9th Annual Bioassay Conference
September 28-30, 2016
- Jan Amstrup, Novo Nordisk A/S
- Juan Arciniega, FDA
- David Avenal, F. Hoffmann-La Roche
- Sue Charlton, Public Health England
- Jey Cheng, Promega R&D
- Florian Cymer, F. Hoffmann-La Roche Ltd.
- Lien Dejager, Ablynx
- Karen Dixen, Novo Norkisk A/S
- Minna Hassinen, Finvector Vision Therapies OY
- David Lansky, Precision Bioassay
- Javier Martin, NIBSC
- Cecil Nick, Parexel
- Sofie Pattijn, ImmunXperts SA
- Marua Prevato, GSK Vaccines
- Pam Proud, Public Health England
- Frank Straube, Novartis Biologics
- Erik Talens, Merck
- Iris Unterrieder, Baxalta
- Paula Urquhart, Covance
- Maryam Varposhti, CinnaGen Medical Biotechnology Research Center
- Thorsten Verch, Merck
- Thomas Wilton, Division of Virology
- Pin Yee Wong, Genentech
- Ann Yellowlees, Quantics
- Debbie Allan, Sartorius Stedim BioOutsource Limited
- Angelique Baclin, GSK Vaccines
- Anne Benoit, GSK Vaccines
- Kate Getliffe, PsiOxus Therapeutics
- Julie Goemaere, GSK Vaccines
- Ulrike Graab, Eurofins BioPharma Product Testing
- Sten Erik Jensen, Statens Serum Institut
- Stephanie Katzenbach, AbbVie Deutschland GmbH & CO KG
- Sebastian Königsberger, Eurofins BioPharma Product Testing
- Christophe Lallemand, Florian Deisenhammer, & Michael G. Tovey
- Christophe Lallemand and Michael G. Tovey Biomonitor
- Eric Muhr, Promega Corporation
- Vanessa Ott, Promega Corporation
- Thorsten Pflanzner, AbbVie
- Dyan Sheehan , Janssen Biologics
- Rachel Shuker, DiscoverX Corp
- Rachel Shuker, DiscoverX Corp
- Rachel Shuker, DiscoverX Corp
- Richard Somberg, Promega Corporation
- Presenter: Debbie Allan, Sartorius Stedim BioOutsource Limited
- Abstract: The biggest challenge in developing a biosimilar monoclonal antibody is to comprehensively characterise the biosimilar and demonstrate comparability to the innovator. The complex nature of monoclonal antibodies frequently results in differences between batches of the innovator, and also between the biosimilar and innovator. The key to regulatory acceptance is to understand these differences in relation to the clinical significance; regulators advocate generating extensive analytical characterisation data to present a “totality of evidence” in regulatory submissions. A key factor of this is understanding the functional significance of structural differences between molecules.
At Sartorius-Stedim BioOutsource, we have explored the impact of differing glycan structures on antibody function using a case study comparing a biosimilar to the innovator molecule. Orthogonal methods were used to define the structural characteristics of the antibody, including glycan analysis (by LC/MS), CD16a binding (by SPR and ELISA), and to investigate how this translates into functional ADCC activity. Correlations between orthogonal methods will be discussed, and an overview provided of how in-depth characterisation of the structure/function relationship can be used to provide a comprehensive understanding of residual differences between biosimilar and innovator molecules.
The data presented highlights the benefits of carrying out the appropriate structural and functional analysis for each stage of biosimilar development and the positive impact this can have on the progress of a biosimilar development program. This analytical approach to assessing similarity between biosimilar and innovator molecules provides biosimilar developers with a comprehensive testing package that aims to provide a totality of evidence fit for regulatory submissions.
Debbie Allan, Martin De Cecco, Terry Gray and Catriona Thomson. Sartorius Stedim BioOutsource Limited, Glasgow, UK
- Speaker: Jan Amstrup , Novo Nordisk A/S
- Abstract: Potency determination is an important part of the quality assessment/control. According to ICH Guideline Q6B, potency must be evaluated by using well-characterized and validated bioactivity assays.
A bioassay is often validated at phase I/II, but during the biologic’s life cycle, the assay often needs optimization and refinement, to fulfil both internal and external requirements.
Here the optimization of a bioassay used at Novo Nordisk A/S for potency determination of a biopharmaceutical protein will be shown. By implementing Design of Experiments DoE in the optimization process, nine assay factors and four factor interactions based on time consumption and complexity of buffers were evaluated. By using DoE the factors were simultaneously evaluated in an efficient and effective manner. The conclusions drawn from statistical analysis of the results obtained from the DoE provided improved assay conditions and settings that were successfully evaluated in a proof of concept assay.
Furthermore, how to implement the optimizations will be discussed with regard to validation status of the assay.
Sandra Fredsted and Jan Amstrup Novo Nordisk A/S, Denmark
- Day 2: Use of DOE as a Development Tool Session
- Speaker: Juan Arciniega, DSc, CBER/FDA
- Abstract: Potency tests aim to ensure, on the basis of the best available scientific information, that vaccines show the expected efficacy. Because stability plays a critical role in vaccine effectiveness, potency is included in all vaccine stability-testing programs. Since all lots entering the National Strategic Stockpile must remain potent throughout protracted storage periods, anthrax vaccines must have reliably long shelf-lives.
Novel anthrax vaccines containing recombinant Protective Antigen (rPA) as the only antigen face a stability issue: purified rPA is particularly susceptible to degradation through non-enzymatic modifications such as deamidation and aggregation.
Spontaneous deamidation of rPA, which occurs at relatively mild temperature, adversely affects vaccine immunogenicity in mice. In addition, rPA forms aggregates in solution after exposure to temperatures ≥40 ºC, losing not only its ability to form lethal toxin (LeTx), but also its immunogenicity.
A macrophage lysis assay (MLA) has been proposed to evaluate rPA quality prior to formulation into a final vaccine bulk containing an adjuvant; MLA quantifies the ability of rPA to form LeTx in vitro when mixed with Lethal Factor.
