Recombinant Human FSH Produced by Ferring (REKOVELLE®) using In vivo Bioassay and In vitro Cell-based Assay

• Speaker: Avi Zrachya, Bio-Technology General Ltd.
• Abstract: Human follicle stimulating hormone (hFSH) stimulates the growth and recruitment of immature ovarian follicles in the ovary. Traditionally, therapeutic FSH preparations are calibrated by the Steelman-Pohley rat in vivo bioassay (Steelman and Pohley, 1953).During the development of REKOVELLE®, the potency was determined by the in vivo bioassay to demonstrate consistency between batches (together with other quality attributes).The potency of REKOVELLE® can be also determined by an in vitro cell-based assay developed by BTG-Ferring, using human embryonic kidney 293 (HEK 293) cells stably expressing the human FSH receptor (hFSH-R). In this model, REKOVELLE® activity is determined by direct measurement of intracellular cyclic adenosine mono phosphate (cAMP) accumulation upon binding of REKOVELLE® to the hFSH-R in reporter cells. The potency of REKOVELLE® is then determined as a function of the intracellular cAMP accumulation and relative to the assigned potency of the in-house reference standard. The in vitro cell-based assay was validated for REKOVELLE® drug substance (DS).As part of the justification to replace the in vivo bioassay with the in vitro cell-based assay, the methods were compared at extreme potency levels. Comparable results were obtained in both potency assays for samples tested at the extreme of the potency values.In view of the results of these studies, the in vitro cell-based assay will replace the in vivo bioassay as a release test for potency determination of REKOVELLE® DS; other analytical methods will continue to confirm the consistency in glycosylation and charged isoform distribution in REKOVELLE® DS.
• (Day 2: Bridging Assays Session)