Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Assay format diversity in pre-clinical immunogenicity risk assessment: Toward a possible harmonization of antigenicity assays.

A major impediment to successful use of therapeutic protein drugs is their ability to induce anti-drug antibodies (ADA) that can alter treatment efficacy and safety in a significant number of patients. To this aim, in silico, in vitro, and in vivo tools have been developed to assess sequence and other liabilities contributing to ADA development at different stages of the immune response. However, variability exists between similar assays developed by different investigators due to the complexity of assays, a degree of uncertainty about the underlying science, and their intended use. The impact of protocol variations on the outcome of the assays, i.e., on the immunogenicity risk assigned to a given drug candidate, cannot always be precisely assessed. Here, the Non-Clinical Immunogenicity Risk Assessment working group of the European Immunogenicity Platform (EIP) reviews currently used assays and protocols and discusses feasibility and next steps toward harmonization and standardization.

T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation-Updated Consensus and Review 2020.

Immune responses to protein and peptide drugs can alter or reduce their efficacy and may be associated with adverse effects. While anti-drug antibodies (ADA) are a standard clinical measure of protein therapeutic immunogenicity, T cell epitopes in the primary sequences of these drugs are the key drivers or modulators of ADA response, depending on the type of T cell response that is stimulated (e.g., T helper or Regulatory T cells, respectively). In a previous publication on T cell-dependent immunogenicity of biotherapeutics, we addressed mitigation efforts such as identifying and reducing the presence of T cell epitopes or T cell response to protein therapeutics prior to further development of the protein therapeutic for clinical use. Over the past 5 years, greater insight into the role of regulatory T cell epitopes and the conservation of T cell epitopes with self (beyond germline) has improved the preclinical assessment of immunogenic potential. In addition, impurities contained in therapeutic drug formulations such as host cell proteins have also attracted attention and become the focus of novel risk assessment methods. Target effects have come into focus, given the emergence of protein and peptide drugs that target immune receptors in immuno-oncology applications. Lastly, new modalities are entering the clinic, leading to the need to revise certain aspects of the preclinical immunogenicity assessment pathway. In addition to drugs that have multiple antibody-derived domains or non-antibody scaffolds, therapeutic drugs may now be introduced via viral vectors, cell-based constructs, or nucleic acid based therapeutics that may, in addition to delivering drug, also prime the immune system, driving immune response to the delivery vehicle as well as the encoded therapeutic, adding to the complexity of assessing immunogenicity risk. While it is challenging to keep pace with emerging methods for the preclinical assessment of protein therapeutics and new biologic therapeutic modalities, this collective compendium provides a guide to current best practices and new concepts in the field.

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. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

How Close Are We to Profiling Immunogenicity Risk Using In Silico Algorithms and In Vitro Methods?: an Industry Perspective.

In silico HLA-binding algorithms and in vitro T cell-based assays as predictive tools for human immunogenicity risk have made inroads in the biotherapeutic drug discovery and development process. Currently, these tools are being used only for candidate selection or characterization and not for making a go/no-go decision for further development. A clear limitation for a broader implementation is the lack of correlation between the predicted T cell epitope content/immune reactivity potential of a biotherapeutic and the subsequent ADA-related clinical immunogenicity outcome. The current state of technologies and their pros and cons were discussed as a part of the 2016 AAPS National Biotechnology Conference in a themed session. A review of the advances in the area and the session talks along with the ensuing discussions are summarized in this commentary.

Immune Suppression During Preclinical Drug Development Mitigates Immunogenicity-Mediated Impact on Therapeutic Exposure.

In the clinical setting, anti-drug antibodies (ADA) against biotherapeutics can influence patient safety and interfere with product efficacy. High immunogenicity has been addressed in clinic by concomitant immune suppression, such as co-administration of methotrexate with enzyme replacement therapy (ERT) and combination tacrolimus/sirolimus treatment for prophylaxis against organ transplant rejection. This study investigates the use of such immune suppressants in mitigating ADA responses to a fully human monoclonal antibody (mAb1) in preclinical animal studies. Three groups of Sprague Dawley rats (n = 18) were treated with low (0.01 mg/kg), moderate (50 mg/kg), or high (300 mg/kg) doses of mAb1. Experimental groups also received either methotrexate or tacrolimus/sirolimus immune suppressive regimens. ELISA-based methods were utilized to measure and characterize ADA and mAb1 pharmacokinetics (PK). Results demonstrated a stepwise increase in immunogenicity with mAb1 dosage. Methotrexate significantly lowered incidence of anti-variable region antibodies at moderate mAb1 dose (P < 0.05), while tacrolimus/sirolimus did likewise at moderate and high doses (P < 0.01) of mAb1. Except for low-dose mAb1 + methotrexate, all immunosuppressed groups displayed more than a 70-fold decrease in ADA magnitude (P < 0.05). This abrogation in ADA response correlated with more mAb1 in circulation by week 4 for moderate- and high-dosed mAb1 groups. These data provide an approach to mitigate preclinical immunogenicity by the use of immunosuppressant regimens. Such preconditioning can support preclinical drug development of human therapeutics that are antigenic to animals. Similar approaches could be investigated for wider application to novel therapeutics.

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation.

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

Impact of anti-drug antibodies in preclinical pharmacokinetic assessment.

The administration of human biotherapeutics is often associated with a higher incidence of immunogenicity in preclinical species. The presence of anti-drug antibodies (ADAs) in the test samples can affect the accurate measurement of therapeutic protein (TP) in bioanalytical methods designed to support pharmacokinetic (PK) and toxicokinetic (TK) assessments. The impact can vary depending on the bioanalytical method platform and study dosing design. The goal of this study is to evaluate the impact of ADA response on the bioanalytical methods in support of PK/TK and the associated study data interpretation. Sprague Dawley rats were administered with four weekly doses of 50 mg/kg TP, a humanized monoclonal antibody. The TP in serum samples was measured using three bioanalytical methods that quantified bound and/or unbound TP to ADA. The ADA response in the animals was classified into negative, low, medium, and high based on the magnitude of the response. The presence of ADA in samples led to discrepant TP measurements between the methods, especially at time points where the TP concentrations were low. This could be due to ADA interference to the accurate measurement of ADA-bound TP concentrations. The TP concentration at last time point (C last) was reduced by 82.8%, 98.6%, and 99.8%, respectively, for samples containing low, medium, and high levels of ADA. The interfering effects of the ADA on bioanalytical methods and exposure were evident as early as 2 weeks post-dosing. This modeling approach can provide the better understanding of ADA impact on PK exposure in multiple doses.

Universal immunoassay applied during early development of large molecules to understand impact of immunogenicity on biotherapeutic exposure.

Immunogenicity testing during early biotherapeutic development is usually limited by resources needed for assay development, validation, and the necessity for unique product-specific controls and reagents. We describe a unique immunoassay [universal indirect species-specific assay (UNISA)] that can be applied during early phase preclinical studies to support pharmacology, pharmacokinetics (PK), and toxicology evaluation during biotherapeutic antibody candidate assessment. UNISA was evaluated across three animal species: mouse, rat, and cynomolgus monkey. For each species, a unique and specific antibody pair was generated consisting of the secondary antibody and the positive control. The secondary antibody is specific for species anti-IgG antibody while demonstrating no cross-reactivity to human antibody-based biotherapeutics. The positive control is comprised of a species-specific anti-human IgG antibody clone specific for binding to the CH2 domain of all human IgG subtypes. Applications of this platform included: (a) identifying the dose with the least immunogenicity risk; (b) characterizing the impact of immunogenicity on PK exposure profiles across multiple antibody candidates and dose regimens; and (c) characterizing the immune response specificity to the idiotype or non-idiotypic region of the biotherapeutic candidate. Due to its use of universal species-specific reagents, UNISA can overcome resource constraints and avoid extensive validation and development time to support immunogenicity testing during the early research and preclinical phase of programs. Enhanced understanding of the impact of the immunogenicity on biotherapeutic exposure and target-related immunomodulatory effects have been made possible with the use of this assay.

Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics.

An immune response to a biotherapeutic can be induced when the therapeutic is processed and presented by antigen presenting cell to T helper cells. This study evaluates the performance of an in vitro assay that can elicit antigen specific effector T cell responses. Two biotherapeutics with known clinical immunogenicity [FPX1 and FPX2] were assessed for their ability to induce antigen-specific IFN-γ secreting T cells in peripheral blood mononuclear cells (PBMC). The 24 amino acid peptide component of FPX1 elicited an antigen-specific response in 16/34 (47%) individual naïve healthy donors. This in vitro effect was consistent with high rate of immunogenicity which was observed when this drug was administered in clinical trials. FPX2 did not induce antigen-specific T cells in vitro, which correlates with the low rate of development of anti-drug antibody responses to this molecule in the clinic. The assay has the potential to predict immunogenicity and help in the selection of biotherapeutics at the early development stage of a clinical candidate.