Quality Control Environment Research Articles

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Abstract 4249: Quantitative PBMC ADCC bioassay and ADCC reporter bioassays for immunotherapy and SARS-CoV-2 monoclonal antibody development

Abstract Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies (mAbs). Quantitative measurement of antibody-dependent cellular cytotoxicity (ADCC) is critically required for understanding the Fc function in mAb drug development. Despite the increasing interest and clinical success of the mAb therapeutic, it has been highly challenging to measure their ADCC activity in a reproducible and quantitative manner due to the lack of consistency in current methods that are based on primary PBMCs or NK cells and use tedious assay procedures. To improve ADCC assay precision so they can be validated as potency assay in cGMP laboratories, we developed reporter based ADCC bioassays using engineered effector cell line stably expressing a luciferase reporter and FcγRIIIa (V or F variant) to replace primary PBMC to overcome the assay variation. The ADCC reporter bioassays have been validated according to ICH guidelines by many laboratories and are demonstrated to be suitable for product release and stability studies in a quality-controlled environment. For early research and antibody characterization, we developed an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells so they can measure the target specific lysis in ADCC. The PBMCs used in the study are isolated from prescreened blood donors and QC tested in ADCC assay. When HiBiT target cells are incubated with an antibody and PBMCs, HiBiT are released to the culture medium where it binds to LgBiT in the detection reagent to form a functional NanoBiT luciferase to generate luminescence signal. This new PBMC ADCC bioassay is simple, homogenous, highly sensitive, and gives a robust assay window. We demonstrate that it can quantitatively measure the potency for mAb drugs in cancer immunotherapy (e.g., rituximab, trastuzumab), and for anti-SARS-CoV-2 spike antibodies in antiviral drug development. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay. In summary, the new PBMC ADCC bioassay using HiBiT target cells can be a valuable tool for early antibody discovery and characterization and also for method bridging study with ADCC reporter bioassay. Citation Format: Pete Stecha, Jun Wang, Denise Garvin, Julia Gilden, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative PBMC ADCC bioassay and ADCC reporter bioassays for immunotherapy and SARS-CoV-2 monoclonal antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4249.

Concurrent 10. Presentation for: Collaborating to bring new technology on developments

Presented on Wednesday 18 May: Session 10 Project delivery certainty is a key a success factor for positive business outcomes. Taking on world first applications across multiple delivery streams comes with risk that requires significant risk reduction to meeting business requirements. The Julimar Development completed this successfully across both drilling and completions, and subsea. Reliable multizone completions with robust sand control have traditionally been a challenge in high-rate gas wells. Cased hole stacked gravel packs can leave high mechanical skin, require multiple trips and are complex operations. While Open Hole Gravel Packs (OHGPs) have provided reliable sand control in such wells, multizone applications have been limited due to the tradeoff between effective gravel placement and zonal isolation. Recent developments in technology have enabled reliable gravel placement and complete zonal isolation. The collaboration between Woodside and Subsea 7 has delivered an ‘industry first’ on the Julimar Project, with the completion of an 18″ Corrosion-Resistant Alloy (CRA) gas transmission flowline installed via reel-lay – the largest diameter insulated CRA pipeline ever reeled. For background, most projects have traditionally used the ‘S-Lay’ method for installing pipe in Australia – the reel-lay method is less common. A key benefit of the reel-lay method is it removes thousands of welds from the offshore installation vessel critical path, transferring them onshore into a safer, quality-controlled environment earlier in the schedule. Pipe joints are welded into ‘stalks’ which are then spooled onto purpose-built reel-lay vessels. Woodside and Subsea 7 were able to jointly demonstrate the safety, quality, technical and cost advantages of this innovative but field-proven reeled pipe-lay technology. The 18″ CRA flowline is a tangible example of the performance that can be delivered through early collaborative engagement and strategic investment in technology and is a step change in Australia and the industry. This joint presentation discusses contracting, design, execution and evaluation of technologies on the Julimar Development. It includes the method followed for reel-lay technology selection, engineering development, post project evaluation and the health and safety benefits. The presentation provides both the Operator and Contractor perspectives on the challenges of implementing new technology in developments in Australia. To access the presentation click the link on the right. To read the full paper click here and also here.

Development, validation and standardization of urinary 5-hydroxyindoleacetic acid determination by LC-MS/MS for implementation in quality-controlled environments

Carcinoid tumors referred to neuroendocrine neoplasms that often are indolent and may not became clinical apparent until there has been metastatic spread. Urinary 5-hydroxyindoleacetic acid (5-HIAA) was recommended as a first-line screening biomarker for the diagnosis and follow-up of carcinoid tumors. The measurement of this analyte is conventionally performed by spectrophotometer or high performance liquid chromatography, and has switched to liquid chromatography-tandem mass spectrometry (LC-MS/MS) recently. In this study, a fast, simple and reliable LC-MS/MS method has been developed and validated for 24 h urinary 5-HIAA determination and the quality assurance referring to post-implementation monitoring has been explored. 50 μL of urine was mixed with 200 μL of a 50% methanol/water solution containing the internal standard 5-HIAA-d5. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. The retention time of 5-HIAA is 2.37 min and a total run time is 4 min. This method was validated for excellent linearity from 0.675 to 43.3 μM with CVs ≤ 6.64% and good recovery in the range of 87.1%-107%. No obvious matrix effect was observed. Intra- and inter-day imprecision were below 3.95% and 4.66% respectively. The reference interval of 24-hour urinary 5-HIAA in Chinese adults was established. The quality assurance could ensure reliable and comparable results in routine clinical testing. Thus, this fast, simple and reliable LC-MS/MS method could be proposed as a tool for clinical testing of urinary 5-HIAA in quality-controlled environments.

Collaborating to bring new technology on developments

Project delivery certainty is a key a success factor for positive business outcomes. Taking on world first applications across multiple delivery streams comes with risk that requires significant risk reduction to meeting business requirements. The Julimar Development completed this successfully across both drilling and completions, and subsea. Reliable multizone completions with robust sand control have traditionally been a challenge in high-rate gas wells. Cased hole stacked gravel packs can leave high mechanical skin, require multiple trips and are complex operations. While Open Hole Gravel Packs (OHGPs) have provided reliable sand control in such wells, multizone applications have been limited due to the tradeoff between effective gravel placement and zonal isolation. Recent developments in technology have enabled reliable gravel placement and complete zonal isolation. The collaboration between Woodside and Subsea 7 has delivered an ‘industry first’ on the Julimar Project, with the completion of an 18″ Corrosion-Resistant Alloy (CRA) gas transmission flowline installed via reel-lay – the largest diameter insulated CRA pipeline ever reeled. For background, most projects have traditionally used the ‘S-Lay’ method for installing pipe in Australia – the reel-lay method is less common. A key benefit of the reel-lay method is it removes thousands of welds from the offshore installation vessel critical path, transferring them onshore into a safer, quality-controlled environment earlier in the schedule. Pipe joints are welded into ‘stalks’ which are then spooled onto purpose-built reel-lay vessels. Woodside and Subsea 7 were able to jointly demonstrate the safety, quality, technical and cost advantages of this innovative but field-proven reeled pipe-lay technology. The 18″ CRA flowline is a tangible example of the performance that can be delivered through early collaborative engagement and strategic investment in technology and is a step change in Australia and the industry. This joint presentation discusses contracting, design, execution and evaluation of technologies on the Julimar Development. It includes the method followed for reel-lay technology selection, engineering development, post project evaluation and the health and safety benefits. The presentation provides both the Operator and Contractor perspectives on the challenges of implementing new technology in developments in Australia.

Size Exclusion Chromatography with Dual Wavelength Detection as a Sensitive and Accurate Method for Determining the Empty and Full Capsids of Recombinant Adeno-Associated Viral Vectors.

