Expression of Peptides as Small

One of the most significant advances in medical history is the discovery and development of antibiotics, which in the middle of last century was flourishing and appeared to be the ultimate solution to the treatment of life-threatening human bacterial diseases. However, lately there has been a huge decline in the rate of discovery of new antimicrobial intervention strategies in parallel with an increasing incidence of multidrug-resistant pathogens; if these circumstances do not change we will continue to approach the end of the antibiotic era. Facing this dark future, scientists are considering new strategies for intervention tailored around the appropriate (selective) stimulation of the host's immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One recent player in such an immunomodulatory strategy is the naturally occurring host defence peptides (HDP) and their synthetic innate defence regulator (IDR) analo...

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, since they are able to kill a wide variety of microbes directly and/or to modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been significantly lowered by developing a successful auto-induction strategy, which has been found to support significantly higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing the production scale from 100 to 1,000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Antimicrobial peptides are the upcoming therapeutic molecules as alternative drugs to the existing antibiotics owing to their potent action against pathogenic microorganisms. In this study, to obtain an antimicrobial peptide with a broad range of activity, the synthetic cationic antimicrobial peptide was designed by using in silico tools viz., antimicrobial peptide database, protparam, hierarchical neural network. Later, the peptide was translated back into a core nucleotide sequence and the gene for the peptide was constructed by overlapping PCR. The amplified gene was cloned into pRSET-A vector and transformed into salt inducible expression host E. coli GJ1158. The expression results show high yields of soluble recombinant fusion peptide (0.52 g/L) from salt-inducible E. coli. The recombinant peptide was purified by the IMAC purification system and cleaved by enterokinase. The digested product was further purified and 0.12 g/L of biologically active recombinant cationic antimicrobial peptide was obtained. In vitro analysis of the purified peptide demonstrated high antimicrobial activity against both Gram positive and Gram negative bacteria devoid of hemolytic activity. Therefore, this synthetic cationic antimicrobial peptide could serves as an promising agent over chemical antibiotics. In this study, a synthetic cationic antimicrobial peptide was designed, cloned and expressed from salt-inducible E. coli GJ1158 using cost effective media in the large scale production of antimicrobial peptide and its biological activity was analysed against different Gram positive and negative organisms.

In this study we used a yeast expression system to express a new antimicrobial peptide dybowskin-2CAMa from the skin cDNA library of Rana amurenisis. The entire coding region of the dybowskin-2CAMa was cloned into the plasmid pPICZα-A and then transformed into competent P. pastoris X33. The expressed dybowskin-2CAMa was purified from the culture supernatant by Sephadex G-25 and YMC*GEL ODS-A chromatography followed by C18 reverse phased HPLC. The purified peptide exhibited a single band of about 2 kDa when resolved by Tricine-SDS-PAGE. Its exact molecular weight was 2456.46 Da which was consistent with the value predicted from its deduced amino acid sequence. Antimicrobial activity assay showed that the recombinant dybowskin-2CAMa could inhibit the growth of a broad spectrum of bacteria, while displaying very low level of hemolytic activity (≤4% relative to Triton X-100), even at concentration of up to 500 µg/mL.

Authors’ address: 1 Department of Biotechnology, Bapatla Engineering College, Bapatla 522101, Guntur, Andhra Pradesh, India. 2 Department of Biotechnology, Acharya Nagarjuna University, Guntur 522510, Andhra Pradesh, India. 3 Department of Biotechnology, R.V.R. & J.C. College of Engineering, Chowdavaram, Guntur 522019, Andhra Pradesh, India. 4 Department of Botany, Govt. Arts Colelge, Ariyalur, Tamilnadu, India. 5 CBST, VIT, Vellore, Tamilnadu, India. † JB Perevali and SR Kotra contributed equally to this work.

The cytoplasmic granules of bovine neutrophils naturally possess indolicidin-a promising cationic antimicrobial peptide as it displays inherent inhibitory activities against a broad type of microbial pathogens. In this study, a shake flask level production and expression optimizations of the indolicidin by the recombinant Escherichia coli C41 (DE3) clones (transformed with pET21a(+) plasmid carrying indolicidin gene) were carried out under standard conditions, as to determine the conditions required for maximal production. It was determined that a concentration of 1 mM of IPTG was effective, the 2×YT with salts and LB media at pH 7.5 with 3-6 h of incubation were required for maximal indolicidin expression.

The cytoplasmic granules of bovine neutrophils naturally possess indolicidin - a promising cationic antimicrobial peptide as it displays inherent inhibitory activities against a broad type of microbial pathogens. In this study, a shake flask level production and expression optimizations of the indolicidin by the recombinant Escherichia coli C41 (DE3) clones (transformed with pET21a(+) plasmid carrying indolicidin gene) were carried out under standard conditions, as to determine the conditions required for maximal production. It was determined that a concentration of 1 mM of IPTG was effective, the 2×YT with salts and LB media at pH 7.5 with 3-6 h of incubation were required for maximal indolicidin expression.

Background: Bactenecin is one of the smallest cationic eukaryotic antimicrobial peptides (AMPs) with a length of 12 residues and a beta-turn structure originated from bovine neutrophil cells. Previous studies reported poor capability of this peptide against Gram-negative bacteria. Objectives: The present study aimed at investigating the bactenecin bactericidal activity and determining the effect of increasing the positive charge of amine and carboxyl terminus and increasing the hydrophobicity of the central part of the antimicrobial peptide. Methods: Similar to the native peptide, three designed variants were employed named BM1, BM2, and BM3 that had increased positive charges, hydrophobicity, and a combination of both, respectively. Conditions for purifying and assaying their antibacterial activity against Gram-negative bacteria were predicted and optimized by APD3 and ExPASy servers. The cloned and expressed peptides in Pichia pastoris GS115 were partially purified by anion and cation exchange chromatography. The final purification rate of AMPs by HPLC was reported to be 70% and peptides were further characterized by LC-mass analysis. Finally, a minimum inhibitory concentration test was conducted. Results: The results implied the more significant effect of positive charges on the performance of these peptides against Escherichia coli.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Cecropin is a cationic antibacterial peptide composed of 35-39 residues. This peptide has been identified as possessing strong antibacterial activity and low toxicity against eukaryotic cells, and it has been claimed that some types of the cecropin family of peptides are capable of killing cancer cells. In this study, the host effect of cloning antibacterial peptide cecropinB2 was investigated. Three different host expression systems were chosen, i.e., Escherichia coli, Bacillus subtilis and Pichia pastoris. Two gene constructs, cecropinB2 (cecB2) and intein-cecropinB2 (INT-cecB2), were applied. Signal peptide and propeptide from Armigeres subalbatus were also attached to the gene construct. The results showed that the best host for cloning cecropinB2 was P. pastoris SMD1168 harboring the gene of pGAPzαC-prepro-cecB2 via Western blot confirmation. The cecropinB2 that was purified using immobilized-metal affinity chromatography resin showed strong antibacterial activity against the Gram-negative strains, including the multi-drug-resistant bacteria Acinetobacter baumannii.

Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.

bor deaux2.fr (C. Cabanne). 1 Abbre vi a tions used: AMP, anti mi cro bial pep tide; MBP, malt ose bind ing pro tein; Leap2, liver-expressed anti mi cro bial pep tide 2; LB, Luria-Ber tan i; IPTG, iso pro pyl b-d-thio ga lac to py ra no side; EDTA, eth yl ene di amine tet ra ace tic acid; PMSF, phen ylmethanesulfo nyl fluo ride; BCA, bi cinch on i nic acid; RP-HPLC, reverse phase high-per for mance liquid chro ma tog ra phy; SDS-PAGE, sodium dode cyl sulfate-poly acryl amide gel elec tro pho re sis; FPLC, fast pro tein liquid chro ma tog ra phy.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Host defence peptides (HDPs) are innate immune effector molecules found in diverse species. HDPs exhibit a wide range of functions ranging from direct antimicrobial properties to immunomodulatory effects. Research in the last decade has demonstrated that HDPs are critical effectors of both innate and adaptive immunity. Various studies have hypothesized that the antimicrobial property of certain HDPs may be largely due to their immunomodulatory functions. Mechanistic studies revealed that the role of HDPs in immunity is very complex and involves various receptors, signalling pathways and transcription factors. This review will focus on the multiple functions of HDPs in immunity and inflammation, with special reference to cathelicidins, e.g. LL-37, certain defensins and novel synthetic innate defence regulator peptides. We also discuss emerging concepts of specific HDPs in immune-mediated inflammatory diseases, including the potential use of cationic peptides as therapeutics for immune-mediated inflammatory disorders.

. Scatterplot Matrix (SigmaPlot v12) Illustrating the peptides experimentally verified percent inhibition (0-100%), in combination with the structure activity relationship estimated charge distribution (-16 -84) and hydrophobicity distribution (16 -126). The scales for the two latter once are only relative, and will have no direct meaning outside of this context. When the three properties are plotted against itself it illustrates the distribution of this property amongst all the peptides in the tested library ).

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.

. Scatterplot Matrix (SigmaPlot v12) Illustrating the peptides experimentally verified percent inhibition (0-100%), in combination with the structure activity relationship estimated charge distribution (-16 -84) and hydrophobicity distribution (16 -126). The scales for the two latter once are only relative, and will have no direct meaning outside of this context. When the three properties are plotted against itself it illustrates the distribution of this property amongst all the peptides in the tested library ).

Innate defense regulators (IDRs) are synthetic immunomodulatory versions of natural host defense peptides (HDP). IDRs mediate protection against bacterial challenge in the absence of direct antimicrobial activity, representing a novel approach to anti-infective and anti-inflammatory therapy. Previously, we reported that IDR-1018 selectively induced chemokine responses and suppressed pro-inflammatory responses. As there has been an increasing appreciation for the ability of HDPs to modulate complex immune processes, including wound healing, we characterized the wound healing activities of IDR-1018 in vitro. Further, we investigated the efficacy of IDR-1018 in diabetic and non-diabetic wound healing models. In all experiments, IDR-1018 was compared to the human HDP LL-37 and HDP-derived wound healing peptide HB-107. IDR-1018 was significantly less cytotoxic in vitro as compared to either LL-37 or HB-107. Furthermore, administration of IDR-1018 resulted in a dose-dependent increase in fibroblast cellular respiration. In vivo, IDR-1018 demonstrated significantly accelerated wound healing in S. aureus infected porcine and non-diabetic but not in diabetic murine wounds. However, no significant differences in bacterial colonization were observed. Our investigation demonstrates that in addition to previously reported immunomodulatory activities IDR-1018 promotes wound healing independent of direct antibacterial activity. Interestingly, these effects were not observed in diabetic wounds. It is anticipated that the wound healing activities of IDR-1018 can be attributed to modulation of host immune pathways that are suppressed in diabetic wounds and provide further evidence of the multiple immunomodulatory activities of IDR-1018.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for largescale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO–Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.