Definition of particle visibility threshold in parenteral drug products - towards standardization of visual inspection operator qualification.

The detectability size threshold of visible particles (″visibility″ size) in the context of visual inspection of parenteral drug products has been an elusive target for several decades. The current common sense, also reflected in official guidelines, dictates that particles of different shapes and morphologies have different ″visibility″ size thresholds, that can range between hundreds and thousands of micrometers. This study demonstrates experimentally for the first time that it is possible to define a single, shape- and morphology- independent detectability size threshold, identical across particles of various types, provided that observation conditions and product attributes are kept constant.

Monitoring of Visible Particles in Parenteral Products by Manual Visual Inspection-Reassessing Size Threshold and Other Particle Characteristics that Define Particle Visibility.

Visible particles are a critical quality attribute for parenteral products and must be monitored. A carefully designed, executed, and controlled drug product manufacturing process including a final 100 % visual inspection and appropriate end-product controls ensures that visible particles are consistently minimized and demonstrates that the injectable DP is practically free from visible particles. Visual inspection, albeit appearing as a simple analytical procedure, requires several technical and operational controls to ensure adequate performance.

Image Classification of Degraded Polysorbate, Protein and Silicone Oil Sub-Visible Particles Detected by Flow-Imaging Microscopy in Biopharmaceuticals Using a Convolutional Neural Network Model.

Degradation of polysorbates in biopharmaceutical formulations can induce the formation of sub-visible particles (SvPs) in the form of free-fatty acids (FFAs) and potentially protein aggregates. Flow-imaging microscopy (FIM) is one of the most common techniques for enumerating and characterizing the SvPs, allowing for collection of image data of the SvPs in the size ranges of two to several hundred micrometers. The vast amounts of data obtained with FIM do not allow for rapid manual characterization by an experienced analyst and can be ambiguous.

Proteomic Analysis of Adenovirus 5 by UHPLC-MS/MS: Development of a Robust and Reproducible Sample Preparation Workflow.

Adenoviruses (AdVs) have recently become widely used therapeutic vectors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. AdVs are large, nonenveloped viruses with an icosahedral capsid formed from several proteins that encloses double-stranded DNA. These proteins are the main components and key players in initial stages of infection by the virus particles, so their heterogeneity and content must be evaluated to ensure product and process consistency.

A novel protocol for in-depth analysis of recombinant adeno-associated virus capsid proteins using UHPLC-MS/MS.

Rationale: In-depth characterization of the three capsid viral proteins (VPs 1, 2, and 3) of adeno-associated viruses (AAVs) is immediately needed to ensure the consistency in gene therapy products and processes. These proteins are typically present at very low concentrations in matrices containing high concentrations of excipients and salts. Thus, there is a need for convenient methods for sample preparation before proteomic analysis.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments.

4-Hydroxynonenal - A Toxic Leachable from Clinically Used Administration Materials.

Introduction: The migration of chemicals from processing materials into biopharmaceuticals can lead to various problems. Leachables from administration materials, with no possibility of further clearance, are of particular concern. Released chemicals can be toxic or react with formulation components, thereby impacting product safety.

Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper.

Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis.

4-Hydroxynonenal is An Oxidative Degradation Product of Polysorbate 80.

Introduction: Polysorbates (PS) are used in biopharmaceuticals to stabilize therapeutic proteins. Oxidative degradation of (poly)unsaturated fatty acids (PUFAs) contained in PS was shown to lead to α,β-unsaturated carbonyls.

Aim: The n-6-PUFA linoleic acid accounts for up to 18% of all FAs contained in multi-compendial grade PS80.

Characterization of Polymeric Syringes Used for Intravitreal Injection.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g.

Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation.

Purpose: Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause.

Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum.

Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation.

Polysorbates are stabilizers typically required in therapeutic protein formulations. On account of their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects polysorbate 20 (PS20) and polysorbate 80 (PS80) against oxidative degradation.

Correction to: In Vivo Stability of Therapeutic Proteins.

A manuscript version without peer-review revisions was mistakenly processed and published.

In Vivo Stability of Therapeutic Proteins.

Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein.

Method to Predict Glass Vial Fogging in Lyophilized Drug Products.

Glass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging's expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry.

Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration.

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur.

Comparing Physical Container Closure Integrity Test Methods and Artificial Leak Methodologies.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety and is thus a key requirement by health authorities. While sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a product's shelf life.

Considerations for the Use of Polysorbates in Biopharmaceuticals.

Purpose: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations.

Impact of non-ideal analyte behavior on the separation of protein aggregates by asymmetric flow field-flow fractionation.

Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here, we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it.

Container Closure Integrity Testing of Prefilled Syringes.

Prefilled syringes (PFSs) are increasingly preferred over vials as container closure systems (CCSs) for injectable drug products when facilitated or self-administration is required. However, PFSs are more complex compared to CCSs consisting of vial, rubber stopper, and crimp cap. Container closure integrity (CCI) assurance and verification has been a specific challenge for PFSs as they feature several sealing areas.

Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance.

A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process.

Subcutaneous Injection Volume of Biopharmaceuticals-Pushing the Boundaries.

Administration into the subcutaneous (SC) tissue is a typical route of delivery for therapeutic proteins, especially for frequent treatments, long-term regimens, or self-administration. It is currently believed that the maximum volume for SC injections is approximately 1.5 mL.

Factors Governing the Accuracy of Subvisible Particle Counting Methods.

A number of new techniques for subvisible particle characterization in biotechnological products have emerged in the last decade. Although the pharmaceutical community is actively using them, the current knowledge about the analytical performance of some of these tools is still inadequate to support their routine use in the development of biopharmaceuticals (especially in the case of submicron methods). With the aim of increasing this knowledge and our understanding of the most prominent techniques for subvisible particle characterization, this study reports the results of a systematic evaluation of their accuracy.