We have previously studied ELISA and a LeTx-neutralization assay (TNA), as part of an immunogenicity test in mice to measure anthrax vaccine potency, in terms of their stability-indicating properties. This animal-based potency test is required for the release of drug product, because the interactions between adjuvant and rPA may have an impact on the antigenic structure. However, efforts should be made to reduce, replace and refine use of animals in testing. Thus, we decided to evaluate the MLA as a potential replacement of immunogenicity.
We studied the effect of exposure of rPA in solution to temperatures outside refrigeration, including two above its melting point (50 and 75 °C), on the formation of aggregates, and subsequently on the relation of this aggregation to the ability of the protein to form active LeTx by MLA, and on its ability to elicit neutralizing antibody response in mice when combined with adjuvant, by ELISA and TNA. rPA treated at 50 °C for as little as 30 min formed aggregates. While MLA showed that rPA lost about 50% of toxin-forming activity when treated at 50 °C for 1 h before formulation into a final vaccine lot, TNA data showed that antibodies elicited by similarly treated rPA formulated into a vaccine were reduced by over a hundred fold, compared to those elicited by untreated antigen. ELISA data, in contrast to TNA data, indicated only a relatively minor reduction in antibody titer elicited by heat-treated rPA.
These findings suggest that although the MLA may not be used to replace immunogenicity as a potency test for anthrax vaccines, and appears somewhat limited in revealing temperature-driven rPA structural changes, it is still an important in-process test to monitor the immunogenic quality of rPA during the manufacturing of new anthrax vaccines. Our results also confirm previous findings that showed the superiority of the TNA over ELISA for use in the immunogenicity test, in terms of its sensitivity to temperature-mediated changes on rPA.
- (Day 1: Regulatory Updates Session)
- Speaker: Cecile Avenal, F. Hoffmann-La Roche
- Abstract: Abstract: In vitro assays used in QC are aimed to translate complex biological in vivo mechanisms of action into quantitative reproducible results. Currently, reporter assays, based on engineered cell lines and offering the advantage to be robust and extremely fast, are more and more preferred to classical cell-based assays. Based on a case-study, we compare here results obtained from three assays covering early binding: by cell-free assay, middle intracellular signaling: by reporter gene assay and late proliferation: by classical cell- based assay biological events.
- Day 1: Method Optimization Session
- Presenter: Angelique Baclin, GSK Vaccines
- Abstract: (Pending)
Angelique Baclin, GSK Vaccines
- Presenter: Angelique Benoit, GSK Vaccines
- Abstract: During the development of an 8-dilutions format ELISA to be used for IgG measurement in clinical testing of phase III-IV vaccines studies, it was noted that the standard signal was bounded by the reader capacity in certain conditions. The consequent poor fitting of the curve in the upper part of the standard led to an increased proportion of invalid plates and to a calculation bias for samples with high concentrations among borderline valid plates. Therefore, a new standard lot of lower concentration was prepared to help meeting the symmetry condition assumed by the 4PL model and, in turn, to allow better fitting and accuracy in the high range of concentrations.
Prior to the evaluation of the results comparison generated by both standards, an analysis of the parallelism between the current and the candidate standard was performed. Following the USP recommendation, we used an equivalence approach. The equivalence criteria for the model parameters were derived from historical data for the current standard. We present the methodology we applied and discuss our results with regard to the initial problem. Finally, we propose to associate the passed / failed result obtained with this methodology with a continuous probability of success, computed using Bayesian statistics.
Anne Benoit, Julie Goemaere, Angélique Baclin. GSK Vaccines, Rixensart, Belgium
- Speaker: Sue Charlton, Public Health England
- Abstract: Successful development of vaccines requires a toolbox of assays to characterise both the product and the patient’s reaction to that product. This can present the bio-assayist with “interesting” challenges. Assays for potency or immunogenicity should measure immune responses that correlate with protection against disease and whilst correlates of protection for existing vaccines can be well defined, this is not always the case for vaccines currently in development. Developing assays for new vaccines may require reagents or controls that are hazardous, poorly defined or not readily available. This talk will attempt to illustrate some of the challenges faced and use examples of approaches used to overcome them.
- Day 1: Vaccine Potency Assay Development Session
- Speaker: Jey Cheng, Promega R&D
- Abstract: Therapeutic antibodies designed to target immune checkpoint receptors function by modulating a patient’s own immune system and are promising strategies to treat cancer. Clinical results showed co-engagement of multiple immune receptors, such as immune inhibitory receptors PD-1 and CTLA4 or PD-1 and TIGIT in combination immunotherapy, elicit much better therapeutic outcomes compared with targeting a single immune receptor. Current methods used to measure the potency of these therapeutic drugs rely on binding assays or primary cell-based assays which are unable to provide a mechanism of action-based measure of drug potency with the precision and accuracy required for use in controlled drug development environment. In this talk, we will describe the development of several bioluminescent bioassays that can quantitatively measure the biological activity of antibodies targeting immune checkpoint receptors (PD-1, TIGIT, CTLA4 and others) individually and in combination. For this, we engineered stable cell lines to serve as T effector cells and artificial antigen presenting cells (aAPCs), which are further developed into Thaw-and-Use format to provide convenience and minimize day-to-day assay variation. The T effector cells stably express the immune checkpoint receptors of interest with one or two luciferase reporters responding to signals from TCR or the immune receptors stimulation. The aAPCs are engineered by co-expressing corresponding immune checkpoint receptor ligand and TCR activator which can activate T effector cells upon direct interaction. When the T effector cell is co-cultured with its aAPC cell, the immune receptor/ligand interactions modulate T effector cell activation and luciferase activity, which are blocked or further activated by immune checkpoint antibodies. Furthermore, the combination bioassay is able to provide a quantitative measure of the synergetic effeAct of two immune checkpoint antibodies on T cell activation. These bioluminescent bioassays reflect the mechanism of action for each antibody drug candidate, and exhibit assay specificity, precision, accuracy, linearity and robustness required for drug potency and stability determination. They will empower current and future immunotherapy drug development.
