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Biomacromolecules are viewed as promising drugs due to their specific functions in biological processes, biocompatibility, and pharmacological efficacy. Injective administration, chosen to avoid intestinal barriers, may in turn lead to immediate decay in the circulation system, unreliable targeting performance, or the induction of immune responses. For some biomacromolecules, chemically modified proteins have been developed for practical use. Various cargo or carrier systems are under development but have been delayed by technical difficulties. We present self-assembled nanocapsules with diameters ranging from 100 to 500 nm that can be deployed in physiological buffers to enclose various substances present in the buffers at the same time. Our amphiphilic nanocapsule, consisting of silole-core dendrimer products as the hydrophobic part and green fluorescent protein (GFP) derivatives as the hydrophilic part, connects and assembles spontaneously when mixed in solutions while engulfing dissolved or dispersed compounds together in a dose-dependent manner and shows unique optical characteristics because the dendrimer products exhibit aggregation-induced emission. Furthermore, the emission of the dendrimer causes considerable fluorescence resonance energy transfer (FRET) to GFP derivatives upon association. We could easily monitor assemblies by FRET states and particle sizes and have confirmed a stable presence in the buffer for at least a month. Further tracking of nanocapsules by fluorescence confirmed efficient uptake into some cancer cells. Nanocapsules based on GFP variants with or without a cell-surface-specific tag demonstrated that the tag improved the potential for specific targeted delivery. There were also indications that the nanocapsules became unstable after cellular uptake in the intracellular environment. We report here the simple preparation of traceable, stable, and biocompatible self-assembled nanocapsules as the basis for a versatile drug delivery system.

The present study was designed to develop a self-micellizing solid dispersion (SMSD) containing Thymoquinone (TQM), a phytonutrient obtained from Nigella sativa seeds, aiming to improve its biopharmaceutical and nephroprotective functions. The apparent solubility of TQM in polymer solutions was used to choose an appropriate amphiphilic polymer that could be used to make an SMSD system. Based on the apparent solubility, Soluplus® was selected as an appropriate carrier, and mixing with TQM, SMSD-TQM with different loadings of TQM (5-15%) was made by solvent evaporation and freeze-drying techniques, respectively, and the formulations were optimized. The optimized SMSD-TQM was evaluated in terms of particle size distribution, morphology, release characteristics, pharmacokinetic behavior, and nephroprotective effects in a rat model of acute kidney injury. SMSD-TQM significantly improved the dissolution characteristics (97.8%) of TQM in water within 60 min. Oral administration of SMSD-TQM in rats exhibited a 4.9-fold higher systemic exposure than crystalline TQM. In a cisplatin-induced (6 mg/kg, i.p. ) acute kidney-damaged rat model, oral SMSD-TQM (10 mg/kg) improved the nephroprotective effects of TQM based on the results of kidney biomarkers and histological abnormalities. These findings suggest that SMSD-TQM might be efficacious in enhancing the nephroprotective effect of TQM by overcoming biopharmaceutical limitations.

Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production., (© 2024 Wiley‐VCH GmbH.)

Background/aims: Provisions of the Inflation Reduction Act mandating drug price negotiation by the Centers for Medicare & Medicaid Services have been criticized as a threat to pharmaceutical innovation. This study models potential impacts of the Inflation Reduction Act on drug approvals based on the differential contributions of large pharmaceutical companies and smaller biotechnology firms to clinical trials and the availability of capital., Methods: This study examined research and development expense, revenue, and new investment (sale of common and preferred stock) by public biopharmaceutical companies and sponsorship of phased clinical trials in ClinicalTrials.gov. Financial data were incorporated in a model that estimates the number of drugs in each phase and approvals from reported phase-specific costs and transition rates, proportional sponsorship of trials by companies of different size, projected reductions in research and development spending based on company size, and three scenarios by which large companies may allocate reductions in research and development spending among clinical phases: (1) research and development proportionally reduced across phases; (2) research and development disproportionally reduced in phases 2-3; and (3) research and development disproportionately reduced in phases 1-2., Results: Financial data were examined for 1378 public biopharmaceutical companies (2000-2018). Research and development expense was associated with revenue for 79 large companies with market capitalization ≥$7 billion with a 10% reduction in revenue reducing research and development expense by 8.4%. For 1299 smaller companies with market capitalization <$7 billion, research and development was associated with new investment, but not revenue. Smaller companies sponsored 55.2% of phase 1, 55.6% of phase 2, and 49.8% of phase 3 trials in ClinicalTrials.gov 2013-2018. In a model of clinical development that apportions clinical trials between large and smaller companies and determines the number of trials based on research and development resources, 400 drugs entering development produced 47.3 approvals (11.83% rate). A 10% reduction in revenue, reflecting the upper boundary of observed changes 2000-2018, with (1) proportional reduction across phases 1-3 produced 45.1 approvals (4.61% reduction); (2) disproportional reduction of phases 2-3 produced 42.8 approvals (9.55% reduction); and (3) disproportional reduction of phases 1-2 produced 46.9 approvals (0.95% reduction)., Conclusion: This work suggests that the drug price negotiation provisions of the Inflation Reduction Act could have little or no impact on the number of drug approvals. While large pharmaceutical companies may reduce research and development spending, continued research and development by smaller companies and strategic allocation of research and development resources by large companies may mitigate any negative effects of the Inflation Reduction Act., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Drs G.V. and F.D.L. received a grant at Bentley University from the West Health Policy Center. Dr F.D.L. has received grants at Bentley University from the National Biomedical Research Foundation, Institute for New Economic Thinking, and the National Pharmaceutical Council. Drs F.D.L. and G.V. had full access to all of the data in this study.

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O -glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O -glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs., Competing Interests: All authors declare no conflict of interest., (© 2024 The Authors.)

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium . Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective and Significance: Reducing the dimensions, when other additives are present, shows potential as a method to improve the dissolution and solubility of biopharmaceutical classification system class II drugs that have poor solubility. In this investigation, the process involved grinding naproxen with nicotinamide with the aim of improving solubility and the rate of dissolution., Methods: Naproxen was subjected to co-milling with urea, dimethylurea, and nicotinamide using a planetary ball mill for a duration of 90 min, maintaining a 1:1 molar ratio for the excipients (screening studies). The co-milled combinations, naproxen in its pure milled form, and a physical mixture were subjected to analysis using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), and solubility assessment. The mixture displaying the highest solubility (naproxen-nicotinamide) was chosen for further investigation, involving testing for intrinsic dissolution rate (IDR) and Fourier-transform infrared spectroscopy (FTIR) after co-milling for both 90 and 480 min., Results and Conclusion: The co-milled combination, denoted as S-3b and consisting of the most substantial ratio of nicotinamide to naproxen at 1:3, subjected to 480 min of milling, exhibited a remarkable 45-fold increase in solubility and a 9-fold increase in IDR. XRPD analysis of the co-milled samples demonstrated no amorphization, while SEM images portrayed the aggregates of naproxen with nicotinamide. FTIR outcomes negate the presence of any chemical interactions between the components. The co-milled sample exhibiting the highest solubility and IDR was used to create a tablet, which was then subjected to comprehensive evaluation for standard attributes. The results revealed improved compressibility and dissolution properties.

The role of mass spectrometry (MS) has become more important in most application domains in recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number of routine quality control analyses to be carried out. The role of MS is, therefore, very different throughout the whole drug development cycle. While it dominates within the drug discovery and development phase, in routine quality control, the role of MS is minor and indispensable only for selected applications. Moreover, its role is very different in the case of analysis of small molecule pharmaceuticals and biopharmaceuticals. Our review explains the role of current MS in the analysis of both small-molecule chemical drugs and biopharmaceuticals. Important features of MS-based technologies being implemented, method requirements, and related challenges are discussed. The differences in analytical procedures for small molecule pharmaceuticals and biopharmaceuticals are pointed out. While a single method or a small set of methods is usually sufficient for quality control in the case of small molecule pharmaceuticals and MS is often not indispensable, a large panel of methods including extensive use of MS must be used for quality control of biopharmaceuticals. Finally, expected development and future trends are outlined., (© 2023 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

The stability of lactate dehydrogenase (LDH) and β-galactosidase (β-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while β-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, β-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and β-gal may be dependent on the interaction with arginine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Attractive self-interactions and reversible self-association are implicated in many problematic solution behaviors for therapeutic proteins, such as irreversible aggregation, elevated viscosity, phase separation, and opalescence. Protein self-interactions and reversible oligomerization of two Fc-fusion proteins (monovalent and bivalent) and the corresponding fusion partner protein were characterized experimentally with static and dynamic light scattering as a function of pH (5 and 6.5) and ionic strength (10 mM to at least 300 mM). The fusion partner protein and monovalent Fc-fusion each displayed net attractive electrostatic self-interactions at pH 6.5 and net repulsive electrostatic self-interactions at pH 5. Solutions of the bivalent Fc-fusion contained higher molecular weight species that prevented quantification of typical interaction parameters ( B 22 and k D ). All three of the proteins displayed reversible self-association at pH 6.5, where oligomers dissociated with increased ionic strength. Coarse-grained molecular simulations were used to model the self-interactions measured experimentally, assess net self-interactions for the bivalent Fc-fusion, and probe the specific electrostatic interactions between charged amino acids that were involved in attractive electrostatic self-interactions. Mayer-weighted pairwise electrostatic energies from the simulations suggested that attractive electrostatic self-interactions at pH 6.5 for the two Fc-fusion proteins were due to cross-domain interactions between the fusion partner domain(s) and the Fc domain.

Forced degradation, also known as stress testing, is used throughout pharmaceutical development for many purposes including assessing the comparability of biopharmaceutical products according to ICH Guideline Q5E. These formal comparability studies, the results of which are submitted to health authorities, investigate potential impacts of manufacturing process changes on the quality, safety, and efficacy of the drug. Despite the wide use of forced degradation in comparability assessments, detailed guidance on the design and interpretation of such studies is scarce. The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. The BioPhorum Development Group Forced Degradation Workstream recently conducted several group discussions and a benchmarking survey to understand current industry approaches for the use of forced degradation studies to assess comparability of protein-based biopharmaceuticals. The results provide insight into the design of forced degradation studies, analytical characterization and testing strategies, data evaluation criteria, as well as some considerations and differences for non-platform modalities (e.g., non-traditional mAbs). This article presents survey responses from several global companies of various sizes and provides an industry perspective and experience regarding the practicalities of using forced degradation to assess comparability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Drug repurposing refers to the process of discovering new therapeutic uses for existing medicines. Compared to traditional drug discovery, drug repurposing is attractive for its speed, cost, and reduced risk of failure. However, existing approaches for drug repurposing involve complex, computationally-intensive analytical methods that are not widely used in practice. Instead, repurposing decisions are often based on subjective judgments from limited empirical evidence. In this article, we develop a novel Bayesian network meta-analysis (NMA) framework that can predict the efficacy of an approved treatment in a new indication and thereby identify candidate treatments for repurposing. We obtain predictions using two main steps: first, we use standard NMA modeling to estimate average relative effects from a network comprised of treatments studied in both indications in addition to one treatment studied in only one indication. Then, we model the correlation between relative effects using various strategies that differ in how they model treatments across indications and within the same drug class. We evaluate the predictive performance of each model using a simulation study and find that the model minimizing root mean squared error of the posterior median for the candidate treatment depends on the amount of available data, the level of correlation between indications, and whether treatment effects differ, on average, by drug class. We conclude by discussing an illustrative example in psoriasis and psoriatic arthritis and find that the candidate treatment has a high probability of success in a future trial., (© 2023 John Wiley & Sons Ltd.)

The biopharmaceutical industry contributes substantially to volatile organic compounds (VOCs) emissions, causing growing concerns and social developmental conflicts. This study conducted an on-site investigation of the process-based emission of VOCs from three biopharmaceutical enterprises. In the workshops of the three enterprises, 26 VOCs were detected, which could be sorted into 4 classes: hydrocarbons, aromatic hydrocarbons, oxygen-containing compounds, and nitrogen-containing compounds. Ketones were the main components of waste gases, accounting for 44.13%-77.85% of the overall VOCs. Process-based source profiles were compiled for each process unit, with the fermentation and extraction units of tiamulin fumarate being the main source of VOC emissions. Dimethyl heptanone, vinyl acetate, diethylamine, propylene glycol methyl ether (PGME), and benzene were screened as priority pollutants through a fuzzy comprehensive evaluation system. Ground level concentration simulation results of the Gauss plume diffusion model demonstrated that the diffusivity of VOCs in the atmosphere was relatively high, indicating potential non-carcinogenic and carcinogenic risks 1.5-2 km downwind. Furthermore, the process-based formation potentials of ozone and secondary organic aerosols (SOAs) were determined and indicated that N-methyl-2-pyrrolidone, dimethyl heptanone, and PGME should be preferentially controlled to reduce the ozone formation potential, whereas the control of benzene and chlorobenzene should be prioritized to reduce the generation of SOAs. Our results provide a basis for understanding the characteristics of VOC emission by biopharmaceutical industries and their diffusion, potentially allowing the development of measures to reduce health risks and secondary pollution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Freezing is commonly encountered during the processing and storage of biomacromolecule products. Therefore, understanding the phase and state transitions in pharmaceutical frozen solutions is crucial for the rational development of biopharmaceuticals. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) was used to analyze solutions containing sodium phosphate buffer, histidine, and trehalose. Upon freezing, crystallization of disodium phosphate hydrogen dodecahydrate (Na 2 HPO 4 ·12H 2 O, DPDH) and histidine was identified using 31 P and 13 C ssNMR, respectively, and confirmed by synchrotron X-ray diffractometry (SXRD). Using histidine as a molecular probe and based on the chemical shifts of atoms of interest, the pH of the freeze concentrate was measured. The unfrozen water content in freeze concentrates was quantified by 1 H single pulse experiments. 13 C-insensitive nuclei enhancement by polarization transfer (INEPT) and cross-polarization (CP) experiments were used as orthogonal tools to characterize the solutes in a "mobile" and a more "solid-like" state in the freeze-concentrated solutions, respectively. The above analyses were applied to a commercial monoclonal antibody (mAb) formulation of dupilumab. This work further establishes ssNMR spectroscopy as a highly capable biophysical tool to investigate the attributes of biopharmaceuticals and thereby provide insights into process optimization and formulation development.

Patients with psoriasis are more likely to experience depression and suicidality compared to non-psoriatic patients, though systemic therapies have been shown to improve depressive symptoms. It is unclear whether or not biologic or oral agents are more effective at improving such depressive symptoms in psoriasis patients, however. We aimed to determine an estimate of the odds of incident depression in psoriasis patients on different systemic therapies by performing a cross-sectional analysis of postmarketing data. The reporting odds ratio (ROR) for 15 different systemic agents was calculated using reports from the Food and Drug Administration Adverse Events Reporting System (FAERS). After excluding brodalumab and apremilast due to high risk of reporting bias, we found oral agents were associated with a significantly higher ROR of depression compared to biologics (OR = 2.42, 95% confidence interval: 1.93-3.04). These results suggest biologics may be more effective at reducing incident depression than oral agents. Future controlled trials are needed to confirm these findings.

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Biopharmaceutical products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Aligning with the quality by design (QbD) framework and realization, recent advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation and related techniques have enhanced biopharmaceutical characterization capabilities and have supported an increased development of biopharmaceutical products. Beyond its routine qualitative characterization, the quantitative feature of LC-MS has unique applications in biopharmaceutical process development and manufacturing. This review describes the recent applications and implications of the advancement of quantitative MS methods in biopharmaceutical process development, and characterization of biopharmaceutical product, product-related variants, and process-related impurities. We also provide insights on the emerging applications of quantitative MS in the lifecycle of biopharmaceutical product development including quality control in the Good Manufacturing Practice (GMP) environment and process analytical technology (PAT) practices during process development and manufacturing. Through collaboration with instrument and software vendors and regulatory agencies, we envision broader adoption of phase-appropriate quantitative MS-based methods for the analysis of biopharmaceutical products, which in turn has the potential to enable manufacture of higher quality products for patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cardiovascular diseases, such as myocardial infarction, ischemic stroke, and pulmonary embolism, are the most common causes of disability and death worldwide. Blood clot hydrolysis by thrombolytic enzymes and thrombectomy are key clinical interventions. The most widely used thrombolytic enzyme is alteplase, which has been used in clinical practice since 1986. Another clinically used thrombolytic protein is tenecteplase, which has modified epitopes and engineered glycosylation sites, suggesting that carbohydrate modification in thrombolytic enzymes is a viable strategy for their improvement. This comprehensive review summarizes current knowledge on computational and experimental identification of glycosylation sites and glycan identity, together with methods used for their reengineering. Practical examples from previous studies focus on modification of glycosylations in thrombolytics, e.g., alteplase, tenecteplase, reteplase, urokinase, saruplase, and desmoteplase. Collected clinical data on these glycoproteins demonstrate the great potential of this engineering strategy. Outstanding combinatorics originating from multiple glycosylation sites and the vast variety of covalently attached glycan species can be addressed by directed evolution or rational design. Directed evolution pipelines would benefit from more efficient cell-free expression and high-throughput screening assays, while rational design must employ structure prediction by machine learning and in silico characterization by supercomputing. Perspectives on challenges and opportunities for improvement of thrombolytic enzymes by engineering and evolution of protein glycosylation are provided., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group A Streptococcus pyogenes C5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group A S. pyogenes known to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed in Escherichia coli cells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

Recombinant proteins (RP) are widely used as biopharmaceuticals, industrial enzymes, or sustainable food source. Yeasts, with their ability to produce complex proteins through a broad variety of cheap carbon sources, have emerged as promising eukaryotic production hosts. As such, the prevalence of yeasts as favourable production organisms in commercial RP production is expected to increase. Yet, with the selection of a robust production host on the one hand, successful scale-up is dependent on a thorough understanding of the challenging environment and limitations of large-scale bioreactors on the other hand. In the present work, several prominent yeast species, including Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis and Kluyveromyces marxianus are reviewed for their current state and performance in commercial RP production. Thereafter, the impact of principal process control parameters, including dissolved oxygen, pH, substrate concentration, and temperature, on large-scale RP production are discussed. Finally, technical challenges of process scale-up are identified. To that end, process intensification strategies to enhance industrial feasibility are summarized, specifically highlighting fermentation strategies to ensure sufficient cooling capacity, overcome oxygen limitation, and increase protein quality and productivity. As such, this review aims to contribute to the pursuit of sustainable yeast-based RP production., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objectives: Solid biopharmaceutical products can circumvent lower temperature storage and transport and increase remote access with lower carbon emissions and energy consumption. Saccharides are known stabilizers in a solid protein produced via lyophilization and spray drying (SD). Thus, it is essential to understand the interactions between saccharides and proteins and the stabilization mechanism., Methods: A miniaturized single droplet drying (MD) method was developed to understand how different saccharides stabilize proteins during drying. We applied our MD to different aqueous saccharide-protein systems and transferred our findings to SD., Results: The poly- and oligosaccharides tend to destabilize the protein during drying. The oligosaccharide, Hydroxypropyl β-cyclodextrin (HPβCD) shows high aggregation at a high saccharide-to-protein molar ratio (S/P ratio) during MD, and the finding is supported by nanoDSF results. The polysaccharide, Dextran (DEX) leads to larger particles, whereas HPBCD leads to smaller particles. Furthermore, DEX is not able to stabilize the protein at higher S/P ratios either. In contrast, the disaccharide Trehalose Dihydrate (TD) does not increase or induce protein aggregation during the drying of the formulation. It can preserve the protein's secondary structure during drying, already at low concentrations., Conclusion: During the drying of S/P formulations containing the saccharides TD and DEX, the MD approach could anticipate the in-process (in) stability of protein X at laboratory-scale SD. In contrast, for the systems with HPβCD, the results obtained by SD were contradictory to MD. This underlines that depending on the drying operation, careful consideration needs to be applied to the selection of saccharides and their ratios., (© 2023. The Author(s).)

Biopharmaceuticals are administered parenterally and therefore sterility is required. Sterility can be obtained via different processes including exposure to steam or dry heat. Sterilisation studies on biopharmaceuticals, which are highly sensitive medicinal products, are scarce. This study investigates the effect of different sterilisation processes on recombinant human insulin in solid state (gamma and e-beam irradiation (w/wo dry ice), nitrogen dioxide (NO 2 )) and in aqueous solution (gamma irradiation (w/wo dry ice, w/wo glycerin)) using ultra-high performance liquid chromatography-diode array detection-mass spectrometry. It is observed that NO 2 substantially degrades the solid samples, while gamma and e-beam irradiation result in lower levels of degradation (mean normalized peak areas of 95.2-96.2 % with respect to the non-sterilised samples). Gamma irradiation of insulin solutions with and without dry ice at 2.5 kGy results in mean normalised peak areas of 85 % and <40 % with respect to the non-sterilised samples, respectively. It is concluded that sterilisation using ionising radiation of liquid biopharmaceuticals with insulin and sterilisation of insulin dry powder using NO 2 is less suitable with the set-ups used here because of substantial degradation. In contrast, evidence is presented in favour of sterilisation of insulin dry powder using ionising radiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Various industries ranging from water purification to pharmaceutical production have experienced multi separation steps that impose more process time and contamination possibility by batch operation. We propose a developed microfluidic particle sorter (miniaturized hydrocyclone) that adopts centrifugal force as it has ability to decline the number of separation steps and the risk of extrinsic contamination in continuous process. While biological industries have not relied on mini hydrocyclones considerably because of low efficiency and microfabrication difficulties, current work has been planned to conquer these obstacles. In this research, biomass separation from fermentation broth by 3 mm hydrocyclones was investigated. The effect of apex size, feed flow rate, hydrocyclone geometry were analyzed numerically in four mini-hydrocyclones. The most efficient mini-hydrocyclone was chosen to be made by elegant additive manufacturing technology and studied experimentally. The separation efficiency was achieved up to 90% while the concentration ratio of heavy stream (apex) to dilute stream (vortex finder) was reached more than twofold. The mini hydrocyclone performance in view of energy target was studied by Euler-Reynolds-Efficiency plots. The 4 μm cut size was achieved that is promising high throughput separation for biological particles.

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

Plants have substantial potential for the development of various biopharmaceuticals. Plants provide a cost-effective and direct source for the production of biopharmaceuticals such as vaccines, antibodies, proteins, enzymes, and hormones. In most cases, purification is an important and expensive step in the production of these substances. The problem can be resolved when it is produced in plants and the whole plant can be consumed. Direct ingestion of plant materials may help in overcoming the purification step. Being produced in seeds, fruits and tubers, it helps in providing more immunization in developing countries at a cheaper rate. Moreover, it can be administered more efficiently than any other dosage forms. This review focuses on various immunization and therapeutic products that are produced in plants along with currently available formulations in each category., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We review medical economics literature presented at the 2023 annual AEA-ASSA convention, the largest gathering of economists worldwide. Pharmacoeconomic papers addressed a wide range of issues, including gender and racial gaps in clinical trials, hospital credit financing, drug rebates, covid-19 vaccine equality, and the opioid epidemic. Yet, they had some common identifiable themes. We examine them in the context of the "twin towers" of biopharmaceutical innovation: discovery and commercialization . Implementation outcomes and relative success of innovative solutions - whether in terms of products and services, structural design and arrangements, or policies - depend on how adequately they respond to questions and challenges that arise in drug discovery and commercialization, and who gains from them. That innovation's beneficiaries might not equally gain from its intended advantages is another unifying theme in the reviewed literature. Against this backdrop, biopharmaceutical innovation can breed new challenges and opportunities. And health policy can perform a critical, leveling function that reduces cost, increases access, and ensures quality of biopharmaceutical solutions.

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae 's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch ® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg -1 . The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi .

With growing regulatory interest in ensuring the integrity and consistency of the higher order structure of biopharmaceutical and vaccine products, validated methods will be required that operate in quality-regulated environments and in compliance with guidelines such as those published by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ICH]. Circular dichroism [CD] is a widely available, rapid and sensitive method to compare the secondary and tertiary structures of samples. For use in quality-regulated analyses multiple factors need to be considered. Firstly, a thorough understanding of the sources of experimental error is required, alongside protocols and reference standards for sample preparation, instrument calibration and performance monitoring. Secondly, validated algorithms to objectively compare spectra are required, with statistical evaluation to assess when values are significantly different and to define compliance criteria. Thirdly, regulatory authorities mandate the process of method assessment and validation to be acceptable for use in regulated environments. In this review I discuss regulatory expectation, the sources and magnitudes of instrument- and sample-derived uncertainties, the use of reference standards to define and correct instrument performance, and the algorithms used to objectively compare spectra. The ideas discussed here support the use of CD not only in quality-regulated environments but also best practice when used in a research environment., (Copyright © 2022. Published by Elsevier B.V.)

This study seeks to analyze the overall impact that biopharmaceutical innovation had on disability, Social Security recipiency, and the use of medical services of U.S. community residents during the period 1998-2015. We test the hypothesis that the probability of disability, Social Security recipiency, and medical care utilization associated with a medical condition is inversely related to the number of drug classes previously approved for that condition. We use data from the 1998-2015 waves of the Medical Expenditure Panel Survey and other sources to estimate probit models of an individual's probability of disability, Social Security recipiency, and medical care utilization. The effect of biopharmaceutical innovation is identified by differences across over 200 medical conditions in the growth in the lagged number of drug classes ever approved. 18 years of previous biopharmaceutical innovation is estimated to have reduced: the number of people who were completely unable to work at a job, do housework, or go to school in 2015 by 4.5%; the number of people with cognitive limitations by 3.2%; the number of people receiving SSI in 2015 by 247 thousand (3.1%); and the number of people receiving Social Security by 984 thousand (2.0%). Previous innovation is also estimated to have caused reductions in home health visits (9.2%), inpatient events (5.7%), missed school days (5.1%), and outpatient events (4.1%). The estimated value in 2015 of some of the reductions in disability, Social Security recipiency, and use of medical care attributable to previous biopharmaceutical innovation ($115 billion) is fairly close to 2015 expenditure on drug classes that were first approved by the FDA during 1989-2006 ($127 billion). However, for a number of reasons, the costs are likely to be lower, and the benefits are likely to be larger, than these figures., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Introduction: The biomedical industry has grown significantly both globally and in China; however, there are still challenges. This study aimed at evaluating the biopharmaceutical sector of China, in terms of ability to innovate, current sales volume, investment, and R&D expenditure, as well as providing a case study detailing the progress and challenges of the industry in Shaanxi province. Method: A cross-sectional mixed-method study design was used to generate a comprehensive profile of the nature of biopharmaceutical innovation capacity and development in China by triangulating country-wide survey and interview data from Shaanxi province. Only biopharmaceutical companies that are currently marketing or conducting research and development were eligible for inclusion, and Shaanxi province was selected for conducting the interviews. Categorical and continuous variables were analyzed descriptively. Interviews were thematically analyzed by using NVivo version 12. Results: The analysis includes responses from 77 biopharmaceutical enterprises; the majority (36, 46.8%) are in Eastern China, followed by 26 (33.8%) in Central China. In 2018, the total sales of biological products amounted to 26.28 billion yuan, and in 2019, a slight increase was observed (30.34 billion); the amount doubled in 2020 to about 67.91 billion yuan. The top three biopharmaceutical products on sale in 2020 were Camrelizumab (5.14 billion yuan), human albumin (4.56 billion yuan), and human immunoglobulin for injection (3.78 billion yuan). Expenditure on R&D has also increased; it amounted to 1657.7 million yuan in 2018, which more than doubled in 2019 to 3572.1 million yuan and further increased to 5857.7 million yuan in 2020. Nonetheless, the progress is not uniform across all provinces, as shown from the results from Shaanxi province, because of lack of local government policies that will impact on the funding, incentives, and market share that motivate the producers. Conclusion: China's biopharmaceutical industry has expand significantly. The increase in sales indicates that there is an increase in demand for biologicals; moreover, R&D funding is increasing. These are key indicators that influence innovation and development. However, the sector's capacity to innovate and develop needs to be improved, particularly in the western region, where research and production are relatively weak., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Kabba, Yao, Yang, Chang, Ji, Jiang, Zhao, Wen and Fang.)

The availability of rapid methods that can accurately define and quantify biopharmaceutical critical quality attributes has been the driving force for the implementation of mass spectrometry techniques throughout the development and production pipeline. While the multi-attribute method (MAM) has become widely adopted and developed, some critical information cannot be monitored through this workflow, such as correct chain assembly or the presence of fragments or aggregates. In this study, we combine intact protein mass spectrometry and the multi-attribute method to create an intact multi-attribute method - or iMAM. Using a CFR Part 11 compliant data system, we evaluated the proposed workflow using several intact analysis approaches under both denaturing and native conditions. As for the standard MAM approach, iMAM involves the generation of an intact protein target workbook which is created from the analysis of a reference sample, with ID confirmation obtained from deconvolution results and chromatographic retention times, while quantitation is obtained from the intensities of the m/z of most abundant charge states. The created processing method is then applied to the analysis of subsequent samples. New peak detection can also be performed, monitoring the number of components revealed after each analysis. The entire data process can be automated to generate a report within the chromatography data system software. Three case studies presented herein show the potential of iMAM for implementation at different stages of the production pipeline, from product development to stability testing and batch release., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein and peptide biopharmaceuticals have had a major impact on the treatment of a number of diseases. There is a growing interest in overcoming some of the challenges associated with biopharmaceuticals, such as rapid degradation in physiological fluid, using nanocarrier delivery systems. Biopharmaceutical nanoclusters (BNCs) where the therapeutic protein or peptide is clustered together to form the main constituent of the nanocarrier system have the potential to mimic the benefits of more established nanocarriers (e.g., liposomal and polymeric systems) whilst eliminating the issue of low drug loading and potential side effects from additives. These benefits would include enhanced stability, improved absorption, and increased biopharmaceutical activity. However, the successful development of BNCs is challenged by the physicochemical complexity of the protein and peptide constituents as well as the dynamics of clustering. Here, we present and discuss common methodologies for the synthesis of therapeutic protein and peptide nanoclusters, as well as review the current status of this emerging field., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Liquid biopharmaceuticals including monoclonal antibodies (mAbs) have been widely acknowledged to undergo various stresses during shipping/handling and long-term storage. Several mechanical stresses including shaking during shipping has been widely known to cause protein aggregation and sub-visible particle (SbVP) formation in liquid biopharmaceutical formulations. However, shaking-induced degradation of freeze-dried (FD) biopharmaceuticals has seldomly been reported in the literature and therefore this type of stress is widely overlooked in industry due to their presumed high stability, especially when the formulations and freeze-drying processes are fully optimized. In this Lessons Learned article, we report an interesting phenomenon in which the optimized FD biopharmaceutical formulations of three typical mAbs showed much degradation and SbVP formation under shaking stress compared with their liquid counterparts. This is a striking deviation to the notion that mAbs are generally more stable in the FD formulations than in the liquid ones. Therefore, shaking stress experiment should be considered a critical stress condition for early-stage selection of liquid versus FD mAb formulations., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibodies have been established as the largest product class of biopharmaceuticals. Since extensive characterization is required for development and quality control of monoclonal antibody, a widely available reference material (RM) is needed. Herein, a humanized IgG1κ monoclonal antibody reference material, RM 6208-a, AIST-MAB, was established by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The monoclonal antibody solution was produced as a pharmaceutical grade using a Chinese hamster ovary-derived cell line. The assigned indicative value represents the concentration of the antibody with a heterotetrameric structure including oligomeric forms, determined by an amino acid analysis using isotope dilution mass spectrometry, and their homogeneity and stability were assessed. In addition to antibody concentration, various physicochemical properties, including peptide mapping data, charge variants, and aggregates, were examined. This RM is intended for use in validation of analytical procedures and instruments such as a system suitability test for quantification of antibody. It is also intended for comparing and evaluating the results of antibody analyses across analytical methods and analytical laboratories such as inter-laboratory comparison. Both the material and the set of data from our study provide a tool for an accurate and reliable characterization of product quality attributes of monoclonal antibodies in biopharmaceutical and metrology communities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kinumi, Saikusa, Kato, Kojima, Igarashi, Noda and Honda.)

Mass spectrometry (MS)-based proteomic measurements are uniquely poised to impact the development of cell and gene therapies. With the adoption of rigorous instrumental performance qualifications (PQs), large-scale proteomics can move from a research to a manufacturing control tool. Especially suited, data-independent acquisition (DIA) approaches have distinctive qualities to extend multiattribute method (MAM) principles to characterize the proteome of cell therapies. Here, we describe the development of a DIA method for the sensitive identification and quantification of proteins on a Q-TOF instrument. Using the improved acquisition parameters, we defined a control strategy and highlighted some metrics to improve the reproducibility of SWATH acquisition-based proteomic measurements. Finally, we applied the method to analyze the proteome of Jurkat cells that here serves as a model for human T-cells. Raw and processed data were deposited in PRIDE (PXD029780).

Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development., Competing Interests: Author MLdL was employed by company Agilent Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yandrofski, Mouchahoir, De Leoz, Duewer, Hudgens, Anderson, Arbogast, Delaglio, Brinson, Marino, Phinney, Tarlov and Schiel.)

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase's properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension ( 1 D) conditions that are combined with MS-friendly RPLC conditions in the second dimension ( 2 D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets., (© 2022. Merck & Co., Inc., Kenilworth, NJ, USA and its affiliates and Davy Guillarme under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Glycoproteins are the dominant category among approved biopharmaceuticals, indicating their importance as therapeutic proteins. Glycoproteins are decorated with carbohydrate structures (or glycans) in a process called glycosylation. Glycosylation is a post-translational modification that is present in all kingdoms of life, albeit with differences in core modifications, terminal glycan structures, and incorporation of different sugar residues. Glycans play pivotal roles in many biological processes and can impact the efficacy of therapeutic glycoproteins. The majority of biopharmaceuticals are based on human glycoproteins, but non-human glycoproteins, originating from for instance parasitic worms (helminths), form an untapped pool of potential therapeutics for immune-related diseases and vaccine candidates. The production of sufficient quantities of correctly glycosylated putative therapeutic helminth proteins is often challenging and requires extensive engineering of the glycosylation pathway. Therefore, a flexible glycoprotein production system is required that allows straightforward introduction of heterologous glycosylation machinery composed of glycosyltransferases and glycosidases to obtain desired glycan structures. The glycome of plants creates an ideal starting point for N - and O -glyco-engineering of helminth glycans. Plants are also tolerant toward the introduction of heterologous glycosylation enzymes as well as the obtained glycans. Thus, a potent production platform emerges that enables the production of recombinant helminth proteins with unusual glycans. In this review, we discuss recent advances in plant glyco-engineering of potentially therapeutic helminth glycoproteins, challenges and their future prospects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Kaaij, van Noort, Nibbering, Wilbers and Schots.)

The aim of this study was to demonstrate the strength of combining immunochemical and biophysical analysis tools for assessing the quality of Sabin inactivated poliovirus vaccine (Sabin-IPV) bulk products. We assessed Sabin-IPV serotypes 1, 2 and 3 from six different manufacturers and evaluated their comparability through biosensor analysis and biophysical characterization methods, including tryptophan fluorescence and asymmetrical flow field-flow fractionation - multi-angle light scattering analysis. These methods enabled us to assess antigenic as well as conformational and structural integrity profiles, respectively. Based on Sabin-IPV samples that were subjected to accelerated storage conditions, we revealed that existing immunochemical methods exhibit remarkably similar trends to the results obtained by the biophysical characterization methods. While the results underpin that the comparability of Sabin-IPV bulk products of different manufacturers is weak, information about their quality can rapidly be obtained by using both immunochemical and biophysical methods. Furthermore, the study highlights that quality assessment of Sabin-IPV can be obtained through biophysical techniques can complement the assessments performed with monoclonal antibodies and suggests that similar techniques could be employed to characterize other enteroviruses., Competing Interests: Conflict of Interest Statement The authors declare there are no other conflicts of interest associated with this article., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniques and formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.

The charge heterogeneity of fusion proteins can vary dramatically compared with more traditional biopharmaceuticals like monoclonal antibodies, making the characterization of fusion proteins a challenge. A single platform method suitable for the analysis of multiple fusion proteins would reduce method development and streamline production workflows. Here, we develop a platform method to characterize the charge heterogeneity of a variety of fusion protein therapeutics using imaged capillary isoelectric focusing (icIEF). We describe the development of the platform method, and analyze 9 fusion protein therapeutics. The results are reproducible in peak group area percentage and apparent pI determination. We compare the platform icIEF method to traditional slab gel IEF, which is still used in many laboratories for the analysis of fusion proteins. The peak patterns obtained from the icIEF method is comparable to the band patterns of the gel IEF. The platform method can also be used as the starting point if further optimization is needed even when high resolution is required. The platform method described in this study can be applied as an identity and purity assay for fusion proteins in the biopharmaceutical industry., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Conflict of InterestThe authors declare no competing interests., (© The Author(s) 2021.)

The COVID-19 pandemic has once again brought the significance of biopharmaceutical and medical technology sectors to the spotlight. Seeing that some of the most critical medical breakthroughs such as the speedy mRNA vaccine development were results of cross-border patenting collaboration, we have proposed in a previous work a new method to identify the cross-border collaborative regional centres in the patent networks, using a clustering comparison approach based on adjusted mutual information (AMI). In this paper, we focus on the UK industrial landscape. We use the UK bioscience and health technology sector statistics from 2015 to 2020 and look into the regional growth of each postcode area. We compare the top growth regions with the cross-border collaborative centres identified using AMI comparison at the postcode area level, and find that both long-term and short-term AMI gains show an increase in the correlation with regional annual growth rates of firm numbers in the studied sectors from 2016 to 2020, and the increase is more consistent with the short-term AMI gain. We also found that areas more central in the long-term cross-regional R&D collaboration demonstrate a stronger association with more developed industrial settings indicated by more firms and, potentially more employment and turnover in the field. However, AMI gains are found to have negative correlations with the industrial growths as a sign of possible trade-offs of being central., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2022.)

Biopharmaceuticals have revolutionized the field of medicine in the types of active ingredient molecules and treatable indications. Adoption of Quality by Design and Process Analytical Technology (PAT) frameworks has helped the biopharmaceutical field to realize consistent product quality, process intensification, and real-time control. As part of the PAT strategy, Raman spectroscopy offers many benefits and is used successfully in bioprocessing from single-cell analysis to cGMP process control. Since first introduced in 2011 for industrial bioprocessing applications, Raman has become a first-choice PAT for monitoring and controlling upstream bioprocesses because it facilitates advanced process control and enables consistent process quality. This paper will discuss new frontiers in extending these successes in upstream from scale-down to commercial manufacturing. New reports concerning the use of Raman spectroscopy in the basic science of single cells and downstream process monitoring illustrate industrial recognition of Raman's value throughout a biopharmaceutical product's lifecycle. Finally, we draw upon a nearly 90-year history in biological Raman spectroscopy to provide the basis for laboratory and in-line measurements of protein quality, including higher-order structure and composition modifications, to support formulation development., (© 2021. The Author(s).)

Methods to optimize the solution behavior of therapeutic proteins are frequently time-consuming, provide limited information, and often use milligram quantities of material. Here, we present a simple, versatile method that provides valuable information to guide the identification and comparison of formulation conditions for, in principle, any biopharmaceutical drug. The subject protein is incubated with a designed synthetic peptide microarray; the extent of binding to each peptide is dependent on the solution conditions. The array is washed, and the adhesion of the subject protein is detected using a secondary antibody. We exemplify the method using a well-characterized human single-chain Fv and a selection of human monoclonal antibodies. Correlations of peptide adhesion profiles can be used to establish quantitative relationships between different solution conditions, allowing subgrouping into dendrograms. Multidimensional reduction methods, such as t-distributed stochastic neighbor embedding, can be applied to compare how different monoclonals vary in their adhesion properties under different solution conditions. Finally, we screened peptide binding profiles using a selection of monoclonal antibodies for which a range of biophysical measurements were available under specified buffer conditions. We used a neural network method to train the data against aggregation temperature, k D , percentage recovery after incubation at 25 °C, and melting temperature. The results demonstrate that peptide binding profiles can indeed be effectively trained on these indicators of protein stability and self-association in solution. The method opens up multiple possibilities for the application of machine learning methods in therapeutic protein formulation.

An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed-batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single-use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed-batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)