BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

          Bispecific antibodies consist of two small-chain fragments variable (scFv) of the antibody that has binding affinities to different target antigens. T cell engagers are a class of bispecific antibodies that selectively recruit and activate T cytotoxic cells in the tumor tissue and mediate cytotoxicity. In T cell engagers, one scFv domain is designed to bind to CD3 molecules in the T cell, and the second scFv domain binds to a tumor-associated antigen expressed in the tumor tissue. Certain tumor-associated antigens are known to express in specific cancer types. These tumor-associated antigens have been targeted for the treatment of these specific cancers.  These tumor antigens include CD19, BCMA, PSMA, HER2, TROP2, etc.            Bispecific antibodies, similar to any protein therapeutics, can evoke the generation of anti-drug antibodies. Therefore, detection and monitoring of anti-drug antibodies is an i...

S urface Plasmon Resonance Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real-time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions. SPR signal is directly dependent on the refractive index of the medium on the sensor chip. The binding of biomolecules results in changes in the refractive index on the sensor surface. In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to a second molecule (the Analyte) is measured under flow. Response is measured in resonance units (RU) and is proportional to the mass on the surface, and for any given interactant, the response is proportional to the number of molecules bound to the surface. Response is recorded and displayed as a sensogram in real time. Principle of Surface Plasmon Resonance (GE) Application of SPR Increase understanding of molecular mechanisms and structure-function relationships Define poten...

Ion exchange chromatography Principle:  Ion exchange chromatography is a technique for separating and purifying the compounds in the mixture based on their net charge. Ion exchange chromatography resin contains negatively or positively charged functional groups covalently bound to a solid support, yielding either a cation or anion exchanger, respectively.  Charged compounds are adsorbed and retained by an ion exchanger having the opposite charge, whereas compounds that are neutral or have the same charge as the media pass through the void volume and are eluted from the column.  The binding of the charged compounds is reversible, and adsorbed compounds are commonly eluted with a salt or pH gradient. Ion exchange media are available in various particle sizes, ionic forms, and purity ranges.  Ion exchange chromatography is used both for preparative and analytical purposes and can separate a large range of molecules from amino acids and nucleotides to large prote...

Anti-drug antibody (ADA) assays are utilized for the detection and characterization of antibodies that are generated against the therapeutic molecules. These therapeutic candidates can be proteins, enzyme, viral packaged gene or RNA for interference or exon skipping. The detection and characterization of ADA against therapeutic candidates are crucial for immunogenicity profiling and investigation of immune response-related safety events. (Ref: Immunogenicity Assessment). The assay sensitivity is the lowest concentration of antibodies that can be consistently detected by the method. The sensitive assay should detect ADAs in the clinical samples before they reach a level that can be associated with the altered PK/PD & safety profile (clinically relevant ADA). Evidence from clinical studies has shown that ADA levels as low as 100 ng/ml may be associated with altered PK/PD or safety profile. Therefore, the Food and Drug Agency (FDA) and the European Medical Agency (EMA) expect the se...

Introduction The multi-tiered bioanalytical approaches that are commonly implemented for the assessment of immunogenicity include detection (screening, confirmation, and titer) and characterization of the anti-drug antibody. The anti-drug antibody (ADA) positive samples during the screening assay are run in a confirmatory assay. A specificity confirmatory assay eliminates any false positives detected on screen assay. The titers are used to obtain relative quantitation of ADA in the samples. The appropriate assays are developed to characterize the neutralizing capacity of antibodies in the ADA positive samples. Furthermore, assays for relevant biomarkers and/or pharmacokinetic measurements should complement ADA characterization and analysis of their in vivo impact. (Buttel et al, 2011) Figure 1- Tier Based Bioanalytical Approach for Immunogenicity Assessment Detection of Antidrug antibodies (Screening and confirmatory assay): Screening assays are the initial step of immun...

Human Gene Therapy for Rare Diseases  CONSIDERATIONS FOR PRECLINICAL STUDIES  A preclinical program that is tailored to the investigational product and the planned early-phase clinical trial contributes to the characterization of the product’s benefit/risk profile for the intended patient population. The overall objectives of a preclinical program for a GT product include:  identification of a biologically active dose range;  recommendations for an initial clinical dose level, dose-escalation schedule, and dosing regimen; establishment of feasibility and reasonable safety of the proposed clinical route of administration (ROA);  support of patient eligibility criteria; and  identification of potential toxicities and physiologic parameters that help guide clinical monitoring for a particular investigational product.  In addition, to justify conducting a pediatric first-in-human clinical trial that is associated with more than a minor increase over minima...

Anti-drug antibody (ADA) assay is a semi-quantitative bioanalytical method that detects the antibodies specific to the drug. The ADA assay utilizes the assay cut point above which the samples are considered positive. The assay cut points are established by analyzing a set of approximately 50 drug-naive individuals with an assumption that these selected samples do not contain any pre-existing antibodies. While this is true for most ADA assays, in some instances, there is a high probability of prevalent pre-existing antibodies when a closely related antigen is exposed prior to drug administration. The pre-exposure of antigens such as adeno-associated viruses (AAVs), Cas9 protein, and PEG molecule account for preexisting antibodies in clinical subjects. When the preexisting antibody is prevalent in the study population, the cut point should be established carefully to enable the detection of the clinically significant ADA. Applying the recommended statistical cut point calculation metho...

Agilent RapidFire 400 Technical Specification The Agilent RapidFire 400 is an integrated autosampler for ultrafast sample cleanup based on automated solid-phase extraction (SPE) and introduction to mass spectrometry. Designed for highest-throughput laboratories, the large capacity, temperature-controlled, 1536 well-plate handling robot allows unattended weekend operation with sample injection times as short as 8 seconds. RapidFire 400 builds on successful previous models, combining more than 14 years of high-throughput mass spectrometry innovation. This system provides reliable high throughput and accurate measurements for the qualification of drug targets like automated ADME in discovery, compound binding assessment, elucidation of metabolic processes, the discovery of novel metabolomics targets, and much more. Fully controlled by a single PC, the RapidFire 400 can be combined with any Agilent LC/Q-TOF or LC/TQ, while integrating the Ultivo LC/TQ into the system dramatically redu...

Anti-Adeno Associated Virus Total Antibody Assay  - A variety of platform has been used for the detection of total anti-AAV antibodies in animals and humans  - Two main formats have been reported in the literature         i)  Direct ELISA Format         ii)  MSD Bridging Assay Format i) Direct ELISA Format - The direct ELISA format has been used to detect the total anti-AAV antibodies, anti-AAV IgG & IgM isotype characterization.  - In these assays, the specific AAV full capsids are used. For example, AAV8 capsids are used for the detection of anti-AAV8 antibodies - The AAV capsid may be directly absorbed into the high binding 96 well plates.  - Alternately, biotin-conjugated AAV may be used as a capture. In this case, streptavidin precoated plates should be used. - The assay is optimized with sequential steps that include: a coating of the plate with AAV capsid, the addition of sample, and finally the add...

 Preclinical evidence to support a prospect of direct benefit is most important when clinical evidence of effectiveness is not available from adult subjects with the same disease. Further details for general considerations in preclinical studies of these investigational GT products are available in a separate guidance document. The Food and Drug Administration issued draft guidance for "Human Gene Therapy for Neurodegenerative Diseases" for the development of a preclinical program for an investigational GT product intended for the treatment of neurodegenerative disease. Objectives of Preclinical Studies: A preclinical program that is tailored to the investigational product and planned early-phase clinical trial contributes to the characterization of the product’s benefit/risk profile for the intended patient population. The overall objectives of a preclinical program for a GT product include:       1) identification of a biologically active dose range;  ...

Introduction Regulatory agencies require a pivotal phase 2/3 study to prove that the outcome of treatment is higher than the potential risk of a proposed therapeutic protein. This study includes evaluation of efficacy and safety of therapeutic proteins for intended use and formulation risk mitigation strategy for adverse/inadvertent events if any. The immunogenicity assessment is the core of assessing safety and efficacy in both clinical and non-clinical studies. Therapeutic proteins (TP) are immunogenic in nature and propensity of immunogenicity is attributed to structure, conformation, and posttranslational modifications of TP. In addition, disease population; individual genetic factors; manufacturing processes, dosages, and route of administration also impact immunogenicity. The clinical outcome of immunogenicity also depends on the affinity against drugs, isotype, amount, and persistence of anti-drug antibodies (ADA) developed, the epitope recognized on the TP, and the ability o...

The robust assays are crucial for facilitating the understanding of immunogenicity and their impact on pharmacokinetics, pharmacodynamics, safety, and efficacy of therapeutic protein products. During development and validation, assay sensitivity, specificity, drug tolerance, precision,  critical reagent stability are assessed. Table 2: FDA guidance for Immunogenicity Testing Assays: Summary of the key assay parameters for developing and validating ADA assays Assay Parameters Description Assessment Method Cut point (Click here for details) -The level of response of the assay that defines the sample response as positive and negative. -Screening, confirmation, and titer cut points are used for three tiers of the assay. - Cut-points are critical to minimize the risk of false-negative  - Drug naïve individuals from the target population should be used to derive cut point, if feasible. - Normal healthy individuals obtained from commercial vendors during develo...