- Day 2: Approaches to Assay Validation Session
- Speaker: Florian Cymer, F. Hoffmann-La Roche Ltd.
- Abstract: Receptors that bind the Fc-portion of IgG type antibodies are critical mediators of effector functions and can also critically influence the serum half-life of therapeutic antibodies. Such mediators like the Fc-gamma receptors (FcγR’s) and the neonatal Fc-receptor (FcRn) are frequently used in bioanalytics during assessments of therapeutic antibodies by Biacore interaction measurements, cell-based binding measurements and cell-based assays on effector functions. Analysts are confronted with a very heterogeneous receptor population, containing a large diversity in terms of posttranslational modifications, e.g., glycan structures and cells expressing both activating and inactivating receptors in different amounts. Receptor glycan structures are known to depend on the cell line used during production and also affect the interaction with its binding partners. Similarly, the very heterogeneous antibody samples pose challenges on the interpretation of the resulting data and supporting analytical methods. Results from cell-based binding experiments, cell-based effector function assessment and biophysical binding data can only be integrated with a deep knowledge of the respective systems. The basic background on Fc-receptors, their complexities in terms of assay design and possible pros and cons of certain assay setups will be discussed.
- Authors: Florian Cymer 1,2, and Hermann Beck 2
1 School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz
2 Pharma Technical Development Analytics Biologics, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland
- (Day 1: More Than One Bioassay? What To Do! Session)
Justification of the Use of Surrogate Potency Assays for Lot Release and stability Testing During Clinical Development of Nanobody®-Based Therapies
- Speaker: Lien Dejager, Ablynx
- Abstract: Nanobodies® are a class of therapeutic proteins based on single-domain antibody fragments that contain the unique structural and functional properties of naturally-occurring heavy chain only antibodies. During clinical development, a potency assay is required to support lot release and stability studies and is used for biological characterization of a compound. Ideally, the potency assay is reflective of the mechanism of action (MoA) of the biological product. Cell-based assays typically have the potential to reflect different aspects of the MoA, although these often show high variability and poor assay performance and mostly require long assay times. Well-controlled assay conditions are often more easily obtained with a simple, surrogate assay. However, as such surrogate assays typically mimic a single aspect of the MoA (e.g. binding), justification of the surrogate assay is generally required for its use in lot release and stability studies.
Through selected case studies, we will discuss our generic justification approach in which the assay performance and stability indicating properties of a surrogate assay and a bio-assay are compared. In order to justify the use of a surrogate assay, the results obtained with this assay should correlate with the results of the bio-assay and the physicochemical characterization of the product-related variants. Therefore, in a first stage during development, mock variants and affinity variants are used. Furthermore, during Phase II clinical development, samples which are exposed to several stress conditions, such as increased temperature, vibration stress, photo stress, etc., are used to demonstrate and compare the stability indicating capacity of both assays. The overall justification strategy consists first of a general assessment if both assays are able to identify similar changes in potency of the stressed test samples. Therefore, equivalence is assessed by checking whether the confidence interval of the ratio of the potency of a test sample to the control sample, per assay, falls within pre-set equivalence limits or not. Secondly, statistical equivalence is evaluated of both assays for all stress samples tested. This equivalence test also requires pre-defined equivalence limits which are based on the variability of a representative historical dataset.
- (Day 1: More Than One Bioassay? What To Do! Session)
- Speaker: Karen Dixen, Novo Norkisk A/S
- Abstract: Changing potency assays during clinical phase 2 studies requires various considerations. This presentation of a case study is about how a carefully worked-out strategy for changing a potency assay with minimum testing was executed. Results from assay validation and statistical equivalence testing will be discussed. Additionally, the handling of ongoing stability programmes as well as timelines and the strategy for registration will be presented.
- Day 1: More Than One Bioassay? What to Do! Session
- Presenter: Kate Getliffe, PsiOxus Therapeutics
- Abstract: PsiOxus is a development stage immuno-oncology company which has developed a Tumour-Specific Immune Gene (T-SIGn) therapy platform based on an oncolytic virus, enadenotucirev. One class of proteins that can be delivered to tumours using this platform is Membrane-integrated T-cell engagers (MiTes), which are encoded by the virus and expressed on the surface of infected tumour cells. This study describes the development of a Cell-ELISA assay for the detection of a MiTe protein expressed on the surface of virus-infected adenocarcinoma cells.
In the Cell Surface ELISA, A549 cells are infected with a MiTe-encoding T-SIGn virus and incubated for 48 hours. The cells (now expressing the MiTe protein) are fixed with formaldehyde and then subjected to standard ELISA steps including blocking and incubating with a biotinylated anti-MiTe antibody, streptavidin conjugated to horse radish peroxidase (HRP) and a detection substrate. The Cell Surface ELISA for our first MiTe-encoding T-SIGn virus was developed and optimized using the following steps:
1) A commercially available Cell-ELISA method, designed to detect intracellular proteins, was adapted to the detection of cell-surface proteins by the removal of permeabilisation steps, quenching steps and all detergents from wash buffers.
2) The cell and virus part of the assay was optimised using a Design of Experiments (DoE) approach, including factors such as cell density and attachment time, virus concentration and infection time and also fixative concentration and fixing time.
3) The ELISA part of the assay was optimised using DoE including factors such as blocking agent concentration and blocking time, antibody concentration, time and temperature and also conjugate (streptavidin-HRP) concentration and incubation time.
4) The factors having most effect on the performance of the assay were combined in a final DoE run to complete the optimization, investigate interactions between cell and virus factors and ELISA factors and determine potential acceptance limits for the assay.
5) The plate layout and dose response curve was optimized to allow parallel line analysis to be used to determine relative potency.
The result was a high throughput cell surface expression assay that is suitable for pre-qualification and subsequent transfer to a Contract Research Organisation for validation.
Dr. Kate Getliffe (Principle Author)
Abingdon, OX14 4SD
- Presenter: Julie Goemaere, GSK Vaccines
- Abstract: (Pending)
- Presenter: Ulrike Graab, Eurofins BioPharma Product Testing Munich GmbH
- Abstract: Golimumab (Simponi®) is a fully human monoclonal IgG1 antibody that inhibits binding of TNFα to its receptor TNFR. We have developed and qualified a SPR binding assay to characterize the binding of Golimumab Fc to FcRI (CD64) which is one component of a set of characterization assays used for comparison of innovator and biosimilar product. A method to characterise the binding of Golimumab (Simponi) to CD64 using SPR has been developed and qualified. As there are no clearly defined validation requirements for SPR based kinetics assays the qualification approach has been based on the ICH guideline for analytical method validation (ICHQ2(R1)). The qualification process was designed to assess repeatability, intermediate precision, baseline and binding stability and specificity. The data was double referenced and a Langmuir 1:1 fitting model applied to determine association and dissociation rate constants.
Ulrike Graab, Michael Willcox, Alexander Knorre. Eurofins BioPharma Product Testing Munich GmbH
The Testing Strategy to Determine the Pharmacological Activity of Gene Therapy Drug Product (rAd-IFN) to Treat Intravesical Non-Muscle Invasive Bladder Cancer
- Speaker: Minna Hassinen, Finvector Vision Therapies OY
- Abstract: rAd-IFN is a recombinant adenoviral gene therapy vector encoding IFNα2b gene for the treatment of refractory non-muscle invasive bladder cancer. The vector transduces the bladder wall cells where IFNα2b gene is expressed leading to death of cancer cells.
The advanced testing strategy to determine the pharmacological activity of rAd-IFN drug product involves three key assays:
1. Infectious titer of the virus, quantitative assay.
2. Expression of the transgene (IFNa2b), semiquantitative assay.
3. Potency (IFNa2b mediated cell killing), quantitative assay.
The infectivity and transgene expression assays have been performed for batch release and stability monitoring of activity during Phase 2 and will remain unchanged in principle for Phase 3 and commercial use.
In the infectivity assay, the cells supporting adenovirus replication are infected with three concentrations of adenovirus and left to produce the virus for two days. The percentage of infected cells is then determined with a flow cytometer utilizing a fluorescently conjugated antibody against an adenoviral structural protein. Samples are analyzed in parallel with a reference standard and infectivity is given as Relative Infectious Units / ml.
In expression assay, the IFNα expression capability of the virus preparation is determined by infecting IFN insensitive cells with the rAd-IFN virus and the concentration of produced IFNα is measured with a commercial IFNα ELISA (enzyme-linked immunosorbent assay) from cell culture supernatants.
For Phase 3, a new potency assay is developed and added to release and stability testing in order to provide evidence that batches of rAd-IFN are able to produce active IFNa2b which has a relevant pharmacological effect. In this assay, cells are transduced using multiple dilutions of reference standard and test samples leading to expression of IFNα2b and subsequent cell death. Cell killing efficiency is determined using colorimetric method measuring dehydrogenase activity of the living cells. Relative potency of test sample is determined against reference standard response curve after testing parallelism by equivalence test.
All activity assays will be fully validated according to ICH Q2 (R1) prior to release testing of Phase 3 clinical study material (Accuracy, Precision, Specificity, Linearity and Range, System Suitability and Robustness).
The three validated assays will provide enhanced quantitative measure of biologic function of the rAd-IFN vector and thus demonstrate the quality and efficacy of drug product batches.
- (Day 2: Product Specific Potency Assay Development Session)
Assay of Inactivated Poliomyelitis Vaccine: Comparison of In vitro vs. In vivo Assays for Potency Determination
- Presenter: Sten Erik Jensen, Statens Serum Institut
- Abstract: Currently, potency determinations of the polio vaccine components in licensed SSI vaccines are made in an in vivo assay in guinea-pigs. The European convention on the protection of vertebrate animals used for experimental and other scientific purposes requires that tests in animals shall not be carried out if a scientifically satisfactory alternative is reasonably and practically available.
A study for waiving of the in vivo potency assay of inactivated poliomyelitis vaccine (IPV) was made with reference to the guideline included in the European Pharmacopoeia method 2.7.20. Samples of IPV containing vaccines, including 2 sub-potent vaccine preparations, were tested in both in vivo (rat) and in vitro (D-antigen ELISA) assays. IPV vaccine representative of the current SSI production method should comply with both the in vivo and the in vitro assays and sub-potent IPV vaccine preparations should fail to comply.
The IPV batch representative of the current production method passed the validation accept criteria for both the in vivo rat assay and the in vitro D-antigen assay. For the 2 sub-potent IPV preparations, one preparation passed the accept criteria for both assay methods, whereas the other preparation passed the criteria for the D-antigen assay but failed to pass the accept criteria for the rat assay (poliovirus type 1 and 3) after 2 assay runs.
The in vivo assay in rats generally measured higher potency in the sub-potent IPV preparations than expected; whereas the measurements by the in vitro D-antigen assay gave results close to the expected potency. In addition, the measurements by the D-antigen assay was much less variable than the in vivo rat assay.
The study did not show perfect agreement between the in vitro D-antigen assay and the in vivo assay in rats, however, it was evident that the in vitro D-antigen assay had an improved ability to detect degradation and instability of IPV compared to the in vivo assay. Thus, based on this study, the in vitro D-antigen assay would be better than the in vivo assay for monitoring consistency of production and for stability studies on IPV in routine quality control.
Sten E. Jensen*, Charlotte Sørensen, Kaare R. Hasløv & Gitte Stawski
Statens Serum Institut (SSI), Division of Vaccine, Quality Control Dept., 5 Artillerivej, DK-2300 Copenhagen S, Denmark
- Presenter: Stephanie Katzenbach, AbbVie Deutschland GmbH & Co KG
- Abstract: The use of cells as a cryopreserved, ready-to-use reagent is a very common practice in high-throughput screening (HTS) of compounds by cell-based assays. This process is considerably less labor intensive, more consistent and allows more flexibility than traditional continuous cell culture which is generally employed in GMP bioassays. The purpose of the present study was to determine if the use of cryopreserved ready-to-use cells could also be adapted to a bioassay intended for lot release and stability testing. The cytotoxicity assay of ABBV-ADC was chosen as one of our pilot projects.
Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Silvia Löblein, Dominik Dorer, Renate Kron and Stephanie Katzenbach. NBE Analytical R&D, AbbVie Deutschland GmbH & Co KG, Ludwigshafen, Germany
- Presenter: Sebastian Königsberger, Eurofins BioPharma Product Testing Munich GmbH
- Abstract: Golimumab (Simponi®) is a fully human monoclonal IgG1 antibody that inhibits binding of TNFα to its receptor TNFR. We have developed and qualified a bioassay to characterize the Golimumab Fab functional activity which is one key component of a set of characterization assays used for comparison of innovator and biosimilar product. In the following, we report the qualification results of this bioassay. The qualification parameters analyzed covered intermediate precision, repeatability, accuracy, linearity and specificity. Golimumab (Simponi®) was used as reference standard at 50%, 67%, 100%, 150% and 200% concentration for the qualification of the bioassay. The method has proven to be accurate and precise concerning intra-assay as well as inter-assay variability. In addition, regression analysis revealed a linear dose response curve. In conclusion, it could be shown that the bioassay method can be used to measure the biological activity of Golimumab (Simponi®.)
Sebastian Königsberger, Stefan Wanninger, Onofrio Zirafi, Alexander Knorre. Eurofins BioPharma Product Testing Munich GmbH
Quantification of Bevacizumab Activity and Anti-Bevacizumab Neutralizing Antibodies in a Cohort of Patients with Glioblastoma
- Presenter: Christophe Lallemand, Florian Deisenhammer, & Michael G. Tovey
- Abstract: A novel highly sensitive VEGF-responsive reporter gene assay has been developed that allows bevacizumab activity to be quantified rapidly and in a highly specific manner. The use of this assay has shown that in a cohort of patients with glioblastoma who respond to therapy there is a close correlation between bevacizumab drug levels determined by ELISA and bevacizumab activity determined using the VEGF-responsive reporter gene assay. In contrast, in secondary non-responders with a decreasing PK profile, bevacizumab drug levels determined by ELISA are consistently higher than bevacizumab activity determined using the reporter gene assay, suggesting that bevacizumab activity is partially neutralized by anti-drug neutralizing antibodies. (NAbs). The results obtained suggest that the use of the VEGF-responsive reporter gene assay may allow the appearance of anti-bevacizumab NAbs to be used as a surrogate maker of treatment failure prior to the clinical signs of disease progression.
Christophe Lallemand, Florian Deisenhammer, & Michael G. Tovey
iLite™ Reporter Gene Assay for Quantification of the Activity of Anti-IL-23 and Anti-IL-23 Neutralizing Antibodies
- Presenter: Christophe Lallemand, and Michael G. Tovey Biomonitor
- Abstract: Interleukin 23 (IL-23) is a heterodimeric pro-inflammatory cytokine that shares a common p40 subunit and a common receptor chain with IL-12. Both cytokines exert however distinct non-redundant biological functions. Conventional assays for IL-23 activity are based on the ability of IL-23 to support the proliferation of cell lines such as the IL-2 dependent human T-cell line Kit 225, which has been reported to partially lose dependence on IL-23, rendering the routine use of such assays problematic.
- Authors: Christophe Lallemand and Michael G. Tovey Biomonitor
- Speaker: David Lansky, Genentech
- Abstract: Fitting dose-response models to bioassay data is complex because of: non-linear relationships between log dose and response; non-constant variance; complexities such as blocks, split-unit, or strip-unit designs; pseudo-replicates; missing data; and unusual observations. Good analysis methods for non-linear models usually include: transformation of the response, weights, or mixed models. Both weights and variance components (in mixed models) are particularly difficult to estimate well from small data sets in the presence of unusual observations. Hence, procedures for detecting, removing, and tracking outliers are particularly important. Many bioassays include grouped preparation of samples (which can be created by serial dilution, use of a multichannel pipette, or shared preliminary dilutions); in these assays it is important to check for both group and observation outliers. Because cells (or animals) used in bioassays are inherently sensitive to various departures from normal procedures, multiple unusual observations in a single assay are common. While there are good methods for multilevel outlier detection (using mixed models) and good methods for detecting multiple outliers in a data set (i.e.; Rosner’s method), methods for simultaneously detecting at multiple levels and detecting multiple outliers are not broadly available. This talk will focus on some steps to address this problem.
- (Day 1: Method Optimization Session)
- Speaker: Javier Martin, NIBSC
- Abstract: (Pending)
- (Day 1: Vaccine Potency Assay Development Session)
Improved T Cell Activation Bioassays to Advance the Development of Bispecific Antibodies and Engineered T Cell Immunotherapies
- Presenter: Eric Muhr, Promega Corporation
- 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.
Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong and Zhi-jie Jey Cheng. Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
- Speaker: Cecil Nick, Parexel
- Abstract: Biological assays play a critical role in the determination of biosimilarity by providing a sensitive method for comparison of potency between the biosimilar and the reference product. Using IgG1 as an example, the potential for various quality attributes, such as structural alterations, glycosylation, deamidation, etc., to impact potency in various ways will be discussed. Biological assays include animal and cell based assays, as well as binding assays, however, animal assays are falling out of use and will not be included in this discussion. Generally, in assessing potency, it is appropriate to apply a range of orthogonal methods in order to ensure that, as far as possible, all subtle differences between biosimilar and reference product are detected. The complexity of this challenge increases with molecules possessing multifunctional properties, such as immunoglobulins. Issues that will be considered include the ability of the assay to detect a meaningful difference and the drivers for this will be discussed. These drivers and their importance will, inter alia, differ depending on the assay platform, the impact of potentially different receptor isotypes within clinical populations and the relevance of the biological attribute to the intended therapeutic effect. These considerations will be illustrated using the potential role of Fc-gamma IIIa binding in respect of a monoclonal anti-TNF alpha. Also, in relation to biosimilarity, immunogenicity assays while not directed to measuring potency have a clear role to play and a brief discussion on this will be included. It could be argued that, in relation to the totality of data used to support biosimilarity, the importance of biological assays transcends that of both structural and clinical data. Regulatory agencies, and notably the FDA, are applying ranking and statistical constraints to best ensure adequate equivalence based on quality attributes and, in this respect, potency assays are always included amongst the highest ranked quality attributes that impact biosimilarity.
- (Day 2: Bioassays for Biosimilar Products)
- Presenter: Vanessa Ott, Promega Corporation
- Abstract: Drug developers and regulatory authorities recognize antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) as important mechanisms of action (MOA) of therapeutic antibodies. Traditional ADCC and ADCP bioassays use primary cells, which are labor intensive and highly variable. Less variable, easy-to-use and consistent analysis of these important MOA is needed in drug development programs.
To meet this need, we have developed a suite of functional cell-based Fc effector reporter bioassays for the following receptors:
- ~ Human FcRIIIa (V158 and F158 variants)
- ~ Human FcRIIa (H131 and R131 variants)
- ~ Human FcRI
- ~ Mouse FcRIV and FcRIII
- Each bioassay is provided in “thaw-and-use” format for a rapid and convenient workflow and further reduction in assay variability. In qualification studies according to ICH guidelines, the bioassays show the specificity, accuracy, precision and linearity enabling their use in antibody screening, characterization, stability and potency studies.
Zhi-jie Jey Cheng, Rich Moravec, Aileen Paguio, Denise Garvin, Frank Fan, and Mei Cong. Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
- Speaker: Sofie Pattijn, ImmunXperts SA
- Abstract: During the last years, significant advancement has been made in the clinical application of cancer immunotherapies. Molecules directed against immune checkpoints and other agonists show great promise for treatment of a variety of malignancies. Next to CTLA-4 and PD-1 blockade, a wide range of therapeutics with the potential to reverse the tumor-induced suppression are under development.
Early evaluation of the effectiveness of candidate therapeutics and combination therapies can be done using mouse models and in vitro bioassays with human immune cells.
Mixed lymphocyte reaction assays using both innate cells and lymphoid cells mimic a real physiological T cell response and are widely used for the potency screening of candidate therapeutics. The use of different allogenic donor combinations can provide additional information on the profile of the responding population.
An important factor for sensitive assays and consistent results is the quality of the primary immune cells. PBMC are isolated and cryopreserved shortly after blood redrawn. All donor preparations are quality controlled and HLA typed and optimized procedures are used to generate functional dendritic cells which are co-cultured with allogenic T cells. Response levels can be evaluated by the assessment of proliferation or measurement of cytokine production.
Next to the MLR assay, other T cell assays such as antigen-specific recall activation assays can be used to evaluate the ability of test molecules to promote T cell responses.
- (Day 2: Product Specific Potency Assay Development Session)
- Presenter: Thorsten Pflanzner, AbbVie
- Abstract: Relative potency assay methods for CMC quality control of new biological entities require statistical evaluation to demonstrate curve similarity between reference standard and sample. We established an equivalence testing approach covering the complete potency assay life cycle from early development until commercialization. This approach is based on the development of generic goal posts to support early assay stages. Upon availability of sufficient assay data generated during release and stability testing the generic goal posts are re-evaluated to establish assay specific goal posts during later stages of the assay life cycle. Generic goal posts for equivalence test performance on four parameter logistic curve fits were established for bioassays and binding assays. Based on 556 historical data sets, goal posts for the curve parameters upper asymptote, slope and dynamic range were determined. Five binding ELISAs and one alternative binding method with colorimetric and fluorometric read-outs, and five bioassays based on colorimetric, fluorometric and luminometric read-outs were evaluated to set-up generic goal posts. Furthermore, for generic goal posting and applicability of goal posts in different laboratories, we observed that equivalence testing of ratios of reference standard and sample is superior to equivalence testing of differences between reference standard and sample because e.g. different plate readers can vary with respect to absolute numbers.
All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Thorsten Pflanzner, Yuanyuan Duan, Stephen Hartman, Judith Dudley, Peter Berner, Martina Kron, Lanju Zhang, Renate Kron
- Speaker: Marua Prevato, GSK Vaccines
- Abstract: Quality by Design principles together with the typical request for vaccine productivity increase during lifecycle are the main drivers for introducing new in vitro assays in replacement of in vivo testing into vaccine characterization and lot-release testing.
In general, the in vitro test has the following advantages: i) a much lower variability as compared to the in vivo method; ii) cost-effectiveness with reduction of time for the test for lot release iii) considerable reduction in the use of animals, in line with the 3R principle. In addition, in vitro tests are generally more sensitive to detect minor o subtle product changes. However, challenges related to the needs of correlation between in vitro and in vivo testing together with expectations from regulatory agencies have to be considered for the replacement of in vivo testing.
Here, examples of the development and the path forward for implementation of in vitro tests for measuring safety and potency attributes for vaccines in different stages of development and commercialization will be discussed.
- (Day 1: Vaccine Potency Assay Development Session)
- Speaker: Pam Proud, Public Health England
- Abstract: A cell-based toxin neutralisation assay has been validated to assess the ability of antibodies in sera to neutralise Bacillus anthracis lethal toxin. The assay has been validated for multiple species to test preclinical and clinical samples and as a component for batch release potency tests for anthrax vaccines.
- Day 2: Approaches to Assay Validation Session
- Presenter: Dyan Sheehan, Janssen Biologics
- Abstract: (Pending)
- Presenter: Rachel Shuker, DiscoverX Corp
- Abstract: Denosumab (Prolia®) is a fully human sequence derived monoclonal antibody used for the treatment in osteoporosis in menopausal woman via inhibition of the RANK (receptor activator of nuclear factor-kappa B) pathway. Companies developing a biosimilar are required to demonstrate that the proposed biosimilar is “highly similar” to the innovator material, using an MOA-based assay.
