Biotherapeutic Candidates Research Articles

Evaluation of probiotic efficacy of indigenous yeast strain, Saccharomyces cerevisiae Y-89 isolated from a traditional fermented beverage of West Bengal, India having protective effect against DSS-induced colitis in experimental mice.

Increasing awareness regarding health promotion and disease prevention has driven inclusion of fermented foods and beverages in the daily diet. These are the enormous sources of beneficial microbes, probiotics. This study aims to isolate yeast strains having probiotic potential and effectivity against colitis. Initially, ninety-two yeast strains were isolated from Haria, an ethnic fermented beverage of West Bengal, India. Primary screening was done by their acid (pH 4) and bile salt (0.3%) tolerance ability. Four potent isolates were selected and found effective against Entamoeba histolytica, as this human pathogen is responsible to cause colitis. They were identified as Saccharomyces cerevisiae. They showed luxurious growth even at 37 oC, tolerance up to 5% of NaCl, resistance to gastric juice and high bile salt (2.0%) and oro-gastrointestinal transit tolerance. They exhibited good auto-aggregation and co-aggregation ability and strong hydrophobicity. Finally, heat map and principal component analysis revealed that strain Y-89 was the best candidate. It was further characterised and found to have significant protective effects against DSS-induced colitis in experimental mice model. It includes improvement in colon length, body weight and organ indices; reduction in disease activity index; reduction in cholesterol, LDL, SGPT, SGOT, urea and creatinine levels; improvement in HDL, ALP, total protein and albumin levels; decrease in coliform count and restoration of tissue damage. This study demonstrates that the S. cerevisiae strain Y-89 possesses remarkable probiotic traits and can be used as a potential bio-therapeutic candidate for the prevention of colitis.

LAP: Liability Antibody Profiler by sequence & structural mapping of natural and therapeutic antibodies.

Antibody-based therapeutics must not undergo chemical modifications that would impair their efficacy or hinder their developability. A commonly used technique to de-risk lead biotherapeutic candidates annotates chemical liability motifs on their sequence. By analyzing sequences from all major sources of data (therapeutics, patents, GenBank, literature, and next-generation sequencing outputs), we find that almost all antibodies contain an average of 3-4 such liability motifs in their paratopes, irrespective of the source dataset. This is in line with the common wisdom that liability motif annotation is over-predictive. Therefore, we have compiled three computational flags to prioritize liability motifs for removal from lead drug candidates: 1. germline, to reflect naturally occurring motifs, 2. therapeutic, reflecting chemical liability motifs found in therapeutic antibodies, and 3. surface, indicative of structural accessibility for chemical modification. We show that these flags annotate approximately 60% of liability motifs as benign, that is, the flagged liabilities have a smaller probability of undergoing degradation as benchmarked on two experimental datasets covering deamidation, isomerization, and oxidation. We combined the liability detection and flags into a tool called Liability Antibody Profiler (LAP), publicly available at lap.naturalantibody.com. We anticipate that LAP will save time and effort in de-risking therapeutic molecules.

Critical reagent considerations for immunogenicity assay development for bispecific biotherapeutic candidates.

Aim: To demonstrate the importance of critical reagent characterization for immunogenicity assay development for multi-specific drugs using two case studies.Methods: Bridging anti-drugantibody (ADA) assay with acid-dissociated samples were used for both cases.Results: In the first case study, the unexpected interference in an ADA assay from clinical samples was identified; a model was created to replicate the issue, and an anti-target antibody was identified to mitigate the target interference. In the second case study, an issue due to non-specific binding of a domain-specific confirmatory reagent was identified, and various mitigation techniques were evaluated.Conclusion: A thorough characterization of the critical reagents helped identify the issues with these ADA case studies and provided strategies for resolving them.

Euonymus alatus (Thunb.) Siebold Prevents Osteoclast Differentiation and Osteoporosis.

Euonymus alatus (Thunb.) Siebold, a traditional medicinal plant, has been used in China and several other Asian countries to address a variety of health concerns. The extensive research conducted on E. alatus is driven by its diverse pharmacological applications. However, its biological effects on osteoclastogenesis and osteoporosis have not been previously studied. In this research, we investigated the impact of an ethanolic extract of E. alatus (EEEA) on osteoclast differentiation and function as well as estrogen deficiency-induced bone loss. We found that EEEA inhibits osteoclast differentiation by downregulating the expression of the receptor activator of nuclear factor-κB ligand (RANKL) in osteoclast-supporting cells and by directly impeding RANKL-mediated signaling pathways for osteoclastogenesis in precursor cells. In addition, EEEA inhibited the bone-resorptive function of mature osteoclasts in vitro. Furthermore, oral administration of EEEA significantly alleviated bone loss in an ovariectomy-induced osteoporosis mouse model. Additionally, we identified phytochemicals in EEEA that have suppressive effects on osteoclast differentiation and bone loss. Collectively, these results suggest that EEEA holds potential as a biotherapeutic candidate for anti-postmenopausal osteoporosis.

Relaxin-Loaded Inhaled Porous Microspheres Inhibit Idiopathic Pulmonary Fibrosis and Improve Pulmonary Function Post-Bleomycin Challenges.

Idiopathic pulmonary fibrosis (IPF) causes worsening pulmonary function, and no effective treatment for the disease etiology is available now. Recombinant Human Relaxin-2 (RLX), a peptide agent with anti-remodeling and anti-fibrotic effects, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, due to its short circulating half-life, optimal efficacy requires continuous infusion or repeated injections. Here, we developed the porous microspheres loading RLX (RLX@PMs) and evaluated their therapeutic potential on IPF by aerosol inhalation. RLX@PMs have a large geometric diameter as RLX reservoirs for a long-term drug release, but smaller aerodynamic diameter due to their porous structures, which were beneficial for higher deposition in the deeper lungs. The results showed a prolonged release over 24 days, and the released drug maintained its peptide structure and activity. RLX@PMs protected mice from excessive collagen deposition, architectural distortion, and decreased compliance after a single inhalation administration in the bleomycin-induced pulmonary fibrosis model. Moreover, RLX@PMs showed better safety than frequent gavage administration of pirfenidone. We also found RLX-ameliorated human myofibroblast-induced collagen gel contraction and suppressed macrophage polarization to the M2 type, which may be the reason for reversing fibrosis. Hence, RLX@PMs represent a novel strategy for the treatment of IPF and suggest clinical translational potential.

Creation of a versatile automated two-step purification system with increased throughput capacity for preclinical mAb material generation

The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.

Minimally invasive, sustained-release relaxin-2 microparticles reverse arthrofibrosis.

Substantial advances in biotherapeutics are distinctly lacking for musculoskeletal diseases. Musculoskeletal diseases are biomechanically complex and localized, highlighting the need for novel therapies capable of addressing these issues. All frontline treatment options for arthrofibrosis, a debilitating musculoskeletal disease, fail to treat the disease etiology-the accumulation of fibrotic tissue within the joint space. For millions of patients each year, the lack of modern and effective treatment options necessitates surgery in an attempt to regain joint range of motion (ROM) and escape prolonged pain. Human relaxin-2 (RLX), an endogenous peptide hormone with antifibrotic and antifibrogenic activity, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, RLX has previously faltered through multiple clinical programs because of pharmacokinetic barriers. Here, we describe the design and in vitro characterization of a tailored drug delivery system for the sustained release of RLX. Drug-loaded, polymeric microparticles released RLX over a multiweek time frame without altering peptide structure or bioactivity. In vivo, intraarticular administration of microparticles in rats resulted in prolonged, localized concentrations of RLX with reduced systemic drug exposure. Furthermore, a single injection of RLX-loaded microparticles restored joint ROM and architecture in an atraumatic rat model of arthrofibrosis with clinically derived end points. Finally, confirmation of RLX receptor expression, RXFP1, in multiple human tissues relevant to arthrofibrosis suggests the clinical translational potential of RLX when administered in a sustained and targeted manner.

Efficacy of Selected Live Biotherapeutic Candidates to Inhibit the Interaction of an Adhesive-Invasive Escherichia coli Strain with Caco-2, HT29-MTX Cells and Their Co-Culture.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.

Spray-Drying Encapsulation of the Live Biotherapeutic Candidate Akkermansia muciniphila DSM 22959 to Survive Aerobic Storage.

Akkermansia muciniphila is regarded as a promising next-generation probiotic or live biotherapeutic candidate. Effective delivery strategies must be developed to ensure high enough viability of the probiotic strain throughout its industrial formulation, distribution chain, shelf-life, and, ultimately, the host’s gastrointestinal tract, where it should exert its beneficial effect(s). Among the possible methodologies, spray-drying is considered industrially attractive regarding its costs, efficiency, and scalability, with the due parameter customization. In this study, spray-drying was explored as a one-step process to encapsulate A. muciniphila DSM 22959, testing the drying settings and three different dairy-based matrices. Microcapsule morphology and size was assessed, and viability throughout storage at 4 or 22 °C and simulated gastrointestinal passage was determined. Akkermansia muciniphila microencapsulation by spray-drying, using 10% skim milk and inlet/outlet temperatures of 150/65 °C, is effective in terms of viability stabilization, both during prolonged aerobic storage and exposure to simulated gastrointestinal passage. Akkermansia muciniphila viability was maintained at around 107 CFU/g up to 28 days at 4 °C under aerobic conditions with viability losses inferior to 1 log reduction. This methodology provides the necessary conditions to efficiently deliver the recommended dose of live A. muciniphila in the human gut as a live biotherapeutic product.

Schistosomajaponicum cystatin suppresses osteoclastogenesis via manipulating the NF‑κB signaling pathway.

Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj-Cys) on macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj-Cys (rSj-Cys) dose-dependently restrained osteoclast formation, with a half-maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj-Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription-quantitative PCR and western blot analysis, it was found that rSj-Cys downregulated the expression levels of osteoclastogenesis-related genes and proteins, by interfering with M-CSF and RANKL-induced NF-κB signaling and downstream transcription factors during early-phase osteoclastogenesis. Overall, the results of the present study revealed that rSj-Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

Rapid structural discrimination of IgG antibodies by multicharge-state collision-induced unfolding.

Immunoglobulin G (IgG) antibodies are an important class of biotherapeutics that target various diseases, such as cancers, neurodegenerative disorders, and autoimmune diseases, yet rapid discrimination of IgG antibodies remains a great challenge due to heterogeneity, flexibility, and large size. Herein, we demonstrate a simplified multicharge-state collision-induced unfolding (CIU) method for rapid differentiation of four IgG isotypes that differ in terms of the numbers and patterns of disulfide bonds, bypassing tedious single charge-state selection in advance. The results presented herein reveal that gas-phase unfolding behaviors have a strong dependence on charge states outside IgG surfaces; therefore, multicharge-state CIU analysis of IgG subtypes could offer a great opportunity to gain deeper insights into their gas-phase structural differentiation. The full discrimination of IgG antibody isoforms that possess different disulfide bond numbers and even subtle disulfide bonding patterns can be achieved based on their charge-dependent gas-phase unfolding behaviors and root-mean square deviation in CIU difference spectra. Taken together, the incorporation of all charge states observed in a native ion mobility-mass spectrometry (IM-MS) experiment to CIU analysis could make this strategy sensitive to more subtle structural discrepancies, facilitating the rapid discrimination and evaluation of innovative structurally similar biotherapeutic candidates with unexplored functions.

Significance of African Diets in Biotherapeutic Modulation of the Gut Microbiome.

Diet plays an essential role in human development and growth, contributing to health and well-being. The socio-economic values, cultural perspectives, and dietary formulation in sub-Saharan Africa can influence gut health and disease prevention. The vast microbial ecosystems in the human gut frequently interrelate to maintain a healthy, well-coordinated cellular and humoral immune signalling to prevent metabolic dysfunction, pathogen dominance, and induction of systemic diseases. The diverse indigenous diets could differentially act as biotherapeutics to modulate microbial abundance and population characteristics. Such modulation could prevent stunted growth, malnutrition, induction of bowel diseases, attenuated immune responses, and mortality, particularly among infants. Understanding the associations between specific indigenous African diets and the predictability of the dynamics of gut bacteria genera promises potential biotherapeutics towards improving the prevention, control, and treatment of microbiome-associated diseases such as cancer, inflammatory bowel disease, obesity, type 2 diabetes, and cardiovascular disease. The dietary influence of many African diets (especially grain-base such as millet, maize, brown rice, sorghum, soya, and tapioca) promotes gut lining integrity, immune tolerance towards the microbiota, and its associated immune and inflammatory responses. A fibre-rich diet is a promising biotherapeutic candidate that could effectively modulate inflammatory mediators’ expression associated with immune cell migration, lymphoid tissue maturation, and signalling pathways. It could also modulate the stimulation of cytokines and chemokines involved in ensuring balance for long-term microbiome programming. The interplay between host and gut microbial digestion is complex; microbes using and competing for dietary and endogenous proteins are often attributable to variances in the comparative abundances of Enterobacteriaceae taxa. Many auto-inducers could initiate the process of quorum sensing and mammalian epinephrine host cell signalling system. It could also downregulate inflammatory signals with microbiota tumour taxa that could trigger colorectal cancer initiation, metabolic type 2 diabetes, and inflammatory bowel diseases. The exploitation of essential biotherapeutic molecules derived from fibre-rich indigenous diet promises food substances for the downregulation of inflammatory signalling that could be harmful to gut microbiota ecological balance and improved immune response modulation.

Converting Immune Cold into Hot by Biosynthetic Functional Vesicles to Boost Systematic Antitumor Immunity.

SummaryImmune cold tumor characterized by low immunogenicity, insufficient and exhausted tumor-infiltrating lymphocytes, and immunosuppressive microenvironment is the main bottleneck responsible for low patient response rate of immune checkpoint blockade. Here, we developed biosynthetic functional vesicles (BFVs) to convert immune cold into hot through overcoming hypoxia, inducing immunogenic cell death, and immune checkpoint inhibition. The BFVs present PD1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the surface, whereas load catalase into their inner core. The TRAIL can specifically induce immunogenic death of cancer cells to initiate immune response, which is further synergistically strengthened by blocking PD1/PDL1 checkpoint signal through ectogenic PD1 proteins on BFVs. The catalase can produce O2 to overcome tumor hypoxia, in turn to increase infiltration of effector T cells while deplete immunosuppressive cells in tumor. The BFVs elicit robust and systematic antitumor immunity, as demonstrated by significant regression of tumor growth, prevention of abscopal tumors, and excellent inhibition of lung metastasis.

Applying MAPPs Assays to Assess Drug Immunogenicity.

Immunogenicity against biotherapeutic proteins (BPs) and the potential outcome for the patient are difficult to predict. In vitro assays that can help to assess the immunogenic potential of BPs are not yet used routinely during drug development. MAPPs (MHC-associated peptide proteomics) is one of the assays best characterized regarding its value for immunogenicity potential assessment. This review is focusing on recent studies that have employed human HLA class II-MAPPs assays to rank biotherapeutic candidates, investigate clinical immunogenicity, and understand mechanistic root causes of immunogenicity. Advantages and challenges of the technology are discussed as well as the different areas of application.

A Nanoparticle-Based Assay To Evaluate Surface-Induced Antibody Instability.

Protein stability against aggregation represents a major quality attribute for the successful development of biopharmaceuticals. Increasing evidence indicates that the formation of protein aggregates in aqueous solutions is often triggered by interactions between proteins and interfaces. Yet, in contrast to a large number of methods available to test protein bulk properties, high-throughput assays to investigate protein instability at interfaces remain much less developed. Major challenges include the control of the amount and type of surfaces, as well as the presence of synergistic effects between interfaces and hydrodynamic flows. Here, we describe and develop a highly controlled surface-mediated stress assay of protein instability based on polymeric nanoparticles. We show that hydrophobic nanoparticles are remarkably powerful in destabilizing large proteins such as antibodies. We further show that this approach can be implemented on a high-throughput microfluidic platform by compartmentalizing the protein solution in picoliter droplets surrounded by an oil phase. Our method allows the evaluation of protein instability at hydrophobic interfaces in a time scale of minutes and requires amounts of the sample in the order of a few hundred micrograms. We demonstrate that our assay represents a good mimic of air-water interfaces and finds application as a screening tool to optimize protein stability toward surface-induced aggregation. We provide a concrete example by identifying the optimal concentration range of Tween 80 that prevents antibody instability in the presence of interfaces. Overall, our hydrophobic nanoparticle surface-mediated stress assay (HNSSA) represents an attractive tool for accelerated tests of protein instability at interfaces under both stagnant and flow conditions, with implications for the optimization of buffer composition and the selection of stable biotherapeutic candidate molecules during early stage development.

Suppressive effects of Streptococcus thermophilus KLDS 3.1003 on some foodborne pathogens revealed through in vitro, in vivo and genomic insights.

Foodborne diseases (FBDs) remain a persistent global challenge and recent research efforts suggest that lactic acid bacteria (LAB) strains can contribute towards their prevention and treatment. This study investigates the genetic properties of Streptococcus thermophilus KLDS 3.1003 as a potential probiotic and health-promoting LAB strain as well as its in vitro and in vivo activities against two foodborne pathogens. In vitro, its antimicrobial activities and tolerance levels in simulated bile salts and acids were determined. The cytotoxic effects of the LAB strain in RAW264.7 cells were also evaluated. For in vivo evaluation, 24 BALB/c mice were orally administered control and trial diets for 14 days. Genomic analyses of this strain's bacteriocin configuration, stress response system and multidrug resistance genes were annotated to validate in vitro and in vivo results. In vitro antimicrobial results show that the cells and CFS of S. thermophilus KLDS 3.1003 could inhibit both pathogens with the former being more effective (P < 0.05). In addition, its cell-free supernatant (CFS) could inhibit the growth of both pathogens, with catalase treatment having the highest effect against it. More so, after 3 h of incubation, survivability levels of S. thermophilus KLDS 3.1003 were significantly high (P < 0.05). LPS-induced RAW264.7 cell activities were also significantly reduced by 108-109 CFU mL-1 of S. thermophilus KLDS. In vivo, significant weight losses were inhibited in the TSTEC group compared to the TSTSA group (P < 0.05). Moreover, pathogen-disrupted blood biochemical parameters like HDL, LDL, TP, TG, AST, ALT and some minerals were restored in the respective prevention groups (TSTEC and TSTSA). Genomic analyses showed that S. thermophilus KLDS 3.1003 has bacteriocin-coding peptides, which accounts for its antimicrobial abilities in vitro and in vivo. S. thermophilus KLDS 3.1003 is also endowed with intact genes for acid tolerance, salt-resistance, cold and heat shock responses and antioxidant activities, which are required to promote activities against the selected foodborne pathogens. This study showed that S. thermophilus KLDS 3.1003 has the genomic capacity to inhibit foodborne pathogens' growth in vitro and in vivo, thus qualifying it as a potential probiotic, antimicrobial and bio-therapeutic candidate.

Isolation and characterization of phage (ETP-1) specific to multidrug resistant pathogenic Edwardsiella tarda and its in vivo biocontrol efficacy in zebrafish (Danio rerio)

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1–1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-β, tnf-α, il-6, and il-10, the chemokine, cxcl-8a, and reactive oxygen species, sod-1. When zebrafish were bath exposed to ETP-1 for 12 days and simultaneously challenged with E. tarda (1.08 × 105 CFU fish−1), the survival rate was higher in phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture.

Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.

Coumarin–gold nanoparticle bioconjugates: preparation, antioxidant, and cytotoxic effects against MCF-7 breast cancer cells

In recent, the conjugation of gold nanoparticles (AuNPs) with biomolecules has shown great potential especially in disease diagnostics and treatment. Taking this in account, we report the methodology involved in the conjugation of coumarin onto the surface of citrate-capped AuNPs by a simple in situ method. Herein, we systematically performed UV–Vis spectroscopy, transmission electron microscopy, dynamic light scattering, and zeta potential measurements to characterize citrate-capped AuNPs and bioconjugates. Our results demonstrate in-depth surface chemistry of bioconjugates with improved surface plasmon resonance (529 nm), morphology (near spherical shape), hydrodynamic diameter (25.3 nm) as well as surface charge (− 35 mV). Furthermore, the bioconjugates displayed dose-dependent response in scavenging free radicals and exhibited cytotoxicity against MCF-7 breast cancer cell lines. In addition, phase-contrast microscopic analysis revealed that bioconjugates promote apoptosis in cancer cells in a time-dependent manner. Overall, we ascertain the fact that this kind of bioconjugation of AuNPs with coumarin further enhances the efficacy of inorganic nanomaterials and thus make them a better bio-therapeutic candidate.

Short communication: antiviral activity of porcine IFN-λ3 against porcine epidemic diarrhea virus in vitro.

A new family of IFNs called type III IFN or IFN-λ has been described, and shown to induce antiviral activity against several viruses in the cell culture. In this study, the molecular cloning, expression, and antiporcine epidemic diarrhea virus (PEDV) activity of porcine IFN-λ3 (poIFN-λ3) were reported. The full-length poIFN-λ3 cDNA sequence encoded 196 amino acids with a 23 amino acid signal peptide. Sequence alignments showed that poIFN-λ3 had an amino acid sequence similarity to Ovis aries (78.1 %), Bos taurus (76.0 %), Tupaia belangeri (71.3 %), Equus caballus (69.9 %), and Homo sapiens (69.9 %). The phylogenetic analysis based on the genomic sequences indicated that poIFN-λ3 is located in the same branch as B. taurus and O. aries IFN-λ3. The poIFN-λ3 without a signal anchor sequence was efficiently expressed in Escherichia coli, and the purified recombinant poIFN-λ3 exhibited significant antiviral effects against PEDV in a dose- and time-dependent manner. This inhibitory effect of poIFN-λ3 on PEDV was observed under three different treatment conditions. The highest inhibition of PEDV was observed in Vero E6 cell cultures pretreated with poIFN-λ3 (prior to PEDV infection). In addition, poIFN-λ3 was able to induce the expression of IFN-stimulated genes, including ISG15, OAS1, and Mx1 in Vero E6 cells. These data demonstrate that poIFN-λ3 has antiviral activity against PEDV and may serve as a useful biotherapeutic candidate to inhibit PEDV or other viruses in swine.