Atanas V. Koulov

The Immunogenicity of Antibody Aggregates in a Novel Transgenic Mouse Model

PurposeProtein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations.MethodsTransgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light.ResultsThe expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic.ConclusionThis mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.

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. Moreover, we summarize the lessons these models have taught us and some of the challenges they present. Finally, we provide recommendations for future research utilizing mouse models to improve our understanding of critical factors that may contribute to protein immunogenicity.

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics.

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. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application.

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. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

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. Larger SC injection volumes are considered to be associated with injection pain and adverse events at the injection site. However, no controlled clinical studies and actual evidence exist to support this assumption. In this review, we discuss current and publically available data related to SC administration volumes. We conclude that injection volumes higher than 3.5 mL are worth exploring if required for the development of efficacious drug treatments. Studying tissue back pressure, injection site leakage, local tolerability, and injection-related adverse events, such as injection pain, should be considered for the development of higher SC injection volumes.

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. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.

Determination of the Density of Protein Particles Using a Suspended Microchannel Resonator

One of the analytical tools for characterization of subvisible particles, which gained popularity over the last years because of its unique capabilities, is the resonance mass measurement technique. However, a challenge that this technique presents is the need to know the exact density of the measured particles in order to obtain accurate size calculations. The density of proteinaceous subvisible particles has not been measured experimentally yet and to date researchers have been using estimated density values. In this paper, we report for a first-time experimental measurements of the density of protein particles (0.2-5 μm in size) using particles created by stressing three different proteins using four different types of stress conditions. Interestingly, the particle density values that were measured varied between 1.28 and 1.33 g/cm(3) and were lower than previous estimates. Furthermore, it was found that although the density of proteinaceous particles was affected to a very low degree by the stress conditions used to generate them, there is relatively larger difference between particles originating from different classes of proteins (e.g., monoclonal antibody vs. bovine serum albumin).

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

ABSTRACT The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

Factors Governing the Precision of Subvisible Particle Measurement Methods – A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe

PurposeThe current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance.MethodsThe performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions.ResultsThe results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results.ConclusionsThe variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control.