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The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical technologies such as turbidimetry and dielectric spectroscopy. We developed batch and perfusion processes at the 2-L scale for the production of BR033, a cecropin-like AMP from Lucilia sericata, in stably-transformed polyclonal Sf-9 cells. This is the first time that BR033 has been expressed as a recombinant peptide. Process analytical technology facilitated the online monitoring and control of cell growth, viability and concentration. The perfusion process increased productivity by ~ 180% compared to the batch process and achieved a viable cell concentration of 1.1 × 10 7  cells/mL. Acoustic separation enabled the consistent retention of 98.5-100% of the cells, viability was > 90.5%. The recombinant AMP was recovered from the culture broth by immobilized metal affinity chromatography and gel filtration and was able to inhibit the growth of Escherichia coli K12. These results demonstrate a successful, integrated approach for the development and intensification of a process from cloning to activity testing for the production of new biopharmaceutical candidates., (© 2022. The Author(s).)

Background: Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. Results: The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His6 tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression. Conclusions: The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are ubiquitous components of the insect innate immune system. The model insect Galleria mellonella has at least 18 AMPs, some of which are still uncharacterized in terms of antimicrobial activity. To determine why G. mellonella secretes a repertoire of distinct AMPs following an immune challenge, we selected three different AMPs: cecropin A (CecA), gallerimycin and cobatoxin. We found that cobatoxin was active against Micrococcus luteus at a minimum inhibitory concentration (MIC) of 120 μ m, but at 60 μ m when co-presented with 4 μ m CecA. In contrast, the MIC of gallerimycin presented alone was 60 μ m and the co-presentation of CecA did not affect this value. Cobatoxin and gallerimycin were both inactive against Escherichia coli at physiological concentrations, however gallerimycin could potentiate the sublethal dose of CecA (0.25 μ m) at a concentration of 30 μ m resulting in 100% lethality. The ability of gallerimycin to potentiate the CecA was investigated by flow cytometry, revealing that 30 μ m gallerimycin sensitized E. coli cells by inducing membrane depolarization, which intensified the otherwise negligible effects of 0.25 μ m CecA. We therefore conclude that G. mellonella maximizes the potential of its innate immune response by the co-presentation of different AMPs that become more effective at lower concentrations when presented simultaneously. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi-resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect-derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost-efficient large-scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large-scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect-derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin-like peptide/intein based purification strategies. All of these aspects are discussed with regard to large-scale processes and costs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:1-11, 2015 [ABSTRACT FROM AUTHOR]

Background: Spodoptera frugiperda (Sf-9) cells are often used to produce recombinant proteins by transient expression. The culture medium contributes to the efficiency of protein production, but the effect of modern insect cell culture media on Sf-9 cells has not been evaluated for different transient expression systems. Accordingly, we compared the baculovirus expression vector system (BEVS) and lipopolyfection (LP) for the production of schistosomal ALDH and the antimicrobial peptide BR033, respectively. We used Sf-9 cells adapted to four commercially available media: TriEx ICM, ExpiSf CDM, Sf-900 II SFM and IS Sf Insect ACF. Results: During cell maintenance, the highest viable cell density was achieved using IS Sf Insect ACF, but the shortest doubling time was observed in TriEx ICM. All four media supported transient expression with standard protocols for LP and BEVS. We confirmed the typical switch in metabolic rates (glucose, lactate and amino acids) after transfection or infection, comparable to previous reports. The maximum protein production rates were 0.26 ± 0.04 amol/(cell h) for LP and 4.63 ± 1.85 amol/(cell h) for BEVS, both achieved using Sf-900 II SFM. Conclusions: The experiments demonstrate that the most suitable medium for transient production in Sf-9 cells depends on the experimental objectives or production framework. We therefore recommend implementing the screening of different media during process development. [ABSTRACT FROM AUTHOR]