The DiscoverX PathHunter bioassay for Denosumab uses a U20S cell line expressing RANK and an IκB reporter protein that is tagged with one fragment of a split β-galactosidase system. When the tagged IκB is exposed to the other half of the split β-galactosidase protein, active β-galactosidase is formed which hydrolyses the substrate and produces a chemiluminescent signal. In the assay, RANKL binds to RANK on the cell surface resulting in NF-κB signalling and IκB degradation, resulting in a decrease in chemiluminescent signal. Denosumab inhibits RANKL-based activation of RANK, leading to an increase in chemiluminescence. The increase in chemiluminescence is directly proportional to the functional activity of denosumab.
The data presented in this poster was obtained from an assay qualification performed at Sartorius Stedim BioOutsource. During the qualification study, data was obtained to demonstrate that the assay is accurate and precise over a linear range of 50 to 150% of reference standard. The assay also showed specificity for denosumab. The methodology is therefore deemed fit for the purposes of evaluating the functional comparability and potency of denosumab biosimilar and denosumab innovator material. The benefits of this commercially available assay are that it is easy-to-use, highly reproducible, and saves months of assay development time, translating into overall cost savings in a biosimilar development program.
Catriona Thomson1, Abhishek Saharia2, Terry Gray1, Debbie Allan1, and Jane Lamerdin2
- 1Sartorius Stedim BioOutsource Limited, Glasgow, UK
- 2DiscoverX Corp, Fremont, USA
- Presenter: Rachel Shuker, DiscoverX Corporation
- Abstract: Cell-based bioassays often pose a hurdle during a rapidly moving biologics development program. High standards for assay accuracy, precision, reproducibility and robustness are additionally put to the test by the use of continuous culture cells that can add to variability and increase the cost and complexity of each assay. This is particularly challenging for anti-VEGF drugs, as the prevalent assay is the proliferation of human umbilical vein endothelial cells (HUVECs), which requires 72-96 hours to run, utilizes primary cells that are difficult to culture and introduces performance variability due to changes in passage number, culture conditions and analyst.
Here, we describe a PathHunter® bioassay that has been developed as a fit-for-purpose potency & stability assay for anti-VEGF drugs. The assay quantifies inhibition of VEGF-A-induced VEGFR2 receptor activation, by measuring an early event in the receptor activation cascade. With its shorter assay time (
Jane Lamerdin, Hanako Daino-Laizure, and Abhi Saharia. DiscoverX Corporation, Fremont, USA
- Presenter: Rachel Shuker, DiscoverX Corporation Ltd
- Abstract: Regulation of immune responses within the body is tightly controlled through a balance of co-stimulatory and co-inhibitory checkpoint receptors, often exploited by many cancers. Molecules that block or activate these checkpoint receptors have proved to be powerful therapeutic agents for cancer and autoimmune diseases. However, developing drugs targeting these checkpoint proteins has proved to be quite challenging as cell-based assays used to screen for functional drugs are often difficult to create, involve the use of human primary cells, and have complicated protocols.The PathHunter® Checkpoint Assays provide an easy-to-use, sensitive, and reproducible cell-based assay for several co-inhibitory and co-stimulatory receptors. These assays support screening for small molecules and biologics, and potency assay development for biologic immunotherapies, with an easy protocol and a rapid response in less than five hours. Data will be presented for assays developed for targets such as GITR, CD137, CTLA-4, PD-1, OX-40, CD40, TWEAK, and LIGHT.
Hitting the Gas: Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Co-Stimulatory Immune Checkpoint Receptors
- Presenter: Richard Somberg, Promega Corporation
- 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) 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 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.
Jun Wang, Michael Beck, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong and Zhi-jie Jey Cheng.
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
- Speaker: Frank Straube, Novartis Biologics
- Abstract: A bioassay determines potency relative to a reference; thus, evaluating potency changes of the reference substance itself over time requires modifications. Several concepts for retesting reference material have been used; each comes with its own advantages and shortcomings. Reference retesting may be performed, e.g., annually, and may be based on absolute values like bioassay EC50. But, although the EC50 is related to potency, it can also be influenced by other factors that may be difficult to identify and to control. An alternative approach is to evaluate reference potency based on the readout relative potency. This eliminates the influence of extraneous factors; on the other hand, testing against another standard requires the assumption that this other standard would not degrade at the same rate and to the same extent as the reference substance to be tested. The presentation will evaluate selected reference retesting concepts together with measures to handle the respective shortcomings.
- Day 2: Reference Material
- Speaker: Erik Talens, Merck
- Abstract: A reference standard is used in biological assays to compare test samples with, such that the bioactivity of the test samples can be determined. For the reference standards in use, it is required to verify and maintain the level of bioactivity over time. This presentation will discuss possible strategies for qualifying and monitoring reference standards both in cases where an International Reference Standards is present and in cases where no International Reference Standard is present.
- Day 2: Reference Material Session
- Speaker: Iris Unterrieder, Baxalta
- Abstract: When transferring an assay to an external contract lab, the client needs to provide the “know-how” on the in-house assay and has to plan the set-up of the analytical method transfer validation, including relevant acceptance criteria. This includes the calculation of the sample size necessary to detect significant differences between the labs by using statistical tools, selection of suitable samples to be used in the scope of the transfer validation, the calculation of the critical and/or relevant difference as an acceptance criterion for the comparability based on actual process capability data as well as the evaluation of the results generated by the transferring and the receiving lab during parallel testing by using statistical tools, which are not always available at a contract lab. This will be discussed based on the example of a bioassay, which was transferred to a Japanese contract lab.