Gene therapy has evolved over the past decade into a promising therapeutic class for treating many intractable diseases. Recombinant adeno-associated virus (AAV) is the most commonly used viral vector for delivering therapeutic genes. Independent of the manufacturing process for AAVs, the clinical materials are inherently heterogeneous and contain both empty and full capsids. Empty capsids can impact the safety and efficacy of AAV products and therefore their level needs to be controlled. Several analytical methods have been reported for this purpose. However, some of these methods have an insufficient assay range, or rely on instruments that cannot be readily implemented in a quality control (QC) environment. In this study, we describe a fast size exclusion chromatography (SEC) assay with dual-wavelength detection (SEC-DW) to directly determine the percent full capsids of AAV samples based on their peak area (PA) ratios. The two detection wavelengths selected to represent encapsidated transgenes and capsid proteins were 260 and 230 nm, respectively, instead of the conventionally used 260 and 280 nm. The use of 230 nm instead of 280 nm to monitor the contribution of the capsid protein results in a linear relationship between the PA260/PA230 ratio and the percent full capsids, unlike the nonlinear relationship observed when the PA260/PA280 ratio is used. As a result, the method exhibits a significantly extended assay range (up to 91% full capsids). The accuracy of the SEC-DW method was confirmed by comparing the results obtained against results from orthogonal high-resolution methods such as analytical ultracentrifugation (AUC) and cryo-electron microscopy and excellent agreement was obtained when common samples were analyzed using different methods. The SEC-DW method runs on a readily accessible high-performance liquid chromatography instrument platform, provides much higher assay throughput compared with AUC and electron microscopy, and can be implemented as a release method in a QC environment or used as a rapid screening tool to support process development and product understanding.

201 Genital Examination Education and Instruction: Lowering Anxiety and Raising Competence

Gynaecologic Teaching Associate (GTA) and Male Urogenital Teaching Associate (MUTA) methodology have been utilized for decades in effective breast, pelvic and urogenital examination clinical skill instruction. This methodology is recognized as the gold standard of instruction when educating learners on the sensitive, invasive clinical skills techniques associated with a genital examination. While research shows it is the most effective way to learn these procedures, outside of the USA and Canada, there are few GTA/MUTA programmes at medical learning institutions.This methodology aims to provide hands-on, standardized instruction in an anxiety-free environment where learners safely practice the clinical skills techniques of breast, pelvic and urogenital examinations and have the unique experience of learning these techniques from the patient’s perspective.The GTA/MUTA is both instructor and live simulated patient, using their own bodies as teaching tools, guiding learners through examination techniques and providing instant feedback. With this unique opportunity for skills acquisition, learners receive step-by-step instruction on an actual person in a quality-controlled environment. In addition to correct palpation techniques, this patient-centred form of instruction addresses the emotional reaction patients may have to these examinations. GTA/MUTA instruction also includes patient education and communication and relaxation techniques. The GTA/MUTA patient empowerment methodology is designed to provide an anxiety-free atmosphere for the learner so that the sensitive nature of the genital examination and the embarrassment often accompanying the examination do not become an obstacle to acquiring safe, effective clinical technique.Decades of research prove that this method lowers learner anxiety and provides exceptional outcomes for learners in a multiplicity of learning criteria, including higher overall scores; superior communication skills; better ability to identify pathology; ‘better interpersonal skills than physician trained with lasting effects that can be demonstrated after clinical exposure’ The methodology has far-reaching implications. The specialized skills of these individuals mean that the teaching method can be brought outside of the well-patient experience; sexual assault providers can practice the trauma examination on live simulated patients; remediation can be provided to practitioners who must relearn techniques to maintain licensure; learners are more empathetic to their patients and more inclined to include their patients in the examination process thus improving patient care. This methodology can be utilized in any setting where invasive examination procedures, patient education and communication must be mastered.

Abstract 506: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the reproducibility of these assays is not adequate and they have major issues on biosafety or weak assay sensitivity. Moreover, the use of primary effector cells makes them prone to donor variability, low-throughput and therefore not suitable for implementation in quality-control drug development. We previously developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing primary PBMC-based ADCC assays and ADCC reporter bioassays, we report the development of an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells for therapeutic monoclonal antibodies with Fc functions. When target cells expressing HaloTag-HiBiT are incubated with an antibody and PBMCs, the target cells are lysed and release HaloTag-HiBiT, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate Bioluminescence signal. In addition, it can enable the quantitative measurement of antibody-mediated target cell lysis via specific HaloTag staining using a fluorescent HaloTag ligand by flow cytometry. We demonstrate that the new PBMC ADCC assay is simple, homogenous, highly sensitive, and gives a robust assay window, and is capable of measuring potency of rituximab, cetuximab or trastuzumab. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Gopal B. Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 506.

MASTODON: An Open-Source Software for Seismic Analysis and Risk Assessment of Critical Infrastructure

Seismic analysis and risk assessment of safety-critical infrastructure like hospitals, nuclear power plants, dams, and facilities handling radioactive materials involve computationally intensive numerical models and coupled multiphysics scenarios. They are also performed in a strict regulatory environment that requires high software quality assurance standards, and in the case of safety-related nuclear facilities, a conformance to the American Society of Mechanical Engineers Nuclear Quality Assurance (NQA-1) standard. This paper introduces the open-source finite-element software, MASTODON (Multi-hazard Analysis of Stochastic Time-Domain Phenomena), which implements state-of-the-art seismic analysis and risk assessment tools in a quality-controlled environment. MASTODON is built on MOOSE (Multi-physics Object-Oriented Simulation Environment), which is a highly parallelizable, NQA-1 conforming, coupled multiphysics, finite-element framework developed at Idaho National Laboratory. MASTODON is capable of fault rupture and source-to-site wave propagation using the domain reduction method, nonlinear site response, and soil-structure interaction analysis, implicit and explicit time integration, automated stochastic simulations, and seismic probabilistic risk assessment. When coupled with other MOOSE applications, MASTODON can also solve strongly and weakly coupled multiphysics problems. This paper presents a summary of the capabilities of MASTODON and some demonstrative examples.

Development, validation and standardization of oromucosal ex-vivo permeation studies for implementation in quality-controlled environments

Tissue-based ex-vivo studies on the oromucosal permeability of drugs are often insufficiently adapted to physiological and clinical conditions, which limits their predictivity. Moreover, the scientific community demands for the standardization of ex-vivo studies, since conceptual limitations (e.g. low sensitivity of analytical methods, insufficient monitoring, different designs) restrict the wide implementation in preclinical drug development.Therefore, an innovative ex-vivo permeation process consisting of novel Kerski diffusion cell coupled to fully automated sampling and sample preparation with LC–MS/MS quantification was developed and standardized. Novel assays for routine examination of tissue integrity and viability were developed and embedded in a comprehensive analytical control system. The high level of standardization and automation reduced the differences of between-run to within-run precision to ≤ 0.27 % CV. Successful validation proved a broad calibration range of 0.93–952.38 ng/mL of the model drug cyclobenzaprine with guideline-compliant relative errors from -7.9–12.6 % (between-run accuracy). Consequently, the method allowed the physiological-clinical alignment of the study conditions to therapeutic doses and the short residence time of intraoral drugs (sampling times 1−60 min).Applicability was demonstrated by assessing the oromucosal permeability for different sublingual cyclobenzaprine hydrochloride formulations representing the excipient selection as a common aspect during galenic development. Thereby, expressive evaluation of the dosage forms was achieved resulting in an improved permeation by replacing croscarmellose into polyvinylpyrrolidone (cumulative amount of 42.6 vs. 112.6 μg/cm²). Thus, the automated permeation process ensured lean, standardized and reproducible assessment of oromucosal permeability within quality-controlled academic and regulatory environments. Simultaneously, the improved ex-vivo predictivity through physiological-clinical adjustments facilitates the reduction of costly in-vivo studies.

