Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins.

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

Bioanalytical approaches to support the development of antibody-oligonucleotide conjugate (AOC) therapeutic proteins.

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterise the ADME properties of an AOC during development to optimise distribution to target tissues, to minimise the impact of biotransformation on exposure, and to characterise the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterisation of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterisation of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties.

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material.

BACKGROUND: Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. RESULTS: Here, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. CONCLUSIONS: We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

Progress in fed-batch culture for recombinant protein production in CHO cells.

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells.

Cell-free protein synthesis system: A new frontier for sustainable biotechnology-based products.

Cell-free protein synthesis (CFPS) system is an innovative technology with a wide range of potential applications that could challenge current thinking and provide solutions to environmental and health issues. CFPS system has been demonstrated to be a successful way of producing biomolecules in a variety of applications, including the biomedical industry. Although there are still obstacles to overcome, its ease of use, versatility, and capacity for integration with other technologies open the door for it to continue serving as a vital instrument in synthetic biology research and industry. In this review, we mainly focus on the cell-free based platform for various product productions. Moreover, the challenges in the bio-therapeutic aspect using cell-free systems and their future prospective for the improvement and sustainability of the cell free systems.

Meeting report of the 3rd European Biotransformation Workshop.

Challenges, strategies and new technologies in the field of biotransformation were presented and discussed at the 3rd European Biotransformation Workshop which was held in collaboration with the DMDG on 5-6 October 2022 in Amsterdam. In this meeting report we summarise the presentations and discussions from this workshop. The topics covered are listed below:Accelerator mass spectrometry (AMS) for the support of microtracer studiesBiotransformation of the novel myeloperoxidase inhibitor AZD4831 in preclinical species and humansAMS in biotransformation studies: unusual case studiesDiscussion on new FDA draft guidance and AMSMultimodal molecular imaging and ion mobility applications in drug discovery and developmentMetabolites in Safety Testing considerations for large molecules.

Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications.

The formation of inclusion bodies (IBs) during expression of recombinant therapeutic proteins using E. coli is a significant hurdle in producing high-quality, safe, and efficacious medicines. The improved understanding of the structure-function relationship of the IBs has resulted in the development of novel biotechnologies that have streamlined the isolation, solubilization, refolding, and purification of the active functional proteins from the bacterial IBs. Together, this overall effort promises to radically improve the scope of experimental biology of therapeutic protein production and expand new prospects in IBs usage. Notably, the IBs are increasingly used for applications in more pristine areas such as drug delivery and material sciences. In this review, we intend to provide a comprehensive picture of the bio-processing of bacterial IBs, including assessing critical gaps that still need to be addressed and potential solutions to overcome them. We expect this review to be a useful resource for those working in the area of protein refolding and therapeutic protein production.

Cell-Free Expression of a Therapeutic Protein Serratiopeptidase.

Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca2+ ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications.

Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress.

Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.

Not your usual drug-drug interactions: Monoclonal antibody-based therapeutics may interact with antiseizure medications.

Therapeutic monoclonal antibodies (mAbs) have emerged as the fastest growing drug class. As such, mAbs are increasingly being co-prescribed with other drugs, including antiseizure medications (ASMs). Although mAbs do not share direct targets or mechanisms of disposition with small-molecule drugs (SMDs), combining therapeutics of both types can increase the risk of adverse effects and treatment failure. The primary goal of this literature review was identifying mAb-ASM combinations requiring the attention of professionals who are treating patients with epilepsy. Systematic PubMed and Embase searches (1980-2021) were performed for terms relating to mAbs, ASMs, drug interactions, and their combinations. Additional information was obtained from documents from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Evidence was critically appraised - key issues calling for clinicians' consideration and important knowledge gaps were identified, and practice recommendations were developed by a group of pharmacists and epileptologists. The majority of interactions were attributed to the indirect effects of cytokine-modulating antibodies on drug metabolism. Conversely, strong inhibitors or inducers of drug-metabolizing enzymes or drug transporters could potentially interact with the cytotoxic payload of antibody-drug conjugates, and ASMs could alter mAb biodistribution. In addition, mAbs could potentiate adverse ASM effects. Unfortunately, few studies involved ASMs, requiring the formulation of class-based recommendations. Based on the current literature, most mAb-ASM interactions do not warrant special precautions. However, specific combinations should preferably be avoided, whereas others require monitoring and potentially adjustment of the ASM doses. Reduced drug efficacy or adverse effects could manifest days to weeks after mAb treatment onset or discontinuation, complicating the implication of drug interactions in potentially deleterious outcomes. Prescribers who treat patients with epilepsy should be familiar with mAb pharmacology to better anticipate potential mAb-ASM interactions and avoid toxicity, loss of seizure control, or impaired efficacy of mAb treatment.

A systematic review on disease-drug-drug interactions with immunomodulating drugs: A critical appraisal of risk assessment and drug labelling.

AIM: Use of immunomodulating therapeutics for immune-mediated inflammatory diseases may cause disease-drug-drug interactions (DDDIs) by reversing inflammation-driven alterations in the metabolic capacity of cytochrome P450 enzymes. European Medicine Agency (EMA) and US Food and Drug Administration (FDA) guidelines from 2007 recommend that the DDDI potential of therapeutic proteins should be assessed. This systematic analysis aimed to characterize the available DDDI trials with immunomodulatory drugs, experimental evidence for a DDDI risk and reported DDDI risk information in FDA/EMA approved drug labelling. METHOD: For this systematic review, the EMA list of European Public Assessment Reports of human medicine was used to select immunomodulating monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) marketed after 2007 at risk for a DDDI. Selected drugs were included in PubMed and Embase searches to extract reported interaction studies. The Summary of Product Characteristics (SPCs) and the United States Prescribing Information (USPIs) were subsequently used for analysis of DDDI risk descriptions. RESULTS: Clinical interaction studies to evaluate DDDI risks were performed for 12 of the 24 mAbs (50%) and for none of the TKIs. Four studies identified a DDDI risk, of which three were studies with interleukin-6 (IL-6) neutralizing mAbs. Based on (non)clinical data, a DDDI risk was reported in 32% of the SPCs and in 60% of the USPIs. The EMA/FDA documentation aligned with the DDDI risk potential in 35% of the 20 cases. CONCLUSION: This systematic review reinforces that the risk for DDDI by immunomodulating drugs is target- and disease-specific. Drug labelling information designates the greatest DDDI risk to mAbs that neutralize the effects of IL-6, Tumor Necrosis Factor alfa (TNF-α) and interleukin-1 bèta (IL-1ß) in diseases with systemic inflammation.

A patent review of the antimicrobial applications of lectins: Perspectives on therapy of infectious diseases.

Patents of lectins with antiviral, antibacterial and antifungal applications were searched and reviewed. Lectins are proteins that reversibly bind to specific carbohydrates and have the potential for therapy of infectious diseases as biopharmaceuticals, biomedical tools or in drug design. Given the rising concerns over drug resistance and epidemics, our patent review aims to add information, open horizons and indicate our view of the future perspectives about the antimicrobial applications of lectins. Patents with publications until December 2020 were retrieved from Espacenet using defined search terms and Boolean operators. The documents were used to identify the geographical and temporal distribution of the patents, characterize their lectins, and classify and summarize their antiviral, antibiotic and antifungal applications. Lectins are promising antiviral agents against viruses with epidemics and drug resistance concerns. Mannose-binding lectins were the most suggested antiviral agents since glycans with mannose residues are commonly involved in viral entry mechanisms. They were also immobilized onto surfaces to trap viral particles and inhibit their spread and replication. Many patents described the extraction, isolation, amino acid and nucleotide sequences, and expression vectors of lectins with antibiotic and/or antifungal activities in terms of MIC and IC50 for in vitro assays. The inventions also included lectins as biological tools in nanosensors for antibiotics susceptibility tests, drug-delivery systems for the treatment of resistant bacteria, diagnostics of viral diseases and as a vaccine adjuvant. Although research and development of new medicines is highly expensive, antimicrobial lectins may be worth investments given the emergence of epidemics and drug resistance. For this purpose, less invasive routes should be developed as alternatives to the parenteral administration of biologics. While anti-glycan neutralizing antibodies are difficult to develop due to the low immunogenicity of carbohydrates, lectins can be produced more easily and have a broad-spectrum activity. Protein engineering technologies may make the antimicrobial applications of lectins more successful.

Effects and mechanisms of animal-free hydrolysates on recombination protein yields in CHO cells.

Chinese hamster ovary (CHO) cells are the commonly used cell lines for producing recombinant therapeutic proteins (RTPs) because they possess post-translational modifications similar to human cells. Culture media are necessary for cell growth, and their quality affects the yields and quality of RTPs. Due to safety concerns for the complex purification of RTPs, the development of serum-free media (SFM) is necessary for CHO cells. To meet the need for CHO cells with higher cell density and RTP productivity with consistent product quality in large-scale suspension cultures, the optimization of SFM through adding some enzymatic animal-free hydrolysates (AFHs) is preferred. The AFHs can improve cell culture performance and product yield of RTPs without affecting their quality. Here, the effect and mechanism of various AFHs in improving CHO cell culture performance and protein expression are reviewed. KEY POINTS: • AFHs that improve the recombinant protein yield of CHO cells are reviewed. • AFHs improve recombinant protein yield via influencing cell performance. • The AFHs do not affect the quality of recombinant protein in CHO cells. • AFHs can provide nutrients, block cell cycle, and reduce oxidative stress.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

In situ detection of protein interactions for recombinant therapeutic enzymes.

Despite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and posttranslational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1, and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model and found proteins involved in protein folding, disulfide bond formation, and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.

Ocular Pharmacokinetics of Intravitreally Injected Protein Therapeutics: Comparison among Standard-of-Care Formats.

The current standard of care for antivascular endothelial growth factor (VEGF) treatment requires frequent intravitreal (IVT) injections of protein therapeutics, as a result of limited retention within the eye. A thorough understanding of the determinants of ocular pharmacokinetics (PK) and its translation across species is an essential prerequisite for developing more durable treatments. In this work, we studied the ocular PK in macaques of the protein formats that comprise today's anti-VEGF standard of care. Cynomolgus monkeys received a single IVT injection of a single-chain variable fragment (scFv, brolucizumab), antigen-binding fragment (Fab, ranibizumab), fragment crystallizable-fusion protein (Fc-fusion, aflibercept), or immunoglobulin G monoclonal antibody (IgG, VA2 CrossMAb). Drug concentrations were determined in aqueous humor samples collected up to 42 days postinjection using immunoassay methods. The ocular half-life (t1/2) was 2.28, 2.62, 3.13, and 3.26 days for scFv, Fab, Fc-fusion, and IgG, respectively. A correlation with human t1/2 values from the literature confirmed the translational significance of the cynomolgus monkey as an animal model for ocular research. The relation between ocular t1/2 and molecular size was also investigated. Size was inferred from the molecular weight (MW) or determined experimentally by dynamic light scattering. The MW and hydrodynamic radius were found to be good predictors for the ocular t1/2 of globular proteins. The analysis showed that molecular size is a determinant of ocular disposition and may be used in lieu of dedicated PK studies in animals.

Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up.

The manufacture of recombinant therapeutics is a fastest-developing section of therapeutic pharmaceuticals and presently plays a significant role in disease management. Yeasts are established eukaryotic host for heterologous protein production and offer distinctive benefits in synthesising pharmaceutical recombinants. Yeasts are proficient of vigorous growth on inexpensive media, easy for gene manipulations, and are capable of adding post translational changes of eukaryotes. Saccharomyces cerevisiae is model yeast that has been applied as a main host for the manufacture of pharmaceuticals and is the major tool box for genetic studies; nevertheless, numerous other yeasts comprising Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Yarrowia lipolytica have attained huge attention as non-conventional partners intended for the industrial manufacture of heterologous proteins. Here we review the advances in yeast gene manipulation tools and techniques for heterologous pharmaceutical protein synthesis. Application of secretory pathway engineering, glycosylation engineering strategies and fermentation scale-up strategies in customizing yeast cells for the synthesis of therapeutic proteins has been meticulously described.