A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

Background And Purpose: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses.

Methods And Results: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles.

Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution.

Stress Factors in Primary Packaging, Transportation and Handling of Protein Drug Products and Their Impact on Product Quality.

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g.

Characterization of Subvisible Particles in Biotherapeutic Prefilled Syringes: The Role of Polysorbate and Protein on the Formation of Silicone Oil and Protein Subvisible Particles After Drop Shock.

Subvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP.

Silicone Oil Particles in Prefilled Syringes With Human Monoclonal Antibody, Representative of Real-World Drug Products, Did Not Increase Immunogenicity in In Vivo and In Vitro Model Systems.

Silicone oil is a lubricant for prefilled syringes (PFS), a common primary container for biotherapeutics. Silicone oil particles (SiOP) shed from PFS are a concern for patients due to their potential for increased immunogenicity and therefore also of regulatory concern. To address the safety concern in a context of manufacturing and distribution of drug product (DP), SiOP was increased (up to ∼25,000 particles/mL) in PFS filled with mAb1, a fully human antibody drug, by simulated handling of DP mimicked by drop shock.

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy.

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification.

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.

Purpose: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO).

Methods: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins.

Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes.

Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges.

The success of clinical and commercial therapeutic proteins is rapidly increasing, but their potential immunogenicity is an ongoing concern. Most of the studies that have been conducted over the past few years to examine the importance of various product-related attributes (in particular several types of aggregates and particles) and treatment regimen (such as dose, dosing schedule, and route of administration) in the development of unwanted immune responses have utilized one of a variety of mouse models. In this review, we discuss the utility and drawbacks of different mouse models that have been used for this purpose.

Immunogenicity of Therapeutic Protein Aggregates.

Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation.

Subvisible (2-100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes.

Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy.

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation.

Physical characterization and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting.

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay.

Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies.

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions.

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K.

Classification of protein aggregates.

Comparison of protein aggregates/self-associated species between laboratories and across disciplines is complicated by the imprecise language presently used to describe them. In this commentary, we propose a standardized nomenclature and classification scheme that can be applied to describe all protein aggregates. Five categories are described under which a given aggregate may be independently classified: size, reversibility/dissociation, conformation, covalent modification, and morphology.

High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations.

Design of experiment and statistical analyses were applied to evaluate the effects of several formulation components on the thermal and colloidal stability for a series of monoclonal antibody (mAb) formulations. The high-throughput assessment of the protein stability was performed by measuring the temperature of hydrophobic exposure (T(h) , thermal stability) and the diffusion interaction parameter (k(D) , colloidal stability). To correlate the measured parameters with protein stability, the propensity to aggregate was tested by exposing the mAb samples to two types of stress: mechanical stress caused by shaking agitation and thermal stress.

Chemical modifications in therapeutic protein aggregates generated under different stress conditions.

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K.

Classification and characterization of therapeutic antibody aggregates.

A host of diverse stress techniques was applied to a monoclonal antibody (IgG(2)) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q.

Screening of monoclonal antibody formulations based on high-throughput thermostability and viscosity measurements: design of experiment and statistical analysis.

The purpose of this study was to demonstrate the utility of combining a design of experiment (DOE) approach with high-throughput formulation screening to identify the main factors affecting protein thermostability and solution viscosity. The optimization of buffer compositions was guided by statistical analysis of the data to obtain the targeted combination of low viscosity and high thermostability. Different monoclonal antibody (mAb) formulation variables were evaluated in the study to achieve optimization of two parameters: (i) thermostability characterized by temperature of hydrophobic exposure and (ii) viscosity.

Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange.

High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions.

We propose a new method to measure the viscosity of concentrated protein solutions in a high-throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature.