Orphan drug development: Challenges, regulation, and success stories.

Rare diseases, also known as orphan diseases, are diseases with low occurrence in the population. Developing orphan drugs is challenging because of inadequate financial and scientific resources and insufficient subjects to run clinical trials. With advances in genome sequencing technologies, emergence of cell and gene therapies, and the latest developments in regulatory pathways, some orphan drugs that have curative potential have been approved. In India, due to its large population and resource crunch, developing orphan drugs is phenomenally challenging. After adopting the Orphan Drug Act, the US-FDA has continuously made advances in regulatory pathways for orphan drugs. Particularly, n-of-one clinical trials have been successful in some cases. India has recently adopted policies that have impacted the long-neglected rare-disease ecosystem; however, there is no clear regulatory path for orphan drug development in India. We have proposed a multi-pronged approach involving close collaboration between the government, regulatory bodies, industries, and patient advocacy groups to boost orphan drug development in India. We believe that rapidly evolving technologies and business models can enable better and faster development of novel orphan drugs in India and other resource-constrained countries.

A survey of awareness of diagnosis and treatment of rare diseases among healthcare professionals and researchers in India.

Rare diseases (RDs) are those that affect a small fraction of the total population. In India, where resources are scarce, the healthcare infrastructure and policy framework are focused on mitigating diseases that affect a large number of people. As a result,many cases ofRDs remain unreported, undiagnosed, and untreated. To understandthe currentlevel of RD awareness among healthcare professionals (HCPs) and researchers, as they are key stakeholders in diagnosis, treatment, policy making, and drug development, we conducted a survey based on identification of an RD, time for diagnosis, treatment options, and relationship with family history and geographic location. The survey was divided into two parts, one for researchers and the other for healthcare professionals, each consisting of 22 questions. We observed that among all our respondents, 31% of researchers and 29% of HCPs did not know the time required for diagnosis of a rare disease they mentioned in the survey. They identified the importance of family history but failed to realize its association with geographic location. The results from the exploratory study can provide information for enabling larger studies to develop recommendations and policies that can improve awareness about RDs in healthcare communities.

Understanding preclinical and clinical immunogenicity risks in novel biotherapeutics development.

Immunogenicity continues to pose a challenge in the development of biotherapeutics like conventional therapeutic-proteins and monoclonal antibodies as well as emerging modalities such as gene-therapy components, gene editing, and CAR T cells. The approval of any therapeutic is based on a benefit-risk evaluation. Most biotherapeutics address serious medical conditions where the standard of care has a poor outcome. Consequently, even if immunogenicity limits the utility of the therapeutic in a sub-set of patients, the benefit-risk assessment skews in favor of approval. Some cases resulted in the discontinuation of biotherapeutics due to immunogenicity during drug development processes, This special issue presents a platform for review articles offering a critical assessment of accumulated knowledge as well as novel findings related to nonclinical risks that extend our understanding of the immunogenicity of biotherapeutics. Some of the studies in this collection leveraged assays and methodologies refined over decades to support more clinically relevant biological samples. Others have applied rapidly advancing methodologies in pathway-specific analyses to immunogenicity. Similarly, the reviews address urgent issues such as the rapidly emerging cell and gene therapies which hold immense promise but could have limited reach as a significant number of the patient population could potentially not benefit due to immunogenicity. In addition to summarizing the work presented in this special issue we have endeavored to identify areas where additional studies are required to understand the risks of immunogenicity and develop appropriate mitigation strategies.

A four-part guide to lung immunology: Invasion, inflammation, immunity, and intervention.

Lungs are important respiratory organs primarily involved in gas exchange. Lungs interact directly with the environment and their primary function is affected by several inflammatory responses caused by allergens, inflammatory mediators, and pathogens, eventually leading to disease. The immune architecture of the lung consists of an extensive network of innate immune cells, which induce adaptive immune responses based on the nature of the pathogen(s). The balance of immune responses is critical for maintaining immune homeostasis in the lung. Infection by pathogens and physical or genetic dysregulation of immune homeostasis result in inflammatory diseases. These responses culminate in the production of a plethora of cytokines such as TSLP, IL-9, IL-25, and IL-33, which have been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Shifting the balance of Th1, Th2, Th9, and Th17 responses have been the targets of therapeutic interventions in the treatment of these diseases. Here, we have briefly reviewed the innate and adaptive i3mmune responses in the lung. Genetic and environmental factors, and infection are the major causes of dysregulation of various functions of the lung. We have elaborated on the impact of inflammatory and infectious diseases, advances in therapies, and drug delivery devices on this critical organ. Finally, we have provided a comprehensive compilation of different inflammatory and infectious diseases of the lungs and commented on the pros and cons of different inhalation devices for the management of lung diseases. The review is intended to provide a summary of the immunology of the lung, with an emphasis on drug and device development.

An Overview of Current Accomplishments and Gaps of COVID-19 Vaccine Platforms and Considerations for Next Generation Vaccines.

Vaccines against SARS-CoV-2 have transformed the course of the COVID-19 pandemic with more than 30 authorizations. More than 2 billion people have been vaccinated with these vaccines developed on very different manufacturing platforms. We have reviewed the unprecedented work done in various aspects of the authorized vaccines and listed three potential improvements: 1) long-term stability at room-temperature conditions; 2) suitability for diverse populations such as infants, elderly, immune-compromised, and those with pre-existing or ongoing diseases; and 3) ability to act against different strains. In this article, we have discussed the current status of COVID-19 vaccines with respect to 1) diversity (strength and breadth) of initial immune responses and long-term immune memory; 2) prime-boost combinations that induce protection against variants; and 3) breakthrough infections. Further, we have listed host, product (critical quality attributes), and viral pathogenic factors that contribute to safety, efficacy, and effectiveness of vaccines. In addition, we have elaborated on the potential to (develop models and) determine the immune correlates that can predict long-term immune memory. The graphical representation of the abstract is provided as Fig. 1.

On the Manufacturers of Biosimilars in Asia.

Over the past 2 decades, biosimilars have created an opportunity for access to affordable medicines globally. The development process includes robust analytical and functional comparability, equivalent pharmacokinetic profile, and demonstration of lack of any meaningful clinical differences. In this brief opinion article, we offer an overview of the major aspects that are involved in biosimilar development and regulatory requirements in Asia in order to facilitate a standardized process that can enable cost-effective development of biosimilars.

Advanced strategies in glycosylation prediction and control during biopharmaceutical development: Avenues toward industry 4.0.

Glycosylation has been shown to define the safety and efficacy of biopharmaceuticals, thus classified as a critical quality attribute. However, controlling glycan heterogeneity has always been a major challenge owing to the multivariate factors that govern the glycosylation process. Conventional approaches for controlling glycosylation such as gene editing and metabolic control have succeeded in obtaining desired glycan profiles in accordance with the Quality by Design paradigm. Nonetheless, the development of smart algorithms and omics-enabled complete cell characterization has made it possible to predict glycan profiles beforehand, and manipulate process variables accordingly. This review thus discusses the various approaches available for control and prediction of glycosylation in biopharmaceuticals. Further, the futuristic goal of integrating such technologies is discussed in order to attain an automated and digitized continuous bioprocess for control of glycosylation. Given, control of a process as complex as glycosylation requires intense monitoring intervention, we examine the current technologies that enable automation. Finally, we discuss the challenges and the technological gap that currently limits incorporation of an automated process in routine bio-manufacturing, with a glimpse into the economic bearing.

