Safety Assessment of Protein A and Derivation of a Parenteral Health-Based Exposure Limit.

Protein A (PA) is a bacterial cell wall component of Staphylococcus aureus whose function is to bind to Immunoglobulin G (IgG). Given its ability to bind IgG as well as its stability and resistance to harsh acidic and basic cleaning conditions, it is commonly used in the affinity chromotography purification of biotherapeutics. This use can result in levels of PA being present in a drug product and subsequent patient exposure. Interestingly, PA was previously evaluated in clinical trials as well as supporting nonclinical studies, resulting in a database that enables the derivation of a health-based exposure limit (HBEL). Given the widespread use of PA in the pharmaceutical industry, the IQ DruSafe Impurities Working Group (WG) evaluated the available information with the purpose of establishing a harmonized parenteral HBEL for PA. Based on this thorough, collaborative evaluation of nonclinical and clinical data available for PA, a parenteral HBEL of 1.2 µg/kg/dose (60 µg/dose for a 50 kg individual) is expected to be health protective for patients when it is present as an impurity in a biotherapeutic.

Limosilactobacillus fermentum SLAM 216- Derived Extracellular Vesicles Promote Intestinal Maturation in Mouse Organoid Models.

Probiotics, when consumed in adequate amounts, can promote the health of the host and beneficially modulate the host's immunity. Particularly during the host's early life, the gut intestine undergoes a period of epithelial maturation in which epithelial cells organize into specific crypt and villus structures. This process can be mediated by the gut microbiota. Recent studies have reported that the administration of probiotics can further promote intestinal maturation in the neonatal intestine. Therefore, in this study, we investigated the effects of extracellular vesicles derived from the Limosilactobacillus fermentum SLAM 216 strain, which is an established probiotic with known immune and anti-aging effects on intestinal epithelial maturation and homeostasis, using mouse small intestinal organoids. As per our findings, treatment with L. fermentum SLAM 216-derived LF216EV (LF216EV) has significantly increased the bud number and size of organoid buds. Furthermore, extracellular vesicle (EV) treatment upregulated the expression of maturation-related genes, including Ascl2, Ephb2, Lgr5, and Sox9. Tight junctions are known to have an important role in the intestinal immune barrier, and EV treatment has significantly increased the expression of genes associated with tight junctions, such as Claudin, Muc2, Occludin, and Zo-1, indicating that it can promote intestinal development. This was supported by RNA sequencing, which revealed the upregulation of genes associated with cAMP-mediated signaling, which is known to regulate cellular processes including cell differentiation. Additionally, organoids exposed to LF216EV exhibited upregulation of genes associated with maintaining brain memory and neurotransmission, suggesting possible future functional implications.

Multi-omics analysis of gut-brain axis reveals novel microbial and neurotransmitter signatures in patients with arteriosclerotic cerebral small vessel disease.

Arteriosclerotic cerebral small vessel disease (aCSVD) is a major cause of stroke and dementia. Although its underlying pathogenesis remains poorly understood, both inflammaging and gut microbiota dysbiosis have been hypothesized to play significant roles. This study investigated the role of gut microbiota in the pathogenesis of aCSVD through a comparative analysis of the gut microbiome and metabolome between CSVD patients and healthy controls. The results showed that patients with aCSVD exhibited a marked reduction in potentially beneficial bacterial species, such as Faecalibacterium prausnitzli and Roseburia intestinalis, alongside an increase in taxa from Bacteroides and Proteobacteria. Integrated metagenomic and metabolomic analyses revealed that alterations in microbial metabolic pathways, including LPS biosynthesis and phenylalanine-tyrosine metabolism, were associated with the status of aCSVD. Our findings indicated that microbial LPS biosynthesis and phenylalanine-tyrosine metabolism potentially influenced the symptoms and progression of aCSVD via pro-inflammatory effect and modulation of systemic neurotransmitters, respectively. These results imply that gut microbiota characteristics may serve as indicators for early detection of aCSVD and as potential gut-directed therapeutic intervention target.

Preference for a Novel Oral Alternative to Parenterally Administered Medications.

Background: Rani Therapeutics is developing a robotic pill (RP), an oral drug delivery platform called RaniPill™ that can deliver a number of biotherapeutics with high bioavailability; eliminating the need for injections. While patients in general prefer oral to injectable therapies, preference for a more frequent oral regimen compared to a less frequent injectable regimen is unknown. Two marketing surveys were conducted to gather data on preference for oral versus injectable therapies. A clinical study gathered data on participant preference for oral pills vs injections before and after swallowing a Mock-RP capsule. Methods: A total of 1689 adults taking injections (mean duration 3-7 years) to treat endocrine or inflammatory conditions were anonymously surveyed online for their preference to administer/prescribe medications orally via the RP. In the clinical study, 150 participants currently taking injections for chronic conditions evaluated the swallowability of a Mock-RP and completed a questionnaire regarding their preferences. Results: Majority of respondents surveyed stated they would be willing to convert to an oral alternative over their current parenteral therapy regardless of drug or disease. In the clinical study, all participants were able to swallow the Mock-RP and 91% indicated their preference for the oral route versus their current parenteral route of drug administration. Survey respondents and those in the clinical study using frequent injections were more willing to select a once-daily capsule compared to those injecting infrequently. Even study participants who inject infrequently (≥monthly: 80%) would prefer a once-daily pill over their injection regimen. Conclusion: Patients taking injections and prescribing physicians strongly prefer oral dosing to parenteral administration of biologics even if dosing frequency with the oral option, such as the RP, is increased.

Microbiological insights and dermatological applications of live biotherapeutic products.

As our understanding of dermatological conditions advances, it becomes increasingly evident that traditional pharmaceutical interventions are not universally effective. The intricate balance of the skin microbiota plays a pivotal role in the development of various skin conditions, prompting a growing interest in probiotics, or live biotherapeutic products (LBPs), as potential remedies. Specifically, the topical application of LBPs to modulate bacterial populations on the skin has emerged as a promising approach to alleviate symptoms associated with common skin conditions. This review considers LBPs and their application in addressing a wide spectrum of dermatological conditions with particular emphasis on three key areas: acne, atopic dermatitis, and wound healing. Within this context, the critical role of strain selection is presented as a pivotal factor in effectively managing these dermatological concerns. Additionally, the review considers formulation challenges associated with probiotic viability and proposes a personalised approach to facilitate compatibility with the skin's unique microenvironment. This analysis offers valuable insights into the potential of LBPs in dermatological applications, underlining their promise in reshaping the landscape of dermatological treatments while acknowledging the hurdles that must be overcome to unlock their full potential.

PUNCH CD3-OLS: a phase 3 prospective observational cohort study to evaluate the safety and efficacy of fecal microbiota, live-jslm (REBYOTA) in adults with recurrent Clostridioides difficile infection.

OBJECTIVE: To evaluate the safety and efficacy of fecal microbiota, live-jslm (RBL; REBYOTA) - the first single-dose, broad consortia microbiota-based live biotherapeutic approved by the United States (US) Food and Drug Administration for preventing recurrent Clostridioides difficile infection (rCDI) in adults following standard-of-care (SOC) antibiotic treatment. DESIGN: PUNCH CD3-OLS was a prospective, phase 3, open-label study, conducted across the US and Canada. Participants were aged ≥18 years with documented rCDI and confirmed use of SOC antibiotics. Participants with comorbidities including inflammatory bowel disease and mild-to-moderate immunocompromising conditions could be enrolled. A single dose of RBL was rectally administered within 24-72h of antibiotic completion. The primary endpoint was the number of participants with RBL- or administration-related treatment-emergent adverse events (TEAEs). Secondary endpoints included treatment success and sustained clinical response, at 8 weeks and 6 months after RBL administration, respectively. RESULTS: Overall, 793 participants were enrolled, of whom 697 received RBL. TEAEs through 8 weeks after administration were reported by 47.3% of participants; most events were mild or moderate gastrointestinal disorders. Serious TEAEs were reported by 3.9% of participants. The treatment success rate at 8 weeks was 73.8%; in participants who achieved treatment success, the sustained clinical response rate at 6 months was 91.0%. Safety and efficacy rates were similar across demographic and baseline characteristic subgroups. CONCLUSIONS: RBL was safe and efficacious in participants with rCDI and common comorbidities. This is the largest microbiota-based live biotherapeutic study to date and findings support use of RBL to prevent rCDI in a broad patient population. CLINICAL TRIAL REGISTRATION: The study is registered at ClinicalTrials.gov (NCT03931941).

Wild-Edible Allium Species from Highlands of Eastern Anatolia: Phytochemical Composition and In Vitro Biological Activities.

This study presents the phytochemical composition, antioxidant (hydrogen atom and single-atom transfer mechanisms), and digestive enzyme inhibitory (alpha-amylase, alpha-glucosidase, and pancreatic lipase) activities of ethanol-based extractions and traditional preparations (infusion) of the leaves of wild-edible Allium species (A. kharputense, A. affine, A. shirnakiense, and A. akaka) from the highlands of Eastern Anatolia. Among the eight extracts analyzed, ethanol extractions of the A. kharputense and A. akaka leaves exhibited better biotherapeutic activities and had the highest bioactive content. The dominant bioactive profile was composed of mainly allicin and phenolic compounds (chlorogenic acid, hesperidin, rutin, isoquercitrin, and quercetin) with small amounts of fatty acids. These data were similar to the biological activities and chemical composition of common Allium species and suggest the utilization of the extracts of wild-edible Allium species in the development of Allium-based biotherapeutics or nutraceuticals.

Protection from environmental enteric dysfunction and growth improvement in malnourished newborns by amplification of secretory IgA.

Environmental enteric dysfunction (EED) is a condition associated with malnutrition that can progress to malabsorption and villous atrophy. Severe EED results in linear growth stunting, slowed neurocognitive development, and unresponsiveness to oral vaccines. Prenatal exposure to malnutrition and breast feeding by malnourished mothers replicates EED. Pups are characterized by deprivation of secretory IgA (SIgA) and altered development of the gut immune system and microbiota. Extracellular ATP (eATP) released by microbiota limits T follicular helper (Tfh) cell activity and SIgA generation in Peyer's patches (PPs). Administration of a live biotherapeutic releasing the ATP-degrading enzyme apyrase to malnourished pups restores SIgA levels and ameliorates stunted growth. SIgA is instrumental in improving the growth and intestinal immune competence of mice while they are continuously fed a malnourished diet. The analysis of microbiota composition suggests that amplification of endogenous SIgA may exert a dominant function in correcting malnourishment dysbiosis and its consequences on host organisms, irrespective of the actual microbial ecology.

Probiotics as a strategy for addressing helminth infections in low-income countries: Working smarter rather than richer.

Helminth infections, which affect approximately 1.5 billion individuals worldwide (mainly children), are common in low- and middle-income tropical countries and can lead to various diseases. One crucial factor affecting the occurrence of these diseases is the reduced diversity of the gut microbiome due to antibiotic use. This reduced diversity compromises immune health in hosts and alters host gene expression through epigenetic mechanisms. Helminth infections may produce complex biochemical signatures that could serve as therapeutic targets. Such therapies include next-generation probiotics, live biotherapeutic products, and biochemical drug approaches. Probiotics can bind ferric hydroxide, reducing the iron that is available to opportunistic microorganisms. They also produce short-chain fatty acids associated with immune response modulation, oral tolerance facilitation, and inflammation reduction. In this review, we examine the potential link between these effects and epigenetic changes in immune response-related genes by analyzing methyltransferase-related genes within probiotic strains discussed in the literature. The identified genes were only correlated with methylation in bacterial genes. Various metabolic interactions among hosts, helminth parasites, and intestinal microbiomes can impact the immune system, potentially aiding or hindering worm expulsion through chemical signaling. Implementing a comprehensive strategy using probiotics may reduce the impact of drug-resistant helminth strains.

A synbiotic of Anaerostipes caccae and lactulose prevents and treats food allergy in mice.

Depletion of beneficial microbes by modern lifestyle factors correlates with the rising prevalence of food allergies. Re-introduction of allergy-protective bacteria may be an effective treatment strategy. We characterized the fecal microbiota of healthy and food-allergic infants and found that the anaerobe Anaerostipes caccae (A. caccae) was representative of the protective capacity of the healthy microbiota. We isolated a strain of A. caccae from the feces of a healthy infant and identified lactulose as a prebiotic to optimize butyrate production by A. caccae in vitro. Administration of a synbiotic composed of our isolated A. caccae strain and lactulose increased luminal butyrate in gnotobiotic mice colonized with feces from an allergic infant and in antibiotic-treated specific pathogen-free (SPF) mice, and prevented or treated an anaphylactic response to allergen challenge. The synbiotic's efficacy in two models and microbial contexts suggests that it may be a promising approach for the treatment of food allergy.

Probiotics and the reduction of SARS-CoV-2 infection through regulation of host cell calcium dynamics.

Calcium is a secondary messenger that interacts with several cellular proteins, regulates various physiological processes, and plays a role in diseases such as viral infections. Next-generation probiotics and live biotherapeutic products are linked to the regulation of intracellular calcium levels. Some viruses can manipulate calcium channels, pumps, and membrane receptors to alter calcium influx and promote virion production and release. In this study, we examined the use of bacteria for the prevention and treatment of viral diseases, such as coronavirus of 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccination programs have helped reduce disease severity; however, there is still a lack of well-recognized drug regimens for the clinical management of COVID-19. SARS-CoV-2 interacts with the host cell calcium (Ca2+), manipulates proteins, and disrupts Ca2+ homeostasis. This article explores how viruses exploit, create, or exacerbate calcium imbalances, and the potential role of probiotics in mitigating viral infections by modulating calcium signaling. Pharmacological strategies have been developed to prevent viral replication and block the calcium channels that serve as viral receptors. Alternatively, probiotics may interact with cellular calcium influx, such as Lactobacillus spp. The interaction between Akkermansia muciniphila and cellular calcium homeostasis is evident. A scientific basis for using probiotics to manipulate calcium channel activity needs to be established for the treatment and prevention of viral diseases while maintaining calcium homeostasis. In this review article, we discuss how intracellular calcium signaling can affect viral replication and explore the potential therapeutic benefits of probiotics.

Microbiota restoration therapies for recurrent Clostridioides difficile infection reach an important new milestone.

Microbiota restoration therapy has become a standard treatment for recurrent Clostridioides difficile infection (rCDI). In this article, we review the studies supporting the licensure of two live biotherapeutic products (LBPs) designed to prevent rCDI and to provide clinicians with a perspective on their differences. PubMed was reviewed on 1 October 2023, for all papers published concerning the current Food and Drug Administration allowance of the use of fecal microbiota transplantation (FMT) and the studies that led to the licensure of RBX2660 (REBYOTA™), generic name, fecal microbiota, live-jslm, and SER-109 (VOWST™), generic name, fecal microbiota spores, live-brpk. OpenBiome continues to produce fecal products for patients with rCDI at their treatment sites, and the American Gastroenterology Association has a National Registry focused on long-term safety of administering fecal microbiota products. The science behind the licensing of fecal microbiota, live-jslm, a consortium of fecal anaerobes found in stool augmented with strains of Bacteroidetes and fecal microbiota spores, live-brpk, a mixture of 50 species of purified Firmicutes spores is reviewed. Both products appear to be safe in clinical trials and effective in reducing rCDI episodes by mechanisms established for FMT, including normalization of α- and ß-diversity of the microbiome and by increasing fecal secondary bile acids. The different makeup of the two LBPs suggests that rCDI responds to a variety of engrafting microbiota which explains why nearly all donors in FMT of rCDI are generally effective. Fecal microbiota, live-jslm has also been shown to successfully treat rCDI in elderly patients with advanced comorbidities. With the licensure of two novel LBPs, we are entering a new phase of microbiota replacement therapy. Having standardized manufacturing and proper monitoring of products, harnessing the microbiome to control and prevent disease has a new beginning.

Licensure of two new live biotherapeutic products to treat recurrent C difficile infection is changing the landscape for treatment of this common and often serious infection Microbiota replacement therapy is the most effective way to prevent multiple recurrences of C difficile infection. The article discusses where fecal microbiota transplantation is available in North America. The major focus is on two recently licensed live biotherapeutic products, RBX2660 (REBYOTA), generic name fecal microbiota, live-jslm and SER-109 (VOWST), generic fecal microbiota spores, live-brpk, manufactured under standardized methods which should be safer and more standardized in response. The article compares the new LBPs for safety, effectiveness, cost to help clinicians make decisions. The licensure and availability of two safe and effective standardized and regulated biotherapies, fecal microbiota, live-jslm and fecal microbiota spores, live-brpk, for preventing rCDI is a critical advance in medical management. Both treatments were shown to cure rCDI, to normalize the microbiome of the treated patients by reducing proportions of proinflammatory Enterobacteriaceae and increasing the α- and ß-diversity of the microbiome, and to convert primary bile acids to C. difficile-inhibiting secondary bile acids in fecal samples. Both products included follow-up studies show durable cure without important short-term adverse events. The two recently licensed LBP differ in a number of ways. Fecal microbiota, live-jslm is a broad consortium of microbiota expected in a healthy donor fecal samples, including all the major phyla including Firmicutes. It is augmented with strains of Bacteroidetes, while fecal microbiota spores, live-brpk is ethanol washed spores exclusively within the phylum of Firmicutes. The fact that both products are effective in preventing rCDI support the idea that bacterial restoration in rCDI can be achieved by transplantation of a variety of different microbiota. This is seen in FMT for rCDI where it is generally accepted that all healthy adults are suitable donors and large number of donors can be included unscreened for microbiome diversity in a stool bank such as OpenBiome. When treating conditions other than CDI, the specific makeup of an LBP may need to be adjusted. One way around the unique microbiome requirements of non-CDI illnesses with dysbiosis is to administer FMT product derived from multiple donors. Evidence developed and presented here indicate that the two new LBPs are effective in treating rCDI, although head-to-head comparisons have not been carried out. fecal microbiota, live-jslm is a more traditional microbiome restoration product employing a full range of microbiota. fecal microbiota spores, live-brpk is novel in design and is based on the selection of Firmicutes spores with a narrower range of bioactivity. The future of microbiota-therapy has gotten brighter with the licensure of fecal microbiota, live-jslm and fecal microbiota spores, live-brpk.

A Comparison of Currently Available and Investigational Fecal Microbiota Transplant Products for Recurrent Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) is an intestinal infection that causes morbidity and mortality and places significant burden and cost on the healthcare system, especially in recurrent cases. Antibiotic overuse is well recognized as the leading cause of CDI in high-risk patients, and studies have demonstrated that even short-term antibiotic exposure can cause a large and persistent disturbance to human colonic microbiota. The recovery and sustainability of the gut microbiome after dysbiosis have been associated with fewer CDI recurrences. Fecal microbiota transplantation (FMT) refers to the procedure in which human donor stool is processed and transplanted to a patient with CDI. It has been historically used in patients with pseudomembranous colitis even before the discovery of Clostridioides difficile. More recent research supports the use of FMT as part of the standard therapy of recurrent CDI. This article will be an in-depth review of five microbiome therapeutic products that are either under investigation or currently commercially available: Rebyota (fecal microbiota, live-jslm, formerly RBX2660), Vowst (fecal microbiota spores, live-brpk, formerly SER109), VE303, CP101, and RBX7455. Included in this review is a comparison of the products' composition and dosage forms, available safety and efficacy data, and investigational status.

Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact.

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

Reevaluating the FDA's warning against the use of probiotics in preterm neonates: A societal statement by ESPGHAN and EFCNI.

The recent advisory issued by the United States Food and Drug Administration, cautioning against the routine administration of probiotics in preterm neonates, has sparked a lively debate within the scientific community. This commentary presents a perspective from members of the Special Interest Group on Gut Microbiota and Modifications within the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and other authors who contributed to the ESPGHAN position paper on probiotics for preterm infants, as well as representatives from the European Foundation for the Care of Newborn Infants. We advocate for a more nuanced and supportive approach to the use of certain probiotics in this vulnerable population, balancing the demonstrated benefits and risks.

Faecalibacterium duncaniae as a novel next generation probiotic against influenza.

The gut-lung axis is critical during viral respiratory infections such as influenza. Gut dysbiosis during infection translates into a massive drop of microbially produced short-chain fatty acids (SCFAs). Among them, butyrate is important during influenza suggesting that microbiome-based therapeutics targeting butyrate might hold promises. The butyrate-producing bacterium Faecalibacterium duncaniae (formerly referred to as F. prausnitzii) is an emerging probiotic with several health-promoting characteristics. To investigate the potential effects of F. duncaniae on influenza outcomes, mice were gavaged with live F. duncaniae (A2-165 or I-4574 strains) five days before infection. Supplementation of F. duncaniae was associated with less severe disease, a lower pulmonary viral load, and lower levels of lung inflammation. F. duncaniae supplementation impacted on gut dysbiosis induced by infection, as assessed by 16S rRNA sequencing. Interestingly, F. duncaniae administration was associated with a recovery in levels of SCFAs (including butyrate) in infected animals. The live form of F. duncaniae was more potent that the pasteurized form in improving influenza outcomes. Lastly, F. duncaniae partially protected against secondary (systemic) bacterial infection. We conclude that F. duncaniae might serve as a novel next generation probiotic against acute viral respiratory diseases.

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70 g/L vs 2.56 g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6 g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1. Fifty-three payload-containing peptides were identified, ranging in mass from 1.8 to 16.9 kDa, and leading to the unambiguous identification of 46 out of 92 possible conjugation sites. In addition, seven peptides were identified containing multiple payloads. The characterization of these types of heterogeneous peptides represents an important step in unraveling the combinatorial nature of lysine-conjugated ADCs.

A review of fecal microbiota, live-jslm for the prevention of recurrent Clostridioides difficile infection.

PURPOSE: To review the composition, preparation, proposed mechanism of action, safety, efficacy, and current place in therapy of Rebyota (fecal microbiota, live-jslm). SUMMARY: As the first agent in a new class of drugs, live biotherapeutic products (LBPs), fecal microbiota, live-jslm offers another therapeutic approach for the prevention of recurrent Clostridioides difficile infection (rCDI). LBPs are given following antibiotic therapy for C. difficile to reintroduce certain bacteria present in the normal microbiome, as a means to reconstitute the microbiome of infected individuals. This review provides a summary of phase 2 and 3 clinical trials, product information, discussion of data limitations, and recommendations for place in therapy. High efficacy rates compared to placebo with sustained response up to 24 months after administration have been reported. The majority of adverse events identified were mild to moderate without significant safety signals. CONCLUSION: Fecal microbiota, live-jslm has consistently been shown in randomized trials to be safe and effective in reducing rCDI. Its approval marks the culmination of decades of work to identify, characterize, and refine the intestinal microbiome to create pharmaceutical products.