- (Day 2: Approaches to Assay Validation Session)
- Speaker: Paula Urquhart, Covance
- Abstract: One of the key tests in the assessment of Biosimilarity is relative potency. Typically cell based Bioasaays used to determine potency are variable and time consuming.Sensitive bioassays in simple kit form (DiscoverX), targeted at Biosimilar molecules, are currently being developed and assessed. In this way, biosimilar molecule testing can produce relative potency results within reduced time frames e.g. <48hrs.The development of a bioassay kit to measure biosimilar molecules of anti-vascular endothelial growth factor (anti-VEGF) will be presented.VEGF signalling is well established as an inducer of cell proliferation and promotes cell migration (1). Uncontrolled regulation has been implicated in the development of disease states such as polycystic ovaries and ovarian cancer in which abnormal angiogenesis occurs (2). Anti-VEGF molecules have been successfully released onto the market e.g. Avastin (Bevacizumab) and Eylea (Aflibercept) for conditions including metastatic colorectal cancer, metastatic kidney cancer and wet age related macular degeneration. Biosimilars of these drugs are now being tested in preparation for release after current patents end.1) The FASEB Journal (1999), 13, 9-22
2) Mol Med Rep. 2016 Apr 25. doi: 10.3892/mmr.2016.5173.
- (Day 2: Bioassays for Biosimilar Products Session)
Development and Validation of Potency Assay for a TNFα Blocking Monoclonal Antibody: Adalimumab as a Case Study
- Speaker: Maryam Varposhti, CinnaGen Medical Biotechnology Research Center, Alborz University of medical sciences, Karaj, Iran
- Abstract: Anti-tumor necrosis factor (anti-TNF) drugs are a class of drugs that are used worldwide to treat inflammatory conditions. This presentation will give an overview on developing a reliable method for in vitro biological activity measurement of Adalimumab. During inflammatory diseases TNFα binds to its receptor on cell surface and induces cell death. The Mode of Action (MoA) of the drug is reducing the inflammation by binding to TNFα and preventing it from binding to its receptor. We have developed a proliferation-based bioassay method for Adalimumab and a proper method for quantitative measurement of living cells. TNFα blocking ability of the drug was compared with a suitable reference material. Method validation was performed according to ICH guidelines and system suitability parameters were defined. Also, a proper statistical data analysis method was set for the experiment. Here we discuss the assay development steps and the changes which were applied during the development process in order to optimize the method.
- (Day 2: Product Specific Potency Assay Development)
- Speaker: Thorsten Verch, Merck
- Abstract: Proteins in complex mixtures are quantified using immunoassays such as ELISAs. Typical ELISA protocols are carried out in 96-well microtiter plates and involve multiple steps, wash separations, and the use of multichannel liquid handling equipment such as pipets and washers. At the end, spectrophotometers that acquire assay signals read signals in a row- or column-wise pattern.
Many publications report two major sources of bias in ELISAs: 1. Bias from extensive serial dilutions to reach the sensitive assay range and 2. Positional effects with lower signal along the edges of the plate.
We addressed dilution bias in a relative potency ELISA by incorporating a fluorescent dye into the first step of the sample and reference preparations. Prior to sample incubation on the assay plate, the dye signal was measured and used later to correct final assay results by the measured dilution bias.
We mitigated positional effects by a block-randomization scheme. Curve points of samples and reference were distributed in a pattern designed to minimize the impact of positional bias on the measured result.
Using dilution correction and block-randomization, we were able to reduce ELISA variability by 50% and also improve accuracy.
Thorsten Verch, Chris Roselle, Mary Retzlaff
- Day 1: Method Optimization Session
- Speaker: Thomas Wilton, Division of Virology
- Abstract: Oral live attenuated poliovirus vaccine (OPV) and inactivated poliovirus vaccine (IPV) play an instrumental role in the Global Poliovirus Eradication Initiative (GPEI). The potency of OPV can be assessed by an in vitro assay which measures the infectivity of monovalent polio vaccine bulk harvests using a 50 % end point technique in microtitre system. This assay is used to determine dilutions and validate titres of samples tested for neurovirulence. The potency of IPV can be assessed by an in vitro assay which is based upon the assessment of the quantity of the D-Antigen (D-Ag) units in an IPV. The D-Ag unit is used as a measure of potency as it is largely expressed on native infectious virions and is the protective immunogen. The indirect ELISA is the most commonly used in vitro test. However the potency of IPV is primarily assessed by an in vivo assay developed in rats. This assay is based on the assessment of the neutralising antibody titer within the sera of rats. With the development of transgenic mice expressing the human poliovirus receptor, immunisation-challenge tests have been developed to assess the potency of IPVs.
- Speaker: Pin Yee Wong, Genentech
- Abstract: This presentation will highlight some of the advances in bioassays such as the incorporation of new technologies and approaches which have significantly increased speed, sensitivity, efficiency, throughput and consistency of potency assays and the bridging experiments or validation performed to support the implementation of these enhancements. Case studies will focus on the development of bioassays for multi-domain proteins, implementation of automation in QC, and implementing use of ready-to-use cryopreserved cells for a legacy method.
- (Day 1: Method Optimization Session)
- Speaker: Ann Yellowlees, Quantics
- Abstract: The demonstration of biosimilarity between a candidate biosimilar product and the innovator product involves comparing the two products for a range of properties. The aim is to show that the (putative) biosimilar is equivalent to the innovator for each property. For any given property assay, demonstrating biosimilarity must therefore be based on demonstrating that a confidence interval for the reportable value, e.g. the relative potency lies entirely within a pre-specified interval – for example 80% – 125%.For relative potency (RP), defined as the ratio of the doses required to achieve the same response, this is only unique (and therefore a useful measure) when the dose-response curves of the two products are parallel – otherwise the ratio varies with the response level. If the curves are not parallel, it is not possible to capture the difference in potency between two products in a single number. This is not necessarily the end of the road for the candidate biosimilar: it may be possible to capture the differences using pair or triplet of numbers, all of which could potentially be used to demonstrate equivalence.The aim of this talk is to explore methods of handling the statistical analysis of reportable values which are relative to a reference standard, where the sample does not behave as a dilution of the reference standard.
- (Day 2: Bioassays for Biosimilar Products Session)