A network of regulatory innovations to improve FDA quality assessments of human drug applications.

A network of regulatory innovations brings a holistic approach to improving the submission, assessment, and lifecycle management of pharmaceutical quality information in the U.S. This dedicated effort in the FDA's Center for Drug Evaluation and Research (CDER) aims to enhance the quality assessment of submissions for new drugs, generic drugs, and biological products including biosimilars. These regulatory innovations include developing or contributing: (i) the Knowledge-Aided Assessment and Structured Application (KASA), (ii) a new common technical document for quality (ICH M4Q(R2)), (iii) structured data on Pharmaceutical Quality/Chemistry, Manufacturing and Controls (PQ/CMC), (iv) Integrated Quality Assessment (IQA), (v) the Quality Surveillance Dashboard (QSD), and (vi) the Established Conditions tool from the ICH Q12 guideline. The innovations collectively drive CDER toward a more coordinated, effective, and efficient quality assessment. Improvements are made possible by structured regulatory submissions, a systems approach to quality risk management, and data-driven decisions based on science, risk, and effective knowledge management. The intended result is better availability of quality medicines for U.S. patients.

Product Quality Research for Developing and Assessing Regulatory Submissions for Generic Cyclosporine Ophthalmic Emulsions.

Approval of the first generic 0.05% cyclosporine ophthalmic emulsion (COE) in the U.S. represents a milestone achievement of the science and research program in the U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER). COE is a locally acting complex drug product indicated to increase tear production in patients whose production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca. The path to approval required overcoming numerous scientific challenges to determining therapeutic equivalence to the reference listed drug. Researchers in CDER's Office of Pharmaceutical Quality and Office of Generic Drugs developed a quality by design approach to understand the effects of process and formulation variables on the product's critical quality attributes, including globule size distribution (GSD), turbidity, viscosity, zeta potential, surface tension, and osmolality. CDER researchers explored multiple techniques to perform physicochemical characterization and analyze the GSD including laser diffraction, nanoparticle tracking analysis, cryogenic transmission electron microscopy, dynamic light scattering, asymmetric field flow fractionation, and two-dimensional diffusion ordered spectroscopy nuclear magnetic resonance. Biphasic models to study drug transfer kinetics demonstrated that COEs with qualitative and quantitative sameness and comparable GSDs, analyzed using earth mover's distance, can be therapeutic equivalents. This body of research facilitated the review and approval of the first U.S. generic COE. In addition, the methods and fundamental understanding developed from this research may support the development and assessment of other complex generics. The approval of a generic COE should improve the availability of this complex drug product to U.S. patients.

Dosage unit uniformity and dissolution testing of extended-release pharmaceutical products marketed in the U.S.

An international sampling study yielded 69 samples of extended-release prescription pharmaceuticals for legal sale in the U.S. Samples included 29 lots of innovator and 40 lots of generic solid oral extended-release drugs manufactured at 16 different facilities and containing 6 different active ingredients. Dosage unit uniformity and dissolution were tested for each lot. All samples met the relevant testing criteria for dosage unit uniformity and dissolution. There were no indications that manufacturer or region impacted a product's acceptability for use by patients. The variability of attributes was used to calculate a process performance index (Ppk) for each facility. Higher Ppk values suggest less variability relative to specification limits. Only two manufacturers fell below a 4-sigma manufacturing benchmark Ppk of 1.33 for dosage unit uniformity: a European manufacturer of a brand drug and an Asian manufacturer of a generic drug. Conversely, all but four manufacturers fell below a 4-sigma benchmark for the minimum Ppk across their product's dissolution timepoints: generic drug manufacturers in India (two), the U.S., and Canada. Compared to the immediate-release products of a previous study, Ppks were generally lower for extended-release products. A retrospective analysis found that manufacturers performing below median Ppks submitted more Field Alert Reports after the end of the sampling period.

An audit of pharmaceutical continuous manufacturing regulatory submissions and outcomes in the US.

Continuous manufacturing (CM) sends materials directly and continuously to the next step of a process, eliminating hold times and reducing processing times. The potential benefits of CM include improved product quality, reduced waste, lower costs, and increased manufacturing flexibility and agility. Some pharmaceutical manufacturers have been hesitant to adopt CM owing to perceived regulatory risks such as increased time to regulatory approval and market entry, more difficulty submitting postapproval changes, and higher inspectional scrutiny. An FDA self-audit of regulatory submissions in the U.S. examined the outcomes, at approval and during the product lifecycle, of continuous manufacturing applications as compared to traditional batch applications. There were no substantial regulatory barriers identified for CM applications related to manufacturing process changes or pre-approval inspections. CM applicants had relatively shorter times to approval and market as compared to similar batch applications, based on the mean or median times to approval (8 or 3 months faster) and marketing (12 or 4 months faster) from submission, translating to an estimated $171-537 M in early revenue benefit.

Industry 4.0 for pharmaceutical manufacturing: Preparing for the smart factories of the future.

Over the last two centuries, medicines have evolved from crude herbal and botanical preparations into more complex manufacturing of sophisticated drug products and dosage forms. Along with the evolution of medicines, the manufacturing practices for their production have advanced from small-scale manual processing with simple tools to large-scale production as part of a trillion-dollar pharmaceutical industry. Today's pharmaceutical manufacturing technologies continue to evolve as the internet of things, artificial intelligence, robotics, and advanced computing begin to challenge the traditional approaches, practices, and business models for the manufacture of pharmaceuticals. The application of these technologies has the potential to dramatically increase the agility, efficiency, flexibility, and quality of the industrial production of medicines. How these technologies are deployed on the journey from data collection to the hallmark digital maturity of Industry 4.0 will define the next generation of pharmaceutical manufacturing. Acheiving the benefits of this future requires a vision for it and an understanding of the extant regulatory, technical, and logistical barriers to realizing it.

An Overview of Drug Substance Manufacturing Processes.

To develop a comprehensive understanding of pharmaceutical drug substance manufacturing (DSM) processes, we conducted a data mining study to examine 50 new drug applications (NDAs) approved in 2010-2016. We analyzed the prevalence of several frequently deployed in-process control (IPC) techniques and postreaction workup procedures, as well as the operational conditions specified for reactions and workups. Our findings show that crystallization and high-performance liquid chromatography (HPLC) were the most commonly used workup steps and in-process controls, respectively, in drug substance manufacturing. On average, each NDA implemented 12.6 in-process controls and 11.3 workups. Operation time for reactions and workup procedures varied from a few minutes to multiple days, though 61% of these were between 1 and 10 h.

Quality Testing of Difficult-to-Make Prescription Pharmaceutical Products Marketed in the US.

Importance: Health care practitioners and patients must have information to support their confidence in the quality of prescription pharmaceuticals. Objective: To determine whether there were clear and substantive differences in major quality attributes between difficult-to-make solid oral dosage form pharmaceutical products marketed in the US. Design, Setting, and Participants: This quality improvement study analyzed US Food and Drug Administration-collected samples of 252 drug products marketed in the US and manufactured in the US, Canada, Europe, India, and the rest of Asia. These drug products were immediate-release solid oral dosage forms considered difficult to make on the basis of product quality history. This sampling included 35 innovator and 217 generic drug samples manufactured by 46 different firms containing 17 different active ingredients. Statistical analysis was performed from February to November 2019. Main Outcomes and Measures: All products were tested within their shelf life on the basis of the legally recognized tests of the US Pharmacopeia for the major quality attributes of dosage unit uniformity and dissolution. These tests measure dosage consistency and drug release, respectively. The consistency of either attribute was used to calculate a process performance index to describe the variability in manufacturing. Results: All 252 drug product samples met the US market standards for dosage unit uniformity and dissolution, although the process performance index (Ppk) for dissolution fell below the level of 4-sigma capability (ie, <1 error per 1600) for 11 different manufacturers and for generics in 4 of 5 regions, including the US. As part of a retrospective analysis, manufacturers performing above the median Ppk for either dissolution or dosage unit uniformity submitted fewer product quality defect reports (mean field alert reports of 0.22 and 0.63, respectively) than those falling at or below the median Ppk for these attributes (mean field alert reports of 2.1 and 1.7, respectively). Conclusions and Relevance: All samples met the US market standards for dosage unit uniformity and dissolution, indicating acceptability for use by patients regardless of manufacturer or region. To our knowledge, this is the largest sampling study of pharmaceutical manufacturers for the US market and these data provide objective insight into the quality of prescription drugs with high manufacturing risks.

The Current Scientific and Regulatory Landscape in Advancing Integrated Continuous Biopharmaceutical Manufacturing.

There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. On the upstream side of biotechnology manufacturing, continuous perfusion cell cultures are fairly well established. However, truly integrated continuous biomanufacturing requires the uninterrupted connection of continuous unit operations (upstream and downstream) with no isolated intermediate or hold steps occurring between them. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing.

A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

The Botanical Drug Substance Crofelemer as a Model System for Comparative Characterization of Complex Mixture Drugs.

Crofelemer is a botanical polymeric proanthocyanidin that inhibits chloride channel activity and is used clinically for treating HIV-associated secretory diarrhea. Crofelemer lots may exhibit significant physicochemical variation due to the natural source of the raw material. A variety of physical, chemical, and biological assays were used to identify potential critical quality attributes (CQAs) of crofelemer, which may be useful in characterizing differently sourced and processed drug products. Crofelemer drug substance was extracted from tablets of one commercial drug product lot, fractionated, and subjected to accelerated thermal degradation studies to produce derivative lots with variations in chemical and physical composition potentially representative of manufacturing and raw material variation. Liquid chromatography, UV absorbance spectroscopy, mass spectrometry, and nuclear magnetic resonance analysis revealed substantial changes in the composition of derivative lots. A chloride channel inhibition cell-based bioassay suggested that substantial changes in crofelemer composition did not necessarily result in major changes to bioactivity. In 2 companion papers, machine learning and data mining approaches were applied to the analytical and biological data sets presented herein, along with chemical stability data sets derived from forced degradation studies, to develop an integrated mathematical model that can identify CQAs which are most relevant in distinguishing between different populations of crofelemer.

Comparative Characterization of Crofelemer Samples Using Data Mining and Machine Learning Approaches With Analytical Stability Data Sets.

There is growing interest in generating physicochemical and biological analytical data sets to compare complex mixture drugs, for example, products from different manufacturers. In this work, we compare various crofelemer samples prepared from a single lot by filtration with varying molecular weight cutoffs combined with incubation for different times at different temperatures. The 2 preceding articles describe experimental data sets generated from analytical characterization of fractionated and degraded crofelemer samples. In this work, we use data mining techniques such as principal component analysis and mutual information scores to help visualize the data and determine discriminatory regions within these large data sets. The mutual information score identifies chemical signatures that differentiate crofelemer samples. These signatures, in many cases, would likely be missed by traditional data analysis tools. We also found that supervised learning classifiers robustly discriminate samples with around 99% classification accuracy, indicating that mathematical models of these physicochemical data sets are capable of identifying even subtle differences in crofelemer samples. Data mining and machine learning techniques can thus identify fingerprint-type attributes of complex mixture drugs that may be used for comparative characterization of products.

Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs.

As the second of a 3-part series of articles in this issue concerning the development of a mathematical model for comparative characterization of complex mixture drugs using crofelemer (CF) as a model compound, this work focuses on the evaluation of the chemical stability profile of CF. CF is a biopolymer containing a mixture of proanthocyanidin oligomers which are primarily composed of gallocatechin with a small contribution from catechin. CF extracted from drug product was subjected to molecular weight-based fractionation and thiolysis. Temperature stress and metal-catalyzed oxidation were selected for accelerated and forced degradation studies. Stressed CF samples were size fractionated, thiolyzed, and analyzed with a combination of negative-ion electrospray ionization mass spectrometry (ESI-MS) and reversed-phase-HPLC with UV absorption and fluorescence detection. We further analyzed the chemical stability data sets for various CF samples generated from reversed-phase-HPLC-UV and ESI-MS using data-mining and machine learning approaches. In particular, calculations based on mutual information of over 800,000 data points in the ESI-MS analytical data set revealed specific CF cleavage and degradation products that were differentially generated under specific storage/degradation conditions, which were not initially identified using traditional analysis of the ESI-MS results.

Advancing pharmaceutical quality: An overview of science and research in the U.S. FDA's Office of Pharmaceutical Quality.

Failures surrounding pharmaceutical quality, particularly with respect to product manufacturing issues and facility remediation, account for the majority of drug shortages and product recalls in the United States. Major scientific advancements pressure established regulatory paradigms, especially in the areas of biosimilars, precision medicine, combination products, emerging manufacturing technologies, and the use of real-world data. Pharmaceutical manufacturing is increasingly globalized, prompting the need for more efficient surveillance systems for monitoring product quality. Furthermore, increasing scrutiny and accelerated approval pathways provide a driving force to be even more efficient with limited regulatory resources. To address these regulatory challenges, the Office of Pharmaceutical Quality (OPQ) in the Center for Drug Evaluation and Research (CDER) at the U.S. Food and Drug Administration (FDA) harbors a rigorous science and research program in core areas that support drug quality review, inspection, surveillance, standards, and policy development. Science and research is the foundation of risk-based quality assessment of new drugs, generic drugs, over-the-counter drugs, and biotechnology products including biosimilars. This is an overview of the science and research activities in OPQ that support the mission of ensuring that safe, effective, and high-quality drugs are available to the American public.

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions.

Glycan microarrays have become a powerful platform to investigate the interactions of carbohydrates with a variety of biomolecules. However, the number and diversity of glycans available for use in such arrays represent a key bottleneck in glycan array fabrication. To address this challenge, we describe a novel glycan array platform based on surface patterning of engineered glycophages that display unique carbohydrate epitopes. Specifically, we show that glycophages are compatible with surface immobilization procedures and that phage-displayed oligosaccharides retain the ability to be recognized by different glycan-binding proteins (e.g. antibodies and lectins) after immobilization. A key advantage of glycophage arrays is that large quantities of glycophages can be produced biosynthetically from recombinant bacteria and isolated directly from bacterial supernatants without laborious purification steps. Taken together, the glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high-throughput analysis of glycan-protein interactions.

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.

The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-linked glycosylation. This machinery has been functionally transferred into Escherichia coli, enabling convenient mechanistic dissection of the N-linked glycosylation process in this genetically tractable host. Here we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the -2 position relative to asparagine. This involved creation of a genetic assay, glycosylation of secreted N-linked acceptor proteins (glycoSNAP), that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that could glycosylate an array of noncanonical acceptor sequences, including one in a eukaryotic N-glycoprotein. These results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-linked glycosylation and for engineering designer N-linked glycosylation biocatalysts.

Directed evolution of Mycobacterium tuberculosis ß-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the ß-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A ß-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to ß-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.

Glycans-by-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates.

There is an urgent need for new tools that enable better understanding of the structure, recognition, metabolism, and biosynthesis of glycans as well as the production of biologically important glycans and glycoconjugates. With the discovery of glycoprotein synthesis in bacteria and functional transfer of glycosylation pathways between species, Escherichia coli cells have become a tractable host for both understanding glycosylation and the underlying glycan code of living cells as well as for expressing glycoprotein therapeutics and vaccines. Here, we review recent efforts to harness natural biological pathways and engineer synthetic designer pathways in bacteria for making complex glycans and conjugating these to lipids and proteins. The result of these efforts has been a veritable transformation of bacteria into living factories for scalable, bottom-up production of complex glycoconjugates by design.

An engineered eukaryotic protein glycosylation pathway in Escherichia coli.

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.