"Advancing the Implementation of Innovative Analytical Technologies in Pharmaceutical Manufacturing - Some Regulatory Considerations".

Analytical technologies and methods play a pivotal role in attribute understanding and control which are essential to the rapidly evolving field of pharmaceutical development and manufacturing. These technologies are advancing quickly, where innovations often involve both new scientific approaches and novel applications of established techniques. In many cases, the lack of harmonized global regulatory expectations presents challenges for the adoption of advanced technologies.

Experimental validation of a parenteral permitted daily exposure value for cleaning-induced degradants from recombinant therapeutic proteins with in vitro immunogenicity assays.

Multiproduct manufacturing of biotherapeutic proteins generate cleaning-induced protein degradants because of extreme pH and temperature conditions during the cleaning process. Cleaning Acceptance limits are calculated based on the maximum allowable carryover (MAC) assessment of the previously manufactured active pharmaceutical ingredient (API) - or drug product - based on the permitted daily exposure (PDE) of the previously manufactured API into the dose of subsequent product. In this study, we tested a previously determined PDE value for cleaning-induced protein degradants of 650 µg/dose.

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.

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS.

State-of-the-art and emerging trends in analytical approaches to pharmaceutical-product commercialization.

The biopharmaceutical landscape continues to evolve rapidly, and associated modality complexity and the need to improve molecular understanding require concomitant advances in analytical approaches used to characterize and release the product. The Product Quality Attribute Assessment (PQAA) and Quality Target Product Profile (QTPP) frameworks help catalog and translate molecular understanding to process and product-design targets, thereby enabling reliable manufacturing of high-quality product. The analytical target profile forms the basis of identifying best-fit analytical methods for attribute measurement and continues to be successfully used to develop robust analytical methods for detailed product characterization as well as release and stability testing.

Observation of Heavy-Chain C-Terminal Amidation in Human Endogenous IgG.

Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C-terminal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS based peptide mapping.

A Mass Spectrometric Characterization of Light-Induced Modifications in Therapeutic Proteins.

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins.

Use of In Vitro Human Skin Models to Assess Potential Immune Activation In Response to Biotherapeutic Attributes and Process-related Impurities.

Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin.

A cross-industry forum on benchmarking critical quality attribute identification and linkage to process characterization studies.

Identification of Critical Quality Attributes (CQAs) and subsequent characterization in process development studies are the key elements of quality by design (QbD) for biopharmaceutical products. Since the inception of ICH Q8R2, several articles have been published on approaches to conducting CQA risk assessments as well as the application to process understanding. A survey was conducted by multiple companies participating in an International Consortium working group on the best practices for identifying CQAs with linkages to process characterization (PC) studies.

Glycan engineering reveals interrelated effects of terminal galactose and core fucose on antibody-dependent cell-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood.

Differential influence on antibody dependent cellular phagocytosis by different glycoforms on therapeutic Monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs), particularly of the IgG1 subclass, are capable of effector function activities that may be important for their mechanism of action. One such effector function activity is Antibody Dependent Cellular Phagocytosis (ADCP), which has been shown to be mediated primarily through the activating FcγR, FcγRIIa, on macrophages and neutrophils. The critical quality attributes that are the most impactful and predictive of ADCP activity, and therefore most suitable to monitor during IgG1 antibody manufacturing, are not well established.

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.

Determination of interactions between antibody biotherapeutics and copper by size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometry (ICP/MS).

The concept of coupling of size-exclusion HPLC with ICP/MS (SEC-ICP/MS) was first applied in this work as a novel approach in the biotechnology field to assess metal binding to Immunoglobulin G (IgG) mAbs. This method can be used to probe the mechanism and biophysical properties of metal-protein interactions to gain a deeper understanding of the potential impact of metals during drug product manufacturing. Two IgG1s and one IgG2 drugs were investigated.

Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity.

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody.

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.

Evaluating Immunogenicity Risk Due to Host Cell Protein Impurities in Antibody-Based Biotherapeutics.

A potential risk factor for immunogenicity of a biotherapeutic is the low levels of host cell protein (HCP) impurities that remain in the product following the purification process. During process development, significant attention has been devoted to removing HCPs due to their potential safety risk. Samples from different purification steps of several monoclonal antibodies (mAbs) purified by one type of platform were evaluated for their residual Chinese Hamster Ovary (CHO) cell-derived HCP content.

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.

Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule.

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.

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.

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.