A bridging assay for detection and characterization of anti-drug antibodies to dostarlimab, a new anti-PD-1 therapeutic monoclonal antibody

Dostarlimab (JEMPERLI) is a humanized anti-programmed death 1 (PD-1) immunoglobulin (Ig)G4-kappa monoclonal antibody that binds to the PD-1 receptor and competitively inhibits binding of its ligands, PD-L1 and PD-L2. Dostarlimab was recently approved in the USA and the European Union. Because dostarlimab is a macromolecular therapeutic, it has the potential to elicit the formation of anti-drug antibodies, which have the capability to impact the drug’s safety and efficacy and to alter pharmacokinetics. The immunogenic potential of dostarlimab remains unknown, and it was therefore necessary to develop analytical assays to detect and characterize anti-drug antibodies as a key component in the mitigation of immunogenicity risk. Here, we present the development and optimization of a 3-tiered electrochemiluminescense bridging assay for the investigation of dostarlimab clinical immunogenicity. In this work, the full details of the method, statistical data analysis and cut point determinations, assay performance during clinical sample analysis, and associated regulatory expectations are discussed. The full validation of this 3-tier anti-drug antibody assay enabled dostarlimab immunogenicity evaluation in clinical studies. Trial registration: Clinicaltrials.gov, NCT02715284. Registered 9 March 2016

A risk-based approach to validation of ion chromatography methods using suppressed conductivity

Ion chromatography (IC) has evolved into one of the most widely used separation techniques of analytical chemistry. Consequently, the number of users of this method is continuously growing. Analysts often assume that widely used guidelines for HPLC method validation in regulated environments routinely apply to IC. This manuscript provides an analysis of the potential shortcomings of traditional approaches to development and validation of IC methods using suppressed conductivity detection and a risk-based alternative approach to these activities. The goal of the alternative approach is a reduction in the risk of erroneous determinations of analytes when IC methods using suppressed conductivity detection are employed.

An industrial case study: QbD to accelerate time-to-market of a drug product

The use of a Quality by Design (QbD) approach in the development of pharmaceutical products is known to bring many advantages to the table, such as increased product and process knowledge, robust manufacturing processes, and regulatory flexibility regarding changes during the commercial phase. However, many companies still adhere to a more traditional pharmaceutical process development—in some cases due to the difficulty of going from a theoretical view of QbD to its actual application. This article presents a real-world case study for the development of an industrial pharmaceutical drug product (oral solid dosage form) using the QbD methodology, demonstrating the activities involved and the gains in obtaining systematic process and product knowledge.

Gamma oryzanol niosomal gel for skin cancer: formulation and optimization using quality by design (QbD) approach

Skin cancer is fifth most diagnosed disease in human population due to ultraviolet radiation (UV) exposure. Gamma oryzanol (OZ) is a natural antioxidant, and it also has skin anti-aging properties. OZ is naturally found in rice bran oil. The main aim of the present work was to optimize OZ niosomal formulation using quality by design approach including one variable at a time and full factorial design. Niosomes were prepared by solvent injection method and characterized for size, polydispersity index, drug entrapment, and transmission electron microscopy. The optimized batch obtained at X1 [drug to span 60 molar ratio (1:5)], X2 [volume of hydration (75 mL)], and X3 [stirring speed (2500 rpm)] to Y1 [average vesicle size (196.6 nm)] and Y2 [entrapment efficiency (78.31%)] as dependent variables. The optimized OZ noisomes were formulated by niosomal gel to provide improved physicochemical stability upon topical application against UV. The niosomal gel was characterized using pH meter, viscometer, Draize test for skin irritancy, ex vivo permeation studies, and stability studies. Ex vivo permeation studies of OZ niosomal gel not only showed fourfold higher permeation but also exhibited better drug retention in dermal layers of skin as compared to OZ gel. Quality Target Product Profile of OZ niosomal formulation was generated. Risk analysis of optimized OZ gel suggested most critical quality attributes (CQAs) and critical process parameters (CPPs) to be characterized as low risk. Thus, γ-oryzanol niosomal gel for topical use can serve as a promising prophylactic treatment in skin cancer, and the developed prototype formulation can be further extended to future newly discovered drugs with similar characteristics. Graphical abstract

Meeting Report: Vaccine Stability Considerations to Enable Rapid Development and Deployment

The Stability Community of the American Association of Pharmaceutical Scientists (AAPS) held a virtual workshop on “Vaccine Stability Considerations to Enable Rapid Development and Deployment”, on March 24-25, 2021. The workshop included distinguished speakers and panelists from across the industry, academia, regulatory agencies, as well as health care leaders. This paper presents a review of the topics covered. Specifically the challenges in accelerating vaccine development and analytical characterization techniques to establish shelf-life were covered. Additionally, vaccine stability modeling using prior knowledge stability models and advanced kinetic analysis played a key in the EUA approaches discussed during the workshop. Finally, the role of stability studies in addressing the challenges of vaccine distribution and deployment during the pandemic were a focus of presentations and panel discussions.Although the workshop did not have any presentation topics directly dedicated to the mRNA vaccines, the techniques discussed are generally applicable. The mRNA vaccine developers were represented in the panel discussions, where experts involved in the EUA approval/deployment stages for this vaccine type could discuss the challenges as applied to their vaccines.

