Littérature scientifique sur le sujet « Peptide production scale-up »

The separation by electrodialysis with ultrafiltration membranes (EDUF), at a semi-industrial scale, of a new whey protein hydrolysate obtained from a whey protein concentrate was assessed. After 6 h of treatment, more than 9 g of peptides were recovered in the peptide recovery fraction, for a recovery yield of 5.46 ± 0.56% and containing 18 major components. Among these components, positively charged peptides, such as ALPMHIR + PHMIR, LIVTQTMK and TKIPAVF, were present, and their relative abundances increased by nearly 1.25 X and up to 7.55 X. The presence of these peptides may be promising, as ALPMHIR has a strong activity against angiotensin-converting enzyme (ACE), and LIVTQTMK has structural properties that could interfere with dipeptidyl peptidase-IV (DPP-IV). Many neutral peptides were also recovered alongside those. Nevertheless, the inhibitory activity against DPP-IV and ACE increased from 2 X and 4 X, respectively, in the peptide recovery fraction compared to the initial hydrolysate, due to the improved content in bioactive peptides. Thus, this new hydrolysate is well-suited for the large-scale production of a peptide fraction with high bioactivities. Furthermore, what was achieved in this work came close to what could be achieved for the industrial production of a bioactive peptide fraction from whey proteins.

<abstract> <p>The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An <italic>Escherichia coli</italic> (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.</p> </abstract>

Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.

Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20–40% v/v), [Lipid] (DDA and MMG, 6–12 mg/mL) and dimethyl sulfoxide [DMSO] (0–10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.

Background: Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems. Methods: : Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2. <P> Results: Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies. Conclusion: Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity.

Abstract Checkpoint inhibitors, cancer vaccines, and adoptive cell therapy exploit T cell mediated immune responses to cancers. Discovering the exact antigens targeted by T cell responses is important for their efficacy. Antigen discovery for ‘orphan’ T cells or TCRs has been a challenging prospect due to high number of possible pMHC specificities. Several current approaches to decipher antigen specificities require prior knowledge of antigen sequences, are unable to scale up, or require production of soluble TCRs. To overcome these drawbacks, we have developed chimeric receptors called Signaling and Antigen-presenting Bifunctional Receptors (SABRs) that allow identification of antigen-presenting cells. SABRs present display pMHC on their extracellular domain, which is recognized by an orphan TCR. Upon recognition, SABRs initiate signaling in the presenting cell using a CD3zeta signaling domain. We transduced reporter cells with SABRs presenting HLA-A2-restricted epitopes from MelanA and NY-ESO-1, and co-incubated them with target cells expressing their cognate TCRs, which resulted in signal transduction only upon correct pMHC-TCR pairing, allowing the presenting cells to express GFP. Second, we showed that SABRs displaying independently expressed peptide and MHC could function similarly. These receptors could present pulsed peptides or endogenously expressed proteins, allowing the uncoupling of peptide and MHC, while retaining their signaling capability. We are currently testing the use of SABR-based antigen libraries to identify novel antigenic specificities targeted by T cells in cancers, infectious diseases, and autoimmune diseases.

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK fromS. pyogenessDT7 overexpressed inE. coli, purification, and biochemical characterization. A gene coding for the SK was cloned fromS. pyogenessDT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed inE. coliBL21 DE3/pESK under the control of the strong promotertacinduced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinantE. coliBL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

Organize the matter on an increasingly small scale is sought in order to increase the performance of materials. In the case of porous materials, such as filtration membranes, a compromise must be found between the selectivity provided by this nanostructuring and a permeability in particular linked to the existing pore volume. In this work, we propose an innovative waterborne approach consisting in co-assembling peptide amphiphiles (PA) which will provide nanostructuring and polyelectrolytes which will provide them with sufficient mechanical properties to sustain water pressure. C16-V3A3K3G-NH2 PA nanocylinders were synthesized and co-assembled with poly(sodium 4-styrenesulfonate) (PSSNa) into porous nano-fibrous network via electrostatic interactions. The ratio between C16-V3A3K3G-NH2 and PSSNa was studied to optimize the material structure. Since spontaneous gelation between the two precursors does not allow the material to be shaped, various production methods have been studied, in particular via tape casting and spray-coating. Whereas self-supported membranes were mechanically weak, co-assemblies supported onto commercial ultrafiltration membranes could sustain water pressure up to 3 bars while a moderate permeability was measured confirming the existence of a percolated network. The produced membrane material falls into the ultrafiltration range with a pore radius of about 7.6 nm.

Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancies worldwide. 45% of patients are diagnosed at a late tumor stage associated with poor survival. For metastatic, unresectable or recurrent (m/uR) HNSCC, immune checkpoint inhibition (ICI) was recently approved as a novel therapeutic option showing significant survival benefits compared to standard chemotherapy-based treatment. However, response to ICI is still limited to a small number of patients calling for further improvement of T cell-based immunotherapies. Peptide-based approaches, which rely on the specific immune recognition of tumor-associated human leukocyte antigen (HLA) presented peptides, represent promising and low side effect treatment options. Peptide vaccination has been shown to enhance and induce long-term anti-tumoral immune responses and even clinical responses in HNSCC patients. However, current vaccines are either monovalent, based on patient-individual tumor-specific mutations or restricted to a single HLA allotype and therefore neither widely applicable nor suitable for reliable studies and large-scale production. In this study, using mass spectrometry (MS) -based immunopeptidome analysis of a large cohort of HNSCC patient (n = 30) tumor and adjacent benign samples, we established a tumor-associated off-the-shelf peptide warehouse for broadly applicable personalized therapies. The malignant dataset, comprising 91651 HLA ligands, was compared to adjacent benign and various benign tissues (www.hla-ligand-atlas.org) to identify tumor-exclusive antigens. Further antigen selection was based on allotype-specific high frequent presentation. In total, 23 frequently presented and tumor-exclusive HNSCC-associated peptides were selected for six of the most common HLA class I allotypes (A*01, A*02, A*24, B*15, B*35, B*40) covering &gt;75% of the world population, as well as five HLA class II presented peptides binding various different HLA class II allotypes. Immunogenicity was validated by IFN-γ ELISPOT screening for spontaneous preexisting T cell responses targeting the respective peptides as well as by in vitro priming experiments of naïve T cells in HNSCC patients and healthy volunteers. Furthermore, immunopeptidome analyses identified these antigens in patient plasma samples providing first evidence for “liquid biopsy” immunopeptidome analysis without the need of primary tumor tissue. A phase I study evaluating safety, immunogenicity as well as first efficacy of this warehouse-based vaccine in combination with ICI in HNSCC patients is currently being set up, with personalized peptide selection based on individual HLA-allotype and MS analysis of patient tumor/plasma sample. In conclusion, we here designed a peptide warehouse that enables a polyvalent and widely applicable but still personalized peptide vaccination in HNSCC patients. Citation Format: Sarah Schroeder, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, Lena Muehlenbruch, Yacine Maringer, Paul-Stefan Mauz, Martin Sailer, Julia Philipp, Sven Becker, Thomas Breuer, Helmut R. Salih, Hans-Georg Rammensee, Hubert Löwenheim, Juliane S. Walz. Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3555.

In the framework of the PhD project of industrial interest, I have been involved in the development and optimization of synthetic procedures to obtain peptides of pharmaceutical interest, both on the laboratory scale and for the industrial production, in the context of the University-Industry Joint Laboratory PeptFarm of the University of Florence. This research develops through two parallel lines. The former is related to the development of a multigram, scalable cGMP-compliant MW-SP synthetic approach for the manufacture of the cyclic peptide Active Pharmaceutical Ingredient Eptifibatide acetate. Additionally, an alternative, patentable synthetic approach has been developed to overcome patent restrictions. On the other hand, the development of an efficient synthetic strategy for the preparation of a library of stapled peptides of pre-clinical interest derived from relaxin hormone was performed, aiming to investigate their biological role. Moreover, the feasibility of an oral administration of serelaxin gastroprotected formulations were investigated. According to the industrial perspective on research needs and opportunities in manufacturing, automation of as many steps as possible within an industrial production frame is pivotal to guarantee safety requirements. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only at the research scale but for large-scale production, as well. The possibility to use microwave-assisted technology on the large scale recently introduced, prompted us to evaluate the possibility to conveniently set-up a safe and fully cGMP-compliant pilot process to produce Eptifibatide acetate Active Pharmaceutical Ingredients (API), a generic hexapeptide, characterized by a single disulfide bridge. We investigated strategies based on the use of the microwave-assisted solid-phase peptide synthesis (MW-SPPS), by the use of a DIC/Oxyma Pure coupling protocol at 90 °C. This fully automated technology, previously accessible only at R&D level, has been recently made available also for the large-scale manufacturing of peptide APIs, taking constantly into account 6 the cost-effectiveness and dangerousness of each procedure. Accordingly, we developed an optimized process at the laboratory scale (1-5 mmol), which was subsequently successfully scaled-up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot-scale plant. The process consists of 5 steps: 1) automated microwave-assisted solid phase synthesis of Eptifibatide linear precursor; 2) cleavage from the resin with concomitant amino acid side-chains deprotection; 3) disulfide-bond formation in solution; 4) purification by flash column chromatography; 5) ion-exchange solid phase extraction. Since the direct scale-up of a kg-scale, cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we preliminary performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result. Moreover, in our optimization process, a reduction in time, solvents and waste have been obtained, ensuring compliance with the quality specifications, according to regulatory agencies requirements (FDA and EMA). Satisfactory results were obtained in terms of Eptifibatide acetate HPLC purity (99.6%) and Yield (22.1%). Additionally, the investigation of an alternative on-resin cyclization strategy for therapeutic peptide industrial production of Eptifibatide acetate has been carried out in parallel with the aim to develop a robust and economically competitive production process avoiding intermediate steps of isolation to preserve the recovery guaranteeing a GMPs quality product, to overcome patent restrictions. A scalable, fully automated approach performed entirely in the same reactor has been developed. We explored and compared four solid-phase disulfide formation approaches (A, B, C, D) between the C-terminal Cys and the N-terminal 3- Mercaptopropionic acid (MPA). These mainly differ one from each other for the final cyclization step, obtained by direct formation of an S-S disulfide bridge (strategies A-B) or via side-chain-to-tail amide bond formation (strategies C-D). Strategy D resulted the best one, thanks to the concomitant reduction of the Stert- butylthio (StBu) Cys protecting group (PG) and disulfide formation with the MPA reducing agent, enjoying the advantage of using an already qualified starting material. This strategy (D) represents an inventive (non-obvious) 7 strategy, (since we were the first to propose MPA to deprotect StBu on cysteine), which proposes for the first time to perform all the processes including disulfide bond formation in a single reactor (novelty), scalable on multigram-scale by Liberty Pro synthesizer (Industrial applicability). Therefore, according to the three patentability criteria required for a new production process: Novelty; inventive and industrial applicability, the present PhD work identifies a new patentable production process.1 In line with the synthesis of conformationally constraint relaxin derivatives, the present work describes the development of an innovative, efficient and reproducible MW-assisted Copper-Catalyzed Azide-Alkyne Cycloaddition (SP MW-CuAAC) performed on solid phase to prepare side-chain-to-side-chain clicked H1-relaxin single B-chain analogues, overcoming the several synthetic drawbacks (aggregation tendency and poor solubility) which hamper relaxins syntheses. All the relevant parameters, that are, resin (PEG-PS vs PS), solvent mixtures (H2O:t-BuOH:DCM 1:1:1, DMSO:DMF 1:2), catalytic system (CuBr vs CuSO4), microwave energy and reaction time were optimized using a systematic approach.2 Two generations of H1-relaxin single B-chain stapled analogues were obtained. First-generation (VR and VIR) and second-generation H1-relaxin single B-chain peptides (VII and VIIR; VIII and VIIIR; IX and IXR) were characterized by different lengths and different positions and orientations of the triazolyl ring, and were designed with the aim to stabilize the α-helix conformation and to expose the binding cassette motif. The α-helicity induced by the side-chain to side-chain stapling obtained was demonstrated by the circular dichroism (CD) performed both in phosphate buffer and in SDS micelle, thanks to the collaboration with Prof. A. Carotenuto (University of Naples Federico II). Moreover, in the frame of the collaboration with Prof. D. Bani (University of Florence) and Prof. A. Hossein (Institute of Neuroscience and Mental Health, University of Melbourne, Australia), H1-relaxin analogues were biologically tested, to verify binding to cells expressing the receptor RXFP1 and activity 8 through cAMP signaling pathway in HEK-293T cells stably expressing the RXFP1 receptor. Moreover, since the major challenge in the development of peptide drugs is to improve their oral bioavailability, we investigated the relative bio-potency of the intact serelaxin molecule (the recombinant form of human H2-relaxin) and the purified porcine one, in comparison with their proteolytic fragments, obtained after treatment with Simulated Intestinal Digestion Fluid (SIF). Signalling events downstream receptor activation in THP-1 human monocytic cells was measured.