Biotherapeutic Products Research Articles

Delivery of Encapsulated Intelligent Engineered Probiotic for Inflammatory Bowel Disease Therapy.

Engineered bacterial therapy holds enormous potential for treating intestinal diseases, employing synthetic biology techniques to achieve localized drug delivery within intestines. However, effective delivery of engineered bacteria to lesion sites and ensuring sustained colonization remain challenging. Here, a mucus encapsulated microsphere gel (MM) delivery system is developed to encapsulate genetically engineered bacteria capable of detecting and treating enteritis. The MM delivery system features an external mucosal coating composed of hyaluronic acid and epigallocatechin gallate, along with internal microspheres of highly biocompatible polyserine modified alginates encapsulating with the engineered probiotics. The MM delivery system effectively protects engineered bacteria harsh environment in stomach and significantly improves intestinal adhesion of the probiotics, extending colonization up to 24h, and does not affect the entry of biomarker or release of Avcystatin. It exhibits notable diagnostic and therapeutic efficacy in inflammatory bowel disease models, thus facilitating the advancement of live biotherapeutic products toward clinical application.

Hybrid Mass Spectrometry Applied across the Production of Antibody Biotherapeutics.

Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline. As an exemplar product, we use the model IgG1 antibody, mAb4. Flexibility, stability, aggregation propensity, and bulk properties are evaluated in relation to perfusion media, purification stages, and formulation solutions. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible solution. Despite presenting similar charge state distributions (CSD) in native MS, mAb4, and Herceptin show distinct unfolding patterns in activated ion mobility mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). Herceptin's greater structural stability and aggregation onset temperature (Tagg) are attributed to heavier glycosylation and kappa-class light chains, unlike the lambda-class light chains in mAb4. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that mAb4 undergoes substantial structural changes during purification, marked by high flexibility, low melting temperature (Tm), and prevalent repulsive protein-protein interactions but transitions to a compact and stable structure in high-salt and formulated environments. Notably, in formulation, the third constant domain (CH3) of the heavy chain retains flexibility and is a region of interest for aggregation. Future work could translate features of interest from comprehensive studies like this to targeted approaches that could be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

Oral Capsule FMT Combined With Bezlotoxumab Is a Successful Rescue Protocol Following Failure of FMT Alone in the Treatment of Recurrent C. difficile Infection.

Evaluate the benefit of adding bezlotoxumab to repeat fecal microbiota transplantation (FMT) in patients with recurrent Clostridioides difficile infections after the failure of FMT alone. The initial failure of FMT in breaking the cycle of recurrent Clostridium difficile(C. difficile) infections is associated with a greater risk of subsequent failure. Our previous analysis showed that FMT failure is associated with delayed repair of fecal microbiota at 1 week after administration. We hypothesized that increasing the symptom-free interval by adding bezlotoxumab would improve the outcomes of a second FMT. A new rescue protocol that combines FMT with bezlotoxumab for patients who previously failed FMT alone was implemented in 2 academic medical centers. The clinical outcomes of a new protocol were captured in a prospective registry. The results were compared in a retrospective analysis of clinical outcomes of prior experience with repeat FMT by itself. All FMT preparations were standardized for dose. Bezlotoxumab administration was synchronized temporally with the second FMT to maximize its duration of action. Our historical cure rate of second FMT in treatment of recurrent C. difficile infection was 48% (15/31 patients). Addition of bezlotoxumab to the second FMT resulted in a cure rate of 89% (24/27 patients). Addition of bezlotoxumab markedly improved the cure rate of the second FMT following initial FMT failure. The rationale for the protocol design highlights the importance of understanding the pharmacokinetics of both bezlotoxumab and FMT. Similar principles may apply to other live biotherapeutic products that are becoming available for prevention of C. difficile infection recurrence.

Probiotics in Health Care: A Critical Appraisal.

Consumption of probiotic products continues to increase, perhaps driven by an interest in gut health. However, the field is filled with controversy, inconsistencies, misuse of terminology, and poor communication. While the probiotic concept is biologically plausible and in some cases mechanistically well established, extrapolation of preclinical results to humans has seldom been proven in well-conducted clinical trials. With noteworthy exceptions, clinical guidance has often been derived not from large, adequately powered clinical trials but rather from comparisons of disparate, small studies with insufficient power to identify the optimal strain. The separation of probiotics from live biotherapeutic products has brought some clarity from a regulatory perspective, but in both cases, consumers should expect scientific rigor and strong supporting evidence for health claims.

Living Engineered Bacterial Therapeutics: Emerging Affordable Precision Interventions.

Live biotherapeutic products (LBPs), including engineered bacteria, are rapidly emerging as potential therapeutic interventions. These innovative therapies can serve as live in situ drug delivery platforms for the direct deposition of therapeutic payloads, including complex biologics, at sites of disease. This approach offers a platform likely to enhance therapeutic efficacy and decrease off-target side effects. LBPs also can likely be distributed at a relatively low price point, as their production can be economically scaled up. LBPs represent an exciting new means for ensuring healthy lives and promoting well-being for all ages, aligning with the World Health Organization's sustainable development goal 3.

Protection against Clostridioides difficile disease by a naturally avirulent strain.

Clostridioides difficile is a leading cause of healthcare infections. Gut dysbiosis promotes C.difficile infection (CDI) and CDIs promote gut dysbiosis, leading to frequent CDI recurrence. Although therapies preventing recurrent CDI have been developed, including live biotherapeutic products, existing therapies are costly and do not prevent primary infections. Here, we show that an avirulent C.difficile isolate, ST1-75, protects mice from developing colitis induced by a virulent R20291 strain when coinfected at a 1:1 ratio. In metabolic analyses, avirulent ST1-75 depletes amino acids more rapidly than virulent R20291 and supplementation with amino acids ablates this competitive advantage, indicating that ST1-75 limits the growth of virulent R20291 through amino acid depletion. Overall, our study identifies inter-strain nutrient depletion as a potentially exploitable mechanism to reduce the incidence of CDI and reveals that the ST1-75 strain may be a biotherapeutic agent that can prevent CDI in high-risk patients.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Barriers to WHO prequalification of similar biotherapeutic insulin.

To identify the barriers preventing manufacturers of similar biotherapeutic human insulin from submitting their products to the World Health Organization (WHO) for prequalification. We used a self-administered questionnaire to collect data from companies producing similar biotherapeutic human insulin. We included questions about the insulin products manufactured, knowledge of WHO prequalification requirements, export of the products and compliance with good manufacturing practices. Companies had the possibility to provide additional relevant information. We sent the questionnaire to 20 manufacturers in total. We evaluated responses and organized the data into themes. We had a response rate of 55% (11/20 companies). Five broad themes emerged: (i)manufacturers and products; (ii)expressions of interest awareness and participation; (iii)need for technical assistance and training; (iv)market and supply chain challenges; and (v)approval for good manufacturing practices. The most important reasons for manufacturers' lack of response to WHO's expression-of-interest invitation were absence of a mechanism to guarantee return on investment, and perceived complexity of prequalification requirements for insulin-similar biotherapeutic products. To encourage greater participation in the WHO prequalification programme, international procurement agencies associated with the programme should consider establishing a platform to enter into advance purchasing agreements with manufacturers. In addition, WHO's Local Production and Assistance Unit should provide companies with ongoing technical assistance on the development of their human insulin products and improvement of their production facilities to comply with the WHO requirements for good manufacturing practices.

End-To-End Automated Intact Protein Mass Spectrometry for High-Throughput Screening and Characterization of Bispecific and Multispecific Antibodies.

Bispecific antibodies (bsAbs) and multispecific antibodies (msAbs) represent a promising frontier in therapeutic antibody development, offering unique capabilities not achievable with traditional monoclonal antibodies. Despite their potential, significant challenges remain due to their increased molecular complexity. One prominent challenge is the correct assembly of light and heavy chains, as improper pairing leads to mispaired or incompletely assembled species that lack therapeutic efficacy and possess undesired properties, impairing the developability, manufacturability, and safety. There is a critical need for rapid, sensitive analytical tools to monitor and control these undesired species and ensure the quality assessment of bsAbs and msAbs. To address this need, we present a novel high-throughput, format-agnostic intact mass workflow that significantly enhances the efficiency of detecting and quantifying biotherapeutic products and related impurities. This workflow integrates automated sample preparation, novel high-resolution rapid mass detection powered by SampleStream-MS, and an advanced data analysis pipeline. It offers increased throughput and data quality while substantially reducing analysis turnover time and labor. This was demonstrated in a pilot program where ∼800 multispecific antibodies were processed in 10 working days. The article details the evaluation and validation of our method, demonstrating its repeatability and intermediate precision in terms of measurement accuracy and relative quantification of various product-related species. We underscore the transformative potential of this end-to-end high-throughput workflow in expediting bispecific and multispecific antibody discovery, optimizing production processes, and ensuring high-quality development and manufacturing for therapeutic antibodies.

Aggregating amyloid resources: A comprehensive review of databases on amyloid-like aggregation

Protein aggregation is responsible for several degenerative conditions in humans, and it is also a bottleneck in industrial protein production and storage of biotherapeutics. Bioinformatics tools have been developed to predict and redesign protein solubility more efficiently by understanding the underlying principles behind aggregation. As more experimental data become available, dedicated resources for storing, indexing, classifying and consolidating experimental results have emerged. These resources vary in focus, including aggregation-prone regions, 3D patches or protein stretches capable of forming amyloid fibrils. Some of these resources also consider the experimental conditions that cause protein aggregation and how they affect the process. This review article explores how protein aggregation databases have evolved and surveys state-of-the-art resources. We highlight their applications, complementarity and existing limitations. Moreover, we showcase the existing symbiosis between amyloid-related databases and predictive tools. To increase the usefulness of our review, we supplement it with a comprehensive list of present and past amyloid databases: https://biogenies.info/amyloid-database-list/.

Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture

Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive 13C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins. CHO cells were cultured in media formulations missing a single amino acid or glucose supplemented with a 13C-tracer of the missing substrate, followed by gas chromatography-mass spectrometry (GC-MS) analysis to track labeled carbon flows and identify by-products. We tracked the sources of all secreted by-products and verified the identity of 45 by-products, majority of which were derived from glucose, leucine, isoleucine, valine, tyrosine, tryptophan, methionine, and phenylalanine. In addition to by-products identified previously, we identified several metabolites including 2-hydroxyisovaleric acid, 2-aminobutyric acid, L-alloisoleucine, ketoisoleucine, 2-hydroxy-3-methylvaleric acid, desmeninol, and 2-aminobutyric acid. When added to CHO cell cultures at different concentrations, certain metabolites inhibited cell growth while others including 2-hydroxy acids, surprisingly, reduced lactate accumulation. In vitro enzymatic analysis indicated that 2-hydroxy acids were metabolized by lactate dehydrogenase suggesting a possible mechanism for lowered lactate accumulation, e.g., competitive substrate inhibition. The 13C-labeling assisted metabolomics pipeline developed and the metabolites identified will serve as a springboard to reduce undesirable by-products accumulation and alleviate inefficient substrate utilization in mammalian cultures used for biomanufacturing and other applications through altered media formulations and pathway engineering strategies.

Fecal Microbiota, Live-jslm (RBL; REBYOTA™) for Recurrent Clostridioides difficile Infection

Clostridioides difficile infection (CDI) is one of the most common causes of health care–associated infections and is often precipitated by recent antibiotic use. CDI occurs after the healthy gut microbiota is disrupted and becomes dysbiotic. Standard-of-care antibiotics for CDI may perpetuate dysbiosis, fueling risk of recurrent CDI (rCDI). New therapeutic options for preventing rCDI include fecal microbiota, live-jslm (RBL), a US Food and Drug Administration–approved, single-dose, rectally administered, microbiota-based live biotherapeutic product. RBL, which is easily given by any health care professional, is hypothesized to assist in restoring dysbiosis and has proven efficacy, safety, and tolerability across diverse patient populations to prevent rCDI.

Regulation of immune responses to food by commensal microbes.

The increasing prevalence of immune-mediated non-communicable chronic diseases, such as food allergies, has prompted a deeper investigation into the role of the gut microbiome in modulating immune responses. Here, we explore the complex interactions between commensal microbes and the host immune system, highlighting the critical role of gut bacteria in maintaining immune homeostasis. We examine how modern lifestyle practices and environmental factors have disrupted co-evolved host-microbe interactions and discuss how changes in microbiome composition impact epithelial barrier function, responses to food allergens, and susceptibility to allergic diseases. Finally, we examine the potential of bioengineered microbiome-based therapies, and live biotherapeutic products, for reestablishing immune homeostasis to prevent or treat food allergies.

From competition to cure: the development of live biotherapeutic products for anticancer therapy in the iGEM competition.

Cancer is a leading cause of mortality globally, often diagnosed at advanced stages with metastases already present, complicating treatment efficacy. Traditional treatments like chemotherapy and radiotherapy face challenges such as lack of specificity and drug resistance. The hallmarks of cancer, as defined by Hanahan and Weinberg, describe tumors as complex entities capable of evolving traits that promote malignancy, including sustained proliferation, resistance to cell death, and metastasis. Emerging research highlights the significant role of the microbiome in cancer development and treatment, influencing tumor progression and immune responses. This review explores the potential of live biotherapeutic products (LBPs) for cancer diagnosis and therapy, focusing on projects from the International Genetically Engineered Machines (iGEM) competition that aim to innovate LBPs for cancer treatment. Analyzing 77 projects from 2022, we highlight the progress and ongoing challenges within this research field.

The 3 Ds: Depression, Dysbiosis, and Clostridiodes difficile.

This paper explores the intricate relationship between depression, gut dysbiosis, and Clostridioides difficile infections, collectively termed "The 3 Ds". Depression is a widespread mental disorder increasing in prevalence. It is recognized for its societal burden and complex pathophysiology, encompassing genetic, environmental, and microbiome-related factors. The consequent increased use of antidepressants has led to growing concerns about their effects on the gut microbiome. Various classes of antidepressants and antipsychotics show antimicrobial activity, potentially leading to shifts in the gut microbiome and contributing to the development of dysbiosis. Dysbiosis, in turn, can predispose individuals to opportunistic infections like C. difficile, a significant healthcare concern due to its high recurrence rates and severe impact on patients' quality of life. Further, the link between antidepressant use and an increased risk of C. difficile infection (CDI) is explored and, finally, the emergence of live biotherapeutic products as novel treatment options for recurrent CDI is discussed.

Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans.

Engineering live biotherapeutic products against fungal pathogens such as Candida albicans has been suggested as a means to tackle the increasing threat of fungal infections and the development of resistance to classical antifungal treatments. One important challenge in the design of live therapeutics is to control their localization inside the human body. The specific binding capability to target organisms or tissues would greatly increase their effectiveness by increasing the local concentration of effector molecules at the site of infection. In this study, we utilized surface display of carbohydrate binding domains to enable the probiotic E. coli Nissle 1917 to adhere specifically to the pathogenic yeast Candida albicans. Binding was quantified using a newly developed method based on the automated analysis of microscopic images. In addition to a rationally selected chitin binding domain, a synthetic peptide of identical length but distinct sequence also conferred binding. Efficient binding was specific to fungal hyphae, the invasive form of C. albicans, while the yeast form, as well as abiotic cellulose and PET particles, was only weakly recognized.

Human microbiome: Impact of newly approved treatments on C. difficile infection.

In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. The primary purposes of this review are to provide a brief overview of the microbiome, discuss the most relevant outcome data and key characteristics of each live microbiome agent, and pose questions for consideration going forward as these agents are integrated into clinical practice. The management of Clostridiodes difficile infection (CDI) remains a difficult clinical conundrum, with recurrent CDI occurring in 15% to 35% of patients and causing significant morbidity and decreased quality of life. For patients with frequent CDI recurrences, fecal microbiota transplantation (FMT) has been demonstrated to have significant benefit but also significant risks, and FMT is not approved by the US Food and Drug Administration (FDA) for that indication. FDA has established a new therapeutic class for agents known as live biotherapeutic products (LBPs) that offer significant advantages over FMT, including standardized screening, testing, and manufacturing as well as known quantities of organisms contained within. Two new live microbiome products within this class were recently approved by FDA for prevention of CDI recurrences in adult patients following treatment for recurrent CDI with standard antimicrobial therapy. Both agents had demonstrated efficacy in registry trials in preventing CDI recurrence but differ significantly in a number of characteristics, such as route of administration. Cost as well as logistics are current obstacles to use of these therapies. Live microbiome therapy is a promising solution for patients with recurrent CDI. Future studies should provide further evidence within yet-to-be-evaluated populations not included in registry studies. This along with real-world evidence will inform future use and clinical guideline placement.

Fecal Microbiota, Live-jslm (RBL; REBYOTA ® ) for Prevention of Recurrent Clostridioides difficile Infection: What Gastroenterology Nurses Need to Know.

Live biotherapeutic products (LBPs) such as fecal microbiota, live-jslm (RBL) are becoming more frequently administered in gastroentintestinal (GI) care settings. As a result, GI nurses must be knowledgeable about and skilled in their administration of LBPs. RBL is a single-dose, rectally-administered, microbiota-based LBP suspension indicated for prevention of recurrent Clostridioides difficile infection (rCDI). RBL contains a diverse set of micro-organisms, including Bacteroides spp., and is believed to restore a healthy gut microbiota to mitigate dysbiosis associated with rCDI. Patients do not require fasting, bowel preparation, or anesthesia prior to RBL administration. Administration takes approximately 5minutes and can be given by any health care professional (e.g., nurses) in any care setting. Nurses also play an important role in educating patients and caregivers about these products and the disease. Collectively, improved familiarity with LBPs and their proper use among nurses can contribute to successful prevention of rCDI in their patients.

Evaluation of the efficacy of a live Escherichia coli biotherapeutic product (asymptomatic bacteriuria E. coli 212).

Recurrent bacterial cystitis, often referred to as recurrent urinary tract infection (UTI), can be difficult to manage and alternative treatments are needed. Intravesicular administration of asymptomatic bacteriuria (ASB) E. coli 212 will not be inferior to antimicrobial treatment for the management of recurrent UTI in dogs. Thirty-four dogs with >1 UTI in the 12 months before presentation. All dogs were deemed normal otherwise based on absence of abnormalities on physical examination, CBC, serum biochemical panel, and abdominal ultrasonography. Dogs were randomized to 1 of 2 treatment groups: Group 1 antimicrobials for 7 days or group 2 intravesicular administration of ASB E. coli 212. Owners were provided a voiding questionnaire regarding their dogs' clinical signs, which was completed daily for 14 days to assess clinical cure. Dogs were examined on days 7 and 14 to assess clinical cure, and urine specimens were submitted for urinalysis and bacterial culture. Clinical cure rates for ASB E. coli 212-treated dogs were not inferior to 7 days of antimicrobial treatment with a 12% margin of difference to determine non-inferiority. No significant difference was found between the treatment groups on days 7 and 14 in the proportion of dogs achieving ≥50% or ≥75% reduction in their clinical score compared with baseline. These data suggest that intravesicular administration of ASB E. coli 212 is not inferior to antimicrobials for the treatment of recurrent UTI in dogs. This biotherapeutic agent could help alleviate the need for antimicrobials for some dogs with recurrent UTI, improving antimicrobial stewardship.

Expanding the toolbox: Novel class IIb microcins show activity against Gram-negative ESKAPE and plant pathogens.

Interspecies interactions involving direct competition via bacteriocin production play a vital role in shaping ecological dynamics within microbial ecosystems. For instance, the ribosomally-produced siderophore bacteriocins, known as class IIb microcins, affect the colonization of host-associated pathogenic Enterobacteriaceae species. Notably, to date, only five of these antimicrobials have been identified, all derived from specific Escherichia coli and Klebsiella pneumoniae strains. We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms. With a customized informatics-driven approach, screening bacterial genomes in public databases with BLAST and manual curation, we have discovered twelve previously unknown class IIb microcins in seven additional Enterobacteriaceae species, encompassing phytopathogens and environmental isolates. We introduce three novel clades of microcins (MccW, MccX, and MccZ), while also identifying eight new variants of the five known class IIb microcins. To validate their antimicrobial potential, we heterologously expressed these microcins in E. coli and demonstrated efficacy against a variety of bacterial isolates, including plant pathogens from the genera Brenneria, Gibbsiella, and Rahnella . Two newly discovered microcins exhibit activity against Gram-negative ESKAPE pathogens, i.e. Acinetobacter baumannii or Pseudomonas aeruginosa , providing the first evidence that class IIb microcins can target bacteria outside of the Enterobacteriaceae family. This study underscores that class IIb microcin genes are more prevalent in the microbial world than previously recognized and that synthetic hybrid microcins can be a viable tool to target clinically relevant drug-resistant pathogens. Our findings hold significant promise for the development of innovative engineered live biotherapeutic products tailored to combat these resilient bacteria.