CAR-T cell therapy in India requires a paradigm shift in training, education and health care processes.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of some kinds of cancers. Hundreds of companies and academic institutions are collaborating to develop gene-modified cell therapies using novel targets, different cell types, and manufacturing processes of autologous and allogenic cell therapies. The individualized, custom-made autologous CAR-T cell production platform remains a significant limiting factor for its large-scale clinical application. In this respect, the advances in standardization and automation of the process can have considerable impact on cost reduction. Development of off-the-shelf, ready-to-use universal killer cells can enable scaling up. Despite the wide use of this cell therapy in the United States, Europe and China, its development is limited in developing countries in Southeast Asia, Africa and Latin America. In this review, we focus on good manufacturing practices-compliant manufacturing requirements, operational logistics, and regulatory processes that need to be considered for high-quality gene-modified cell therapies from an Indian perspective. We also list the potential strategies to overcome challenges associated with translation to affordability and scalability.

Biopharmaceutical Industry Capability Building in India: Report from a Symposium.

The biopharmaceutical industry is evolving with a shift in focus from recombinant proteins and antibodies towards more complex cell and gene therapies. To be competitive globally, biomanufacturers need to focus on aligning with global standards with regard to drug quality, reducing manufacturing failures and delivering drugs to market quickly. Building these capabilities requires a multifaceted approach that includes improvements in operations, quality compliance, and control strategies. To address these needs, the US Pharmacopeia (USP), the Department of Biotechnology (DBT) India, and the Confederation of Indian Industry (CII) held a symposium to discuss the requirements and gaps in the biotechnology and pharmaceutical sectors in India and other developing countries. A panel of experts from academia, manufacturing, and governmental agencies identified several drivers needed for capability building, including a skilled workforce, public-private partnerships, advanced manufacturing technologies, novel biologics, and favorable policies. This article summarizes the recommendations put forward by this panel.

Assessment of Functional Characterization and Comparability of Biotherapeutics: a Review.

The development of monoclonal antibody (mAb) biosimilars is a complex process. The key to their successful development and commercialization is an in-depth understanding of the key product attributes that impact safety and efficacy and the strategies to control them. Functional assessment of mAb is a crucial part of the comparability of biopharmaceutical drugs. The development of a relevant and robust functional assay requires an interdisciplinary approach and sufficient flexibility to balance regulatory concerns as well as dynamics and variability during the manufacturing process. Although many advanced tools are available to study and compare the potency and bioactivity of the protein, most of these techniques suffer from major shortcomings that limit their routine use. These include the complexity of the task, establishment of the relevance of the chosen method with the mechanism of action (MOA) of the biosimilar, cost and extended time of analysis, and often the ambiguity in interpretation of the resulting data. To overcome or to address these challenges, the use of multiple orthogonal state-of-the-art techniques is a necessary prerequisite.

Understanding Quality Paradigm Shifts in the Evolving Pharmaceutical Landscape: Perspectives from the USP Quality Advisory Group.

Recent changes in the pharmaceutical industry have led to significant paradigm shifts in the pharmaceutical quality environment. Globalization of the pharmaceutical industry, increasingly rapid development of novel therapies, and adoption of new manufacturing techniques have presented numerous challenges for the established regulatory framework and quality environment and are impacting the approaches utilized to ensure the quality of pharmaceutical products. Regulators, industry, and standards-setting organizations have begun to recognize the need to rely more on integrated risk-based approaches and to create more nimble and flexible standards to complement these efforts. They also increasingly have recognized that quality needs to be built into systems and processes throughout the lifecycle of the product. Moreover, the recent COVID-19 crisis has emphasized the need to adopt practices that better promote global supply chain resilience. In this paper, the USP Quality Advisory Group explores the various paradigm shifts currently impacting pharmaceutical quality and the approaches that are being taken to adapt to this new environment. Broad adoption of the Analytical Procedure Lifecycle approach, improved data management, and utilization of digital technologies are identified as potential solutions that can help meet the challenges of these quality paradigm shifts. Further discussion and collaboration among stakeholders are needed to pursue these and other solutions that can ensure a continued focus on quality while facilitating pharmaceutical innovation and development.

Predicting the Severity of Disease Progression in COVID-19 at the Individual and Population Level: A Mathematical Model.

The impact of COVID-19 disease on health and economy has been global, and the magnitude of devastation is unparalleled in modern history. Any potential course of action to manage this complex disease requires the systematic and efficient analysis of data that can delineate the underlying pathogenesis. We have developed a mathematical model of disease progression to predict the clinical outcome, utilizing a set of causal factors known to contribute to COVID-19 pathology such as age, comorbidities, and certain viral and immunological parameters. Viral load and selected indicators of a dysfunctional immune response, such as cytokines IL-6 and IFNα which contribute to the cytokine storm and fever, parameters of inflammation D-Dimer and Ferritin, aberrations in lymphocyte number, lymphopenia, and neutralizing antibodies were included for the analysis. The model provides a framework to unravel the multi-factorial complexities of the immune response manifested in SARS-CoV-2 infected individuals. Further, this model can be valuable to predict clinical outcome at an individual level, and to develop strategies for allocating appropriate resources to manage severe cases at a population level.

Predicting the severity of disease progression in COVID-19 at the individual and population level: A mathematical model.

The impact of COVID-19 disease on health and economy has been global, and the magnitude of devastation is unparalleled in modern history. Any potential course of action to manage this complex disease requires the systematic and efficient analysis of data that can delineate the underlying pathogenesis. We have developed a mathematical model of disease progression to predict the clinical outcome, utilizing a set of causal factors known to contribute to COVID-19 pathology such as age, comorbidities, and certain viral and immunological parameters. Viral load and selected indicators of a dysfunctional immune response, such as cytokines IL-6 and IFNα, which contribute to the cytokine storm and fever, parameters of inflammation d-dimer and ferritin, aberrations in lymphocyte number, lymphopenia, and neutralizing antibodies were included for the analysis. The model provides a framework to unravel the multi-factorial complexities of the immune response manifested in SARS-CoV-2 infected individuals. Further, this model can be valuable to predict clinical outcome at an individual level, and to develop strategies for allocating appropriate resources to mitigate severe cases at a population level.

Modulation of granulocyte colony stimulating factor conformation and receptor binding by methionine oxidation.

Biosimilars offer an avenue for potential cost savings and enhanced patient access to various emerging therapies in a budget neutral way. Biosimilars of the granulocyte colony stimulating factor (GCSF) are an excellent example in this regard with as many as 18 versions of the drug being currently approved across globe for treatment of neutropenia. Here, we identified oxidation of the various methionine residues in GCSF as a key heterogeneity that adversely impact its efficacy. In agreement with earlier studies, it was found that oxidation of Met 122 and Met 127 significantly contributes toward reduction of GCSF efficacy, measured using binding affinity to the GCSF receptor. The combination of molecular dynamics simulation along with structural characterization studies established that oxidation of Met 127 and Met 122 brings about a small local conformational change around the B-C loop in GCSF structure due to slight displacement of Asp113 and Thr117 residues. The simulation studies were validated using fluorescence quenching experiments using acrylamide as quencher and site-directed mutagenesis by replacing Met 122 and Met 127 residues with alanine. The results of this study lead to an enhanced mechanistic understanding of the oxidation in GCSF and should be useful in protein engineering efforts to design stable, safe, and efficacious GCSF product. In addition, the structure-function information can provide targets for protein engineering during early drug development and setting specifications of allowable limits of product variants in biosimilar products.

Failure Mode and Effects Analysis (FMEA) for Immunogenicity of Therapeutic Proteins.

Biotherapeutic drugs made by cell-based systems are revolutionizing the practice of medicine. The next generation of biotherapeutics include recombinant proteins, monoclonal antibodies, viral vector expressed proteins, and cell therapies. Immunogenicity associated adverse events is one of the major risks for these biologics. Accurate and precise measurement of the immunogenicity of biologics is a critical component during all phases of drug development. We have utilized the principles of Failure Mode and Effects Analysis (FMEA) in performing assessment of risk of immunogenicity. The multi-dimensional approach involves: i) listing all the potential risks by likelihood of occurrence and severity as part of quality target product profile. ii) ascribing the causes by identifying the risks at each stage of development. iii) predicting the effects. iv) determining the risk mitigation strategy. v) implementing a monitoring process. vi) developing templates for data collection. vii) timely reporting and. viii) life cycle management. FMEA is a continuous process that works throughout the lifecycle of the product or the process and keeps on getting updated with new insights and knowledge.

Recommendations for Enhancing Quality and Capability of Indian Biopharmaceutical Industry: Summary of a Workshop.

The biopharmaceutical industry is undergoing an evolutionary phase with the rise of advanced manufacturing technologies. The regulatory and customer requirements are shifting towards the development of personalized or targeted medicines. With this changing landscape, industry must evaluate the relevance of quality management systems. Over the past two decades, Indian companies have played a significant role in generating access and reducing costs of medicines. The quality management systems that enable the development and manufacturing of biopharmaceuticals require companies to adapt to regulatory requirements of process development, clinical trials, production, and life cycle management. To better understand the status and potential opportunities to enhance the quality management systems of manufacturing biopharmaceuticals, a workshop was organized by United States Pharmacopeia (USP) and Association of Biotechnology Led Enterprises (ABLE). This paper summarizes the recommendations by the panel and participants of the workshop to industry stakeholders, governance bodies, and policymakers. Following points were proposed to strengthen the culture of quality processes in Indian biopharmaceutical industry: i) Inculcating a culture of quality; ii) Effective training programs on quality processes; iii) Focus on quality beyond compliance; iv) Focus on automation and digitization. v) Enhance processes for pharmacovigilance and product life cycle management. vi) Understanding global regulatory processes.

Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins.

Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA. Presence of neutralizing antibodies (Nab) in samples will inhibit insulin induced receptor phosphorylation and consequently lead to a reduction in the percentage of phosphorylated insulin receptor. Additionally, usage of human insulin receptor overexpressing recombinant CHO cell line further improved the assay sensitivity by reducing the fixed drug (EC50) concentration used for induction of receptor phosphorylation. To ensure adequate free drug tolerance a pre-treatment step was introduced, where serum samples underwent acid dissociation and charcoal extraction before drug incubation. In order to distinguish ADA positive samples containing true Nab from samples containing non-antibody phosphorylation inhibitory serum factors, a confirmatory tier was integrated based on immunodepletion using protein AGL mix. Assay parameters including determination of screening and confirmatory cut-points, intra and inter assay precision, selectivity, specificity and stability were assessed during validation in accordance with recent regulatory guidelines and white papers. The advantage of selecting insulin receptor phosphorylation as assay endpoint made the assay capable of detecting Nab against insulin and insulin analogues.

Development and validation of an electrochemiluminescent ELISA for quantitation of oral insulin tregopil in diabetes mellitus serum.

AIM: Tregopil, a novel PEGylated human insulin is in clinical development for oral delivery in diabetes treatment. The aim of the study was to develop and validate a sensitive and specific ELISA method for quantitating Tregopil in diabetes subjects on basal Glargine, since most commercially available insulin kits either do not detect Tregopil or show significant reactivity to Glargine. METHODS: An electrochemiluminescent ELISA was developed and validated for Tregopil quantitation in diabetes serum. RESULTS: The method has a LLOQ of 0.25 ng/ml, shows minimum cross-reactivity to Glargine and was successfully tested using a subset of samples from Tregopil-dosed Type 1 diabetes mellitus patients. CONCLUSION: The ELISA method is sensitive and can be used to support accurate measurement of Tregopil with no cross-reactivity to Glargine and its metabolites in clinical studies.

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.