Abstract 944: Quantitative cell-based reporter bioassays for functional characterization of immunotherapeutic biologics targeting co-stimulatory and co-inhibitory immune checkpoint receptors

Abstract The human immune system is regulated by a complex network of inhibitory and stimulatory immune checkpoint receptors that regulate T Cell function. These receptors are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune disorders. Novel therapies employing a variety of strategies have been developed that rely on releasing the brakes on the immune system, with the aim of eliminating tumor cells even more efficiently. 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, TIM-3, TIGIT) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) receptors, individually and in combination. A major challenge in the development of antibody-based 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 required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. PD-1, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These bioassays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals from TCR and immune checkpoint receptors. Thus, these bioassays reflect mechanisms of action for drug candidates designed for each immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. The bioassays are prequalified according to ICH guidelines and show the precision, accuracy and linearity required for routine use in potency and stability studies. Here we describe the application of MoA-based immune checkpoint receptor bioassays as tools for biologics drug discovery, development, potency and stability studies. Citation Format: Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Gopal Krishnan, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based reporter bioassays for functional characterization of immunotherapeutic biologics targeting co-stimulatory and co-inhibitory immune checkpoint receptors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 944.

Assay of moment resisting precast and monolithic frames using ABAQUS software

Precast structures are concrete structures made in a factory and cured under proper supervision. It is fabricated in a quality-controlled environment and then shifted to the site ready to the erection. In a precast factory, the focus is made on different aspects like batching, testing, rebar work. Precast concrete applications include pipe culverts, facades, retaining walls, stadiums, and buildings. The present study aims to analyze the failure of the beam-column joint with dry mechanical and wet connections in a moment-resisting frame using the ABAQUS software. The dry mechanical connections include J-bolt and cleat angle with stiffener placed with M40 grade concrete and wet connection of M50 grade fiber concrete at frame joints. A numerical method in the finite element analysis software was developed and calibrated for results. The most important observations obtained are spatial displacement patterns, load vs displacement of J-bolt connection, cleat angle with stiffener connection, wet precast connections and a monolithic frame. Other parameters like interaction, convergence is also analyzed to find out the best-precast connection.

Abstract 4113: Quantitative cell-based bioassays to advance immunotherapy programs targeting immune checkpoint receptors

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 disorders. The Nobel prize for Medicine in 2018 was awarded for seminal studies on the role of immune checkpoint targets in T cell activation and furthermore, combining different strategies to release the brakes on the immune system with the aim of eliminating tumor cells even more efficiently. Immunotherapies designed to block co-inhibitory receptors (e.g. PD-1, CTLA-4, TIGIT) 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, TIM-3) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) receptors individually and in combination. A major challenge in the development of antibody-based 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 required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. PD-1, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals. Thus, the bioassays reflect mechanisms of action for the drug candidates designed for each immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. Here we describe the application of MoA-based immune checkpoint receptor bioassays as tools for biologics drug discovery, development, potency and stability studies. Citation Format: Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Gopal Krishnan, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays to advance immunotherapy programs targeting immune checkpoint receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4113.

Quality management for academic laboratories: burden or boon? Professional quality management could be very beneficial for academic research but needs to overcome specific caveats.

EMBO Reports (2018) e47143 Basic and translational biomedical research explores biological and pathophysiological mechanisms with the aim of developing novel therapies, preventive measures, and diagnostics to improve human health. Disappointingly, however, most new therapies fail when they are tested in clinical trials. Although the causes of this “translational attrition” are diverse and often rooted in the complexity of the underlying biology, it has also become clear that methodology is a major issue. The “translational roadblock”, along with what has been dubbed a “reproducibility crisis” [1], has fueled discussions about the reliability and reproducibility of biomedical research in general. There is strong evidence that weaknesses in planning, conducting, analyzing, and (non)reporting of research [2], as well as misidentification or contamination of reagents, biologicals, and cell lines [3], are prevalent factors. Meta‐research has shown that these problems can lead to an inflation of effect size and false positives and consequently decrease the reproducibility and predictiveness of research results. At the same time, the increasing methodological complexity combined with the immense proliferation in research outputs greatly complicates the production and evaluation of reliable evidence. Pressure to publish and hypercompetition for resources further compromise the robustness and rigor of research. Arguably, basic and translational biomedical research has a quality problem. In the 1970s, US cars had major quality problems. By comparison, Japanese cars were much more reliable. The introduction of rigorous quality management in the production process was largely credited for this competitive advantage, which helped the Japanese car industry to dominate the market for decades to come. By now, most industries, including US car manufacturers, the health and pharmaceutical industries as well as clinical medicine, have established sophisticated quality management systems (QMS) on which they spend several percent of their budget. Clearly, these investments pay off as companies …

Analytical Method Development Using Transmission Raman Spectroscopy for Pharmaceutical Assays and Compliance with Regulatory Guidelines—Part II: Practical Implementation Considerations

Transmission Raman spectroscopy is a relatively new technique for quantitative analysis of pharmaceutical products, either during manufacturing or as a finished product test. As with any new analytical tool, several requirements need to be met for widespread application. These include assessment of technical capability, integration with quality and manufacturing processes and successful deployment in a quality-controlled environment. In the first paper of a two-part series, regulatory guidelines and method development were discussed for the creation of transmission Raman spectroscopic methods for content uniformity (CU), assay and drug product identity (ID) applications. In this part II, the practicalities of method development are addressed, and an example of the development of a quantitative method for the determination of drug content uniformity in individual tablet cores using partial least-squares is presented.

Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products

Mass spectrometry performed in non-denaturing conditions (native MS), and its hyphenation to ion mobility spectrometry (IM-MS), have gained interest for the qualitative and quantitative characterization of intact monoclonal antibody-related (mAb) products. However, one main drawback is the manual sample preparation, which hampers its routine use in high throughput automated environments. Size exclusion chromatography (SEC) appears as an interesting technique to perform online buffer exchange in an automated way. We present here an exhaustive and systematic evaluation of the possibilities and versatility of SEC direct hyphenation to native MS or IM-MS (SEC-nativeMS/IM-MS) for the characterization of a variety of mAb-formats (IgGs, ADCs, bispecific mAbs and Fc-fusion proteins). First, online SEC-native MS allows automated sample preparation, resulting in high resolution mass spectra and improved mass accuracies (<80 ppm) compared to manual buffer exchange procedures. When hyphenated to ion mobility, SEC-native IM-MS can deliver conformational characterization through collision cross section (CCS) measurements within few minutes without affecting mAb structures. Finally, benefits of online SEC-nativeIM-MS compared to standalone SEC-UV or native MS techniques are demonstrated for higher order structure characterization of mAb forced degraded samples. While SEC provides separation of high/low molecular weight species from the main mAb peak along with precise quantification of the species, native MS affords complementary unambiguous identification of SEC peaks, even when poor SEC separation is achieved. The synergic online coupling of SEC to native MS/IM-MS is envisioned to definitely push native MS approaches at the forefront of mAb characterization in quality-controlled environments and as multiple monitoring method.

Analytical Method Development Using Transmission Raman Spectroscopy for Pharmaceutical Assays and Compliance with Regulatory Guidelines—Part I: Transmission Raman Spectroscopy and Method Development

Transmission Raman spectroscopy is a relatively new technique for quantitative analysis of pharmaceutical products, either during manufacturing or as a finished product test. As with any new analytical tool, several things need to be accomplished for widespread application use—assessment of technical capability, fit with quality and manufacturing processes and successful deployment in a quality-controlled environment. The technical capability has been discussed previously, but the practicalities of method development and submission to regulatory authorities have not. In this, one of a series of two papers, the considerations for regulatory submission are discussed. In the second paper, practical method development will be presented and examples provided for data analysis and method development processes.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment.

Abstract 5610: Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy

Abstract Immune checkpoint receptors play a critical role in maintaining immune homeostasis and are genetically and functionally associated with autoimmune disease, cancer and persistent viral infections. Blockade of immune checkpoints (e.g., PD-1 and CTLA-4) has emerged as a promising new approach to enhance anti-tumor immune responses. While immunotherapies directed against PD-1 and CTLA-4 are showing unprecedented efficacy in the treatment of cancer, many patients and tumor types remain refractory to these therapies. This has resulted in a broadening of immunotherapy research and development to include additional immune checkpoint receptors (e.g., LAG-3, TIGIT, CD112R) targeted individually or in combination with other immunotherapy strategies. A major challenge in the development of biologics that target immune checkpoints 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 the quality of data that is 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 T cell receptor and immune checkpoint target(s). These mechanism of action-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. Citation Format: Jamison Grailer, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2017-5610