Integrated micro-scale protein a chromatography and Low pH viral inactivation unit operations on an automated platform.

High throughput process development (HTPD) is established for time- and resource- efficient chromatographic process development. However, integration with non-chromatographic operations within a monoclonal antibody (mAb) purification train is less developed. An area of importance is the development of low pH viral inactivation (VI) that follows protein A chromatography. However, the lack of pH measurement devices at the micro-scale represents a barrier to implementation, which prevents integration with the surrounding unit operations, limiting overall process knowledge. This study is based upon the design and testing of a HTPD platform for integration of the protein A and low pH VI operations. This was achieved by using a design and simulation software before execution on an automated liquid handler. The operations were successfully translated to the micro-scale, as assessed by analysis of recoveries and molecular weight content. The integrated platform was then used as a tool to assess the effect of pH on HMWC during low pH hold. The laboratory-scale and micro-scale elution pools showed comparable HMWC across the pH range 3.2-3.7. The investigative power of the platform is highlighted by evaluating the resources required to conduct a hypothetical experiment. This results in lower resource demands and increased labor efficiency relative to the laboratory-scale. For example, the experiment can be conducted in 7 h, compared to 105 h, translating to labor hours, 3 h and 28 h for the micro-scale and laboratory-scale, respectively. This presents the opportunity for further integration beyond chromatographic operations within the purification sequence, to establish a fit-to-platform assessment tool for mAb process development.

Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant ß-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.

Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin.

Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd.

Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture.

Lysobactin (katanosin B) is a macrocyclic depsipeptