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Upon reformatting of an antibody to single-chain variable fragment format, a region in the former variable/constant domain interface of the heavy chain becomes accessible for preexisting (PE) anti-drug antibody (ADA) binding. The region exposed because of this reformatting contains a previously hidden hydrophobic patch. In this study, mutations are introduced in this region to reduce PE ADA reactivity and concomitantly reduce the hydrophobic patch. To enhance our understanding of the importance of individual residues in this region with respect to PE ADA reactivity, a total of 50 molecules for each of two antibodies against different tumor-associated antigens were designed, produced, and characterized by an arsenal of biophysical methods. The aim was to identify suitable mutations that reduce, or completely eliminate, PE ADA reactivity to variable fragments, without compromising biophysical and pharmacodynamic properties. Computational methods were used to pinpoint key residues to mutate and to evaluate designed molecules in silico, in order to reduce the number of molecules to produce and characterize experimentally. Mutation of two threonine residues, Thr101 and Thr146 in the variable heavy domain, proved to be critical to eliminate PE ADA reactivity. This may have important implications in optimizing early drug development for antibody fragment-based therapeutics.

Since the birth of biotechnology, hundreds of biotherapeutics have been developed and approved by the US Food and Drug Administration (FDA) for human use. These novel medicines not only bring significant benefit to patients but also represent precision tools to interrogate human disease biology. Accordingly, much has been learned from the successes and failures of hundreds of high-quality clinical trials. In this review, we discuss general and broadly applicable themes that have emerged from this collective experience. We base our discussion on insights gained from exploring some of the most important target classes, including interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), IL-6, IL-12/23, IL-17, IL-4/13, IL-5, immunoglobulin E (IgE), integrins and B cells. We also describe current challenges and speculate about how emerging technological capabilities may enable the discovery and development of the next generation of biotherapeutics.

Embryofetal toxicity studies are conducted to support inclusion of women of childbearing potential in clinical trials and to support labeling for the marketed pharmaceutical product. For biopharmaceuticals, which frequently lack activity in the rodent or rabbit, the nonhuman primate is the standard model to evaluate embryofetal toxicity. These studies have become increasingly challenging to conduct due to the small number of facilities capable of performing them and a shortage of sexually mature monkeys. The low number of animals per group and the high rate of spontaneous abortion in cynomolgus monkeys further complicate interpretation of the data. Recent FDA guidance has proposed a weight of evidence (WoE) approach to support product labeling for reproductive toxicity of products intended to be used for the treatment of cancer (Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations), an approach that has also supported the approval of biotherapeutics for non-cancer indications. Considerations to determine the appropriateness and content of a WoE approach to support product labeling for embryofetal risk include known class effects in humans; findings from genetically modified animals with or without drug administration; information from surrogate compounds; literature-based assessments about the developmental role of the pharmaceutical target; and the anticipated exposure during embryofetal development. This paper summarizes the content of a session presented at the 42nd annual meeting at the American College of Toxicology, which explored the conditions under which alternative approaches may be appropriate to support product labeling for reproductive risk, and how sponsors can best justify the use of this approach. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semiautomated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads [ABSTRACT FROM AUTHOR]

ABSTRACTBeyond potency, a good developability profile is a key attribute of a biological drug. Selecting and screening for such attributes early in the drug development process can save resources and avoid costly late-stage failures. Here, we review some of the most important developability properties that can be assessed early on for biologics. These include the influence of the source of the biologic, its biophysical and pharmacokinetic properties, and how well it can be expressed recombinantly. We furthermore present in silico, in vitro, and in vivo methods and techniques that can be exploited at different stages of the discovery process to identify molecules with liabilities and thereby facilitate the selection of the most optimal drug leads. Finally, we reflect on the most relevant developability parameters for injectable versus orally delivered biologics and provide an outlook toward what general trends are expected to rise in the development of biologics.

Renal cell carcinoma (RCC) is increasing in incidence and one third of newly diagnosed cases already are metastatic. The metastatic spread of solid tumors renders RCC incurable by surgical resection and consequently more difficult to treat. New molecular-targeted therapies have played a pivotal role in RCC treatment. Unfortunately, tumors frequently develop resistance to these targeted therapies by activating bypass pathways in which alternative signaling or biochemical pathways are activated in response to targeted inhibition of a signaling pathway, allowing cancer cells to continue to survive. Although the advent of immunotherapy with checkpoint inhibitors has led to significant changes in the treatment landscape for advanced RCC, many issues remain to be resolved. For these reasons, there is an urgent need to develop novel therapies and new treatment paradigms for patients with RCC. Much research has been performed thus far in identifying novel targets and treatment strategies in RCC and many of these currently are under investigation and/or in clinical trials. In this article, we discuss therapeutic options in the management of RCC with a focus on the new therapeutic approaches currently investigated in research and for use in the clinic. We divide these potential novel therapies into five groups: nonbiologics, small-molecule drugs, biologics, immunomodulatory therapies, and peptide drugs. We also present some therapeutics and treatment paradigms.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer’s disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation’s long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze–thaw cycles at –80 °C/25 °C or –80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

• Natural products mostly derived from plants, animal and microbial resources. • Low-income countries mostly rely on natural products as a primary health support. • Ethnomedicines expounds better superiority over contemporary drugs. • Natural products need therapeutic authentication for the drug development. • Genetic engineering and modern practices expedite the synthesis of the natural product. Since ancient times, natural medicines have had paramount importance in bolstering biotherapeutics to treat various diseases. The World Health Organization (WHO) revealed that more than 80% of the population of developing countries relies on traditional medicines, predominately herbal medicine, for their immediate medications. The drugs derived from medicinal plants have tremendous diversity with superfluous potency for managing communicable and non-communicable diseases, which diminishes the burden of modern pharmacopoeias in low and middle-developed countries. With the increasing importance and prevalence of herbal drugs, the appropriate evaluations are being implemented for their utilization. Most herbal medicines are prescribed by practical shreds of evidence and recommended in crude and semi-standardized forms. The inadequacy in pharmacological evaluation, preclinical and clinical examination of herbal drugs impedes their integration into contemporary medicinal practices. The preclinical investigation, prominently in-vivo and in-vitro studies, explores various attributes consisting of cell cytotoxicity, cell-cell interactions, intracellular activity, cell-environment interaction, gene expression studies, and metabolomics fingerprints of induced natural drugs. These pre-clinical evaluations and robust evidence consent to the safe and long-term utilization of herbal medicine to treat hideous diseases. Further, several modern practises are being considered for the precise and effective production of bioactive compounds at the commercial level. With this connection, this review illustrates the prominent sources of natural drugs, their pre-clinical assessments, the development of active drug molecules, and their commercialization in low-, middle-, and high-income countries. [ABSTRACT FROM AUTHOR]

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents. [ABSTRACT FROM AUTHOR]

The last three decades have seen the biotherapeutic drug market evolve from promising concept to market dominance in a range of clinical indications. This growth has been spurred by the success of established drug classes like monoclonal antibodies, but also by the introduction of biosimilars, and more recently, multiple novel cell and gene therapies. Biotherapeutic drug development presents many unique challenges, but unintended immune responses are among the most common reasons for program attrition. Anti-drug antibodies can impact the safety and efficacy of drug products, and related immune responses, like the cytokine release syndrome that occurred in the infamous TGN-1412 clinical trial, can be challenging to predict with nonclinical models. For this reason, it is important that development programs proceed with a scientifically grounded and measured approach to these responses. This process begins at the discovery stage with the application of “quality by design,” continues into the clinic with the development of quality assays and management strategies, and culminates in the effective presentation of this information in regulatory documents. This review provides an overview of some of the key strategic and regulatory considerations for biotherapeutics as they pertain to immunogenicity and related responses.

Developing ultra-high concentration biotherapeutics drug products can be challenging due to increased viscosity, processing, and stability issues. Excipients used to alleviate these concerns are traditionally evaluated at lower protein concentrations. This study investigates whether classically known modulators of stability and viscosity at low (<50 mg/mL) to high (>50 – 150 mg/mL) protein concentrations are beneficial in ultra-high (>150 mg/mL) concentration protein formulations and drug products. This study evaluates the effect of arginine monohydrochloride, proline, and lysine monohydrochloride on viscosity and concentratability at different high and ultra-high protein concentrations using a monoclonal antibody, mAbN, formulation as a candidate protein system. The effect of excipients on the viscosity and concentratability (rate and extent) was different at high versus ultra-high protein concentrations. These results highlight that classical excipients in literature known to modulate protein interactions at low protein concentrations and reduce viscosity at high protein concentrations may need to be evaluated at target protein concentrations in a product-specific manner while developing ultra-high concentration biologics drug products. [ABSTRACT FROM AUTHOR]

The last three decades have seen the biotherapeutic drug market evolve from promising concept to market dominance in a range of clinical indications. This growth has been spurred by the success of established drug classes like monoclonal antibodies, but also by the introduction of biosimilars, and more recently, multiple novel cell and gene therapies. Biotherapeutic drug development presents many unique challenges, but unintended immune responses are among the most common reasons for program attrition. Anti-drug antibodies can impact the safety and efficacy of drug products, and related immune responses, like the cytokine release syndrome that occurred in the infamous TGN-1412 clinical trial, can be challenging to predict with nonclinical models. For this reason, it is important that development programs proceed with a scientifically grounded and measured approach to these responses. This process begins at the discovery stage with the application of "quality by design," continues into the clinic with the development of quality assays and management strategies, and culminates in the effective presentation of this information in regulatory documents. This review provides an overview of some of the key strategic and regulatory considerations for biotherapeutics as they pertain to immunogenicity and related responses. [ABSTRACT FROM AUTHOR]

Subcutaneous (SC) delivery of biotherapeutics is well established as a route of administration across many therapeutic areas and has been shown to be effective and well-tolerated. It can offer several advantages over intravenous administration. This notwithstanding, there remain critical development issues and knowledge gaps in SC drug delivery. To articulate and address these issues, the SC Drug Delivery and Development Consortium was convened in 2018 as a pre-competitive collaboration of industry experts in drug delivery, device development, and commercialization. In this review, we outline the Consortium's vision and mission in advancing the development of patient-centered biotherapeutics and establishing a collaborative organization that facilitates open sharing of information and gives voice to diverse viewpoints from SC experts across industries and disciplines. Additionally, we describe the current landscape and challenges associated with SC administration of therapeutic proteins (specifically monoclonal antibodies) and offer insights into potential solutions to these challenges within the context of 8 problem statements developed by the Consortium to highlight key gaps, unmet needs, and actionable issues. Current and future opportunities to accelerate progress in the field through technological advances and the development of drug delivery tools are also discussed. Unlabelled Image • Subcutaneous (SC) delivery of biotherapeutics is increasingly a preferred choice. • There remain critical knowledge gaps and development issues in the SC industry. • The SC Drug Delivery and Development Consortium identified 8 problem statements. • The Consortium seeks to foster collaborations and advance tools and technologies. [ABSTRACT FROM AUTHOR]

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de‐immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de‐immunization strategies reduce the risk of anti‐drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human–cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation‐prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de‐immunized), compared with only 336 hours (adalimumab) or 336–672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes. [ABSTRACT FROM AUTHOR]

The success of inhibitor-based therapeutics is largely constrained by the acquisition of therapeutic resistance, which is partially driven by the undruggable proteome. The emergence of proteolysis targeting chimera (PROTAC) technology, designed for degrading proteins involved in specific biological processes, might provide a novel framework for solving the above constraint. A heterobifunctional PROTAC molecule could structurally connect an E3 ubiquitin ligase ligand with a protein of interest (POI)-binding ligand by chemical linkers. Such technology would result in the degradation of the targeted protein via the ubiquitin-proteasome system (UPS), opening up a novel way of selectively inhibiting undruggable proteins. Herein, we will highlight the advantages of PROTAC technology and summarize the current understanding of the potential mechanisms involved in biotherapeutics, with a particular focus on its application and development where therapeutic benefits over classical small-molecule inhibitors have been achieved. Finally, we discuss how this technology can contribute to developing biotherapeutic drugs, such as antivirals against infectious diseases, for use in clinical practices. [Display omitted] • This review provides an overview of the mechanism and research progress of PROTAC in targeted therapies. • The constituents and applications of different nonconventional PROTACs are discussed. • The reported PROTAC delivery systems have been categorized and described. [ABSTRACT FROM AUTHOR]

Biological drugs comprise a wide field of different modalities with respect to structure, pharmacokinetics and pharmacological function. Considerable non-clinical experience in the development of proteins (e.g. insulin) and antibodies has been accumulated over the past thirty years. In order to improve the efficacy and the safety of these biotherapeutics, Fc modifications (e.g. Fc silent antibody versions), combinations (antibody-drug conjugates, protein-nanoparticle combinations), and new constructs (darpins, fynomers) have been introduced. In the last decade, advanced therapy medicinal products (ATMPs) in research and development have become a considerable and strongly growing part of the biotherapeutic portfolio. ATMPs consisting of gene and cell therapy modalities or even combinations of them, further expand the level of complexity, which already exists in non-clinical development strategies for biological drugs and has thereby led to a further diversification of expertise in safety and PKPD assessment of biological drugs. It is the fundamental rationale of the BioSafe meetings, held yearly in the EU and in the US, to convene experts on a regular basis and foster knowledge exchange and mutual understanding in this fast growing area. In order to reflect at least partially the variety of the biotherapeutics field, the 2016 EU BioSafe meeting addressed the following topics in six sessions: (i) In vitro Meets in vivo to Leverage Biologics Development (ii) New developments and regulatory considerations in the cell and gene therapy field (iii) CMC Challenges with Biologics development (iv) Minipigs in non-clinical safety assessment (v) Opportunities of PKPD Assessment in Less Common Administration Routes In the breakout sessions the following questions were discussed: (i) Cynomolgus monkey as a reprotoxicology Species: Impact of Immunomodulators on Early Pregnancy Maintenance (ii) Safety Risk of Inflammation and Autoimmunity Induced by Immunomodulators (iii) Experience with non-GMP Material in Pivotal Non-clinical Safety Studies to Support First in Man (FiM) Trials (iv) Safety Assessment of Combination Products for Non-oncology [ABSTRACT FROM AUTHOR]

Enhancing the functional repertoire of probiotics is a promising approach to cope with the inexorable rise of antibiotic-resistant pathogens and the rather slow development of new antibiotics. Probiotics that deliver novel therapeutics efficiently and with site specificity are emerging living therapeutics that may transform existing paradigms of disease diagnosis and prevention. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) are an emerging new class of targeted therapeutics for cancer that use antibodies to deliver cytotoxic drugs to cancer cells. There are two FDA approved ADCs on the market and over 30 ADCs in the clinical pipeline against a number of different cancer types. The structure of an ADC is very complex with multiple components and considerable efforts are ongoing to determine the attributes necessary for clinical success. Understanding the pharmacokinetics of an ADC and how it impacts efficacy and toxicity is a critical part of optimizing ADC design and delivery i.e., dose and schedule. This review discusses the pharmacokinetic considerations for an ADC and tools and strategies that can be used to evaluate molecules at the preclinical stage. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) analysis provides important binding characteristic information for an antibody to its binding partner, such as binding specificity and affinity (K D). In recent years, SPR has been increasingly used in biosimilar development as part of the comparative analytical similarity assessment. Although there is no systematic study describing how to qualify SPR assays, there are various SPR result types (outputs) that have been used for assay qualification in publicly available regulatory documents. The mixed usage of SPR output can cause confusion and can be misleading when comparing binding attributes among antibody molecules. In this report, using a recombinant huIgG1 (mAb 1) antibody as an example, we performed assay qualification strictly based on the nature of the biomolecular interaction. We recommend that K D should be used as the output of assay qualification when the K D can be measured accurately by SPR. When K D cannot be accurately determined in a SPR setting, sensorgram comparison and Parallel Line Analysis (PLA) can be used to qualify the assay. We emphasize the importance of setting up appropriate SPR assay conditions for target and/or Fc receptor interactions to ensure the assay qualification parameters, such as accuracy and repeatability, to meet the criteria acceptable for regulatory filings. With increasing numbers of biotherapeutics being developed, the methods and guidelines provided here can help to align SPR application between the drug development industry and regulatory authorities which will benefit the scientific communities involved in biotherapeutic drug development. [Display omitted] • Binding activity of mAb 1 to its target and four of human FcgRs using SPR method optimized for each interaction. • Assay qualification was performed using K D values to calculate accuracy, repeatability and intermediate precision. • When K D cannot be determined, sensorgram comparison and Parallel Line Analysis (PLA) maybe used to qualify the assay. • We emphasize the importance of setting up appropriate SPR assay conditions to ensure the assay qualification parameters to meet the criteria. • The methods and guidelines provided may help to align SPR applications among the industry and regulatory authorities. [ABSTRACT FROM AUTHOR]

For biotherapeutics directed against soluble targets, most often monoclonal antibodies (mAbs), their therapeutic efficacy theoretically is driven by the magnitude and duration of free target suppression. However, for soluble targets of rapid turnover and low abundance, it can be technically challenging to directly measure the lowering of free target following treatment with biologics. The opportunities, challenges, and practical approaches to assess free and bound soluble targets and the utility of free and bound target measurements as biomarkers for target engagement and efficacy are covered in this review. In particular, case examples are presented to illustrate the interplay between drug and free/bound target, and how an integrated bioanalytical and pharmacokinetic/target engagement/pharmacodynamic (PK/TE/PD) modeling approach can be used to assess the target engagement for biologics directed against soluble targets with rapid turnover. Important caveats of the modeling approach in the absence of free target measurements are also discussed. [ABSTRACT FROM AUTHOR]

[Display omitted] Peptides, an emerging modality within the biopharmaceutical industry, are often delivered subcutaneously with evolving prospects on oral delivery. Barrier biology within the subcutis or gastrointestinal tract is a significant challenge in limiting absorption or otherwise disrupting peptide disposition. Aspects of peptide pharmacokinetic performance and ADME can be mitigated with careful molecular design that tailors for properties such as effective size, hydrophobicity, net charge, proteolytic stability, and albumin binding. In this review, we endeavor to highlight effective techniques in qualifying physicochemical properties of peptides and discuss advancements of in vitro models of subcutaneous and oral delivery. Additionally, we will delineate empirical findings around the relationship of these physicochemical properties and in vivo (animal or human) impact. We conclude that robust peptide characterization methods and in vitro techniques with demonstrated correlations to in vivo data are key routines to incorporate in the drug discovery and development to improve the probability of technical and commercial success of peptide therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, there has been increasing interest in using LC–MS/MS to measure protein drugs (biotherapeutics) in plasma/serum to support pharmacokinetic/toxicokinetic (PK/TK) studies. A number of strategies have been proposed to quantify different types of protein drug candidates in biological matrices. However, the application of LC–MS/MS in biotherapeutics is still very limited in regulated bioanalysis due to practical challenges. In this manuscript, we propose a “data-dependent” approach for the LC–MS/MS support of protein drug candidates, focusing on operational aspects. We have developed and validated a fast, simple and reliable LC–MS/MS method to quantify a protein drug candidate in mouse serum. This method can fit into our current workflow for routine small molecule LC–MS/MS assays with an overall sample preparation time of less than two hours. The method has been demonstrated to be accurate, precise and rugged, and it has been successfully applied to analyze samples from toxicology studies. The results were verified using a ligand binding assay and tested by standard-addition sample reanalysis. In addition, general recommendations for developing and validating an LC–MS/MS assay based on surrogate peptide(s) of protein drug candidates are also proposed for regulated bioanalysis. [Copyright &y& Elsevier]

Aggregation is a common hurdle faced during the development of antibody therapeutics. In this study, we explore the potential aggregation liabilities of the Fab (fragment antigen-binding) from a human IgG1κ antibody via multiple elevated temperature molecular dynamic simulations, analogous to accelerated stability studies performed during formulation development. Deformation and solvent exposure changes in response to thermal stress were monitored for individual structural domains (VH, VL, CH1 and CL), their interfaces (VH:VL and CH1:CL), edge beta-strands and sequence-predicted aggregation-prone regions (APRs). During simulations, domain interfaces deformed prior to the unfolding of individual domains. However, interfacial beta-strands retained their secondary structure and remained solvent protected longer than all other strands or loops. Thus, APRs located in interfacial beta-strands are effectively blocked from self-association. Structural deformations were also observed in complementarity-determining regions, edge beta-strands and adjoining framework beta-strands, which increased their solvent-accessible surface area and exposed APRs in these regions. From the analysis of these structural changes, two potential aggregation liabilities were identified in the VH domain of this Fab. Insights gained from this investigation should be useful in devising a rational structure-based strategy for the design and selection of antibody candidates with high potency and improved developability. [ABSTRACT FROM AUTHOR]

Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity. [ABSTRACT FROM AUTHOR]

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach. [ABSTRACT FROM PUBLISHER]

Abstract: Recent advances in our understanding of disease biology, biomarkers, new therapeutic targets, and innovative modalities have each fueled a dramatic expansion in the development of novel human therapeutics. Many are biotechnology-derived biologics possessing high selectivity and affinity for their intended target; as such they often pose challenges in the development path to approval. One challenge is the selection of the first-in-human (FIH) dose. This process has come under increased scrutiny as a result of a FIH trial with a super-agonist monoclonal antibody (TGN1412), which resulted in significant injury to healthy volunteers. Regulatory agencies have responded with supplemental guidance for the development of novel therapeutics. The intent of this paper is to provide experience-based insight, with relevant examples, for those planning the first administration of novel biopharmaceuticals in humans. [ABSTRACT FROM AUTHOR]

To analyze contribution of short aggregation-prone regions (APRs), which may self-associate via cross-β motif and were earlier identified in therapeutic mAbs, towards antigen recognition via structural analyses of antibody-antigen complexes. A dataset of 29 publically available high-resolution crystal structures of Fab-antigen complexes was collected. Contribution of APRs towards the surface areas of the Fabs buried by the cognate antigens was computed. Propensities of amino acids to occur in APRs and to be involved in antigen binding were compared. Coincidence between APRs and individual CDR loops was examined. All Fabs in the dataset contain at least one APR in CDR loops and adjacent framework β-strands. The average contribution of APRs towards buried surface area of Fabs is 16.0 ± 10.7%. Aggregation and antigen recognition may be coupled via aromatic residues (Tyr, Trp), which occur with high propensities in both APRs and antigen binding sites. APRs are infrequent in the heavy chain CDR 3 (H3) loops (7%), but are frequent in H2 loops (45%). Co-incidence of APRs with antigen recognition sites can potentially lead to the loss of function upon aggregation. Rational structure-based design or selection strategies are suggested for biotherapeutics with improved druggability while maintaining potency. [ABSTRACT FROM AUTHOR]