Preparation of O/W nano-emulsion containing nettle and fenugreek extract and cumin essential oil for evaluating antidiabetic properties

The oil-in-water (O/W) nano-emulsion (NE) is expanded to enhance the bioavailability of hydrophobic compounds. The NE can be prepared by herbal extract and essential oil as herbal medicines for antidiabetic treatment. In the present study, the O/W NE was prepared by fenugreek extract (FE), nettle extract (NE), and cumin essential oil (CEO) using tween 80 and span 80 surfactants in an ultrasonic bath, at room temperature within 18 min. The antidiabetic property was evaluated by determining glucose absorption using cultured rat L6 myoblast cell line (L6) myotubes and insulin secretion using the cultured mouse pancreatic beta-cell (RIN-5) for NEs. The samples were investigated by dynamic light scattering (DLS) to examine the size distribution and size, zeta potential for the charge determination, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to investigate morphology and size. The rheological properties were studied by viscosity. The sample stability was evaluated at different temperatures and days by DLS and SEM analyses. The cytotoxicity of samples was explored by MTT assay for HEK293 human cell line as a specific cell line originally derived from human embryonic kidney cells at three different concentrations for three periods of time. The NEs with nanometer-size were observed with antidiabetic properties, low cytotoxicity, and suitable stability. This study provides definitive evidence for the NE as a plant medicine with antidiabetic properties. The NE can be a good candidate for biomedical applications.

Designing an ideal alcohol-based hand sanitizer: in vitro antibacterial responses of ethanol and isopropyl alcohol solutions to changing composition

This study aimed to achieve an in vitro quantification of the effects of composition and formulation factors on the killing rates of alcohol-based hand sanitizers. The killing rates of 85% ethyl alcohol (ET) and isopropyl alcohol (IPA) were studied under different conditions such as pH, electrolyte concentration, or inclusion of herbal extracts (cucumber, carrot, and aloe vera), a quaternary ammonium compound, or thickener over different time intervals. Changes in the activities were retested after 3 months as an indication of stability. From two-way ANOVA, both the time of exposure and the sanitizer type affected the activity against Staphylococcus aureus (P = 0.001 for both alcohols), whereas for Escherichia coli, time of exposure was significant (P = 0.027), while sanitizer type was less significant (P = 0.063). Extreme pHs, the presence of ions, and the inclusion of additives such as benzalkonium chloride (BAC), plant extracts, or carbomer impacted the 3-month activity of the samples differently. Important differences existing in the activities of ET and IPA, as a function of formulation factors or use conditions have been quantified using in vitro methods. Formulations should best be tailored for particular purposes and the all-purpose hand sanitizer may not exist. Graphic Abstract

Tritium labeling of antisense oligonucleotides via different conjugation agents

A novel approach to tritium-labeled antisense oligonucleotides (ASO) was established by conjugating N-succinimidyl propionate, as well as maleimide-derivatives, to the 3′-end of ASOs targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat1) containing amino- or sulfhydryl-linkers. In vitro stability and Malat1 RNA reduction studies demonstrated that N-ethylmaleimide (NEM) could be used as a stable tag while maintaining the desired target interaction. The corresponding radioactive label conjugation using [3H]-NEM resulted in tritium-labeled ASOs with a high molar specific activity of up to 17 Ci/mmol. Single-dose in vivo studies in mice were carried out to compare [3H]-ASOs with their unlabeled counterpart ASOs, with and without conjugation to N-acetylgalactosamine (GalNAc), for tissue and plasma concentrations time profiles. Despite the structural modification of the labeled ASOs, in vitro target interaction and in vivo pharmacokinetic behaviors were similar to that of the unlabeled ASOs. In conclusion, this new method provides a powerful technique for fast and safe access to tritium-labeled oligonucleotides, e.g., for pharmacokinetic, mass balance, or autoradiography studies. Graphical abstract

A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)

Dostarlimab is a humanized anti–PD-1 monoclonal antibody. Dostarlimab (JEMPERLI; TSR-042) was recently approved in the USA and in the EU. The presence of neutralizing antibodies (NAbs) is a cause for concern because they block the therapeutic function of the antibody and reduce drug efficacy. Therefore, programs developing therapeutic biologics need to develop and validate assays that adequately assess the presence of NAbs in the serum of patients treated with biologic therapies. Presented here is the development and validation of a competitive ligand-binding assay that specifically detects anti-dostarlimab NAbs in human serum. Precision, sensitivity, hook effect, selectivity, assay robustness, stabilities, and system suitability were evaluated. In addition, drug tolerance of the assay with the implementation of a drug removal process was investigated. The cut point factor for the detection of NAbs in human serum at a 1% false-positive rate was determined. The assay’s precision, sensitivity, hook effect, selectivity, robustness, and drug interference were tested and found to be acceptable. With system suitability and stability established, this assay has been used to evaluate NAbs to guide the development of dostarlimab.Trial registration: Clinicaltrials.gov, NCT02715284. Registered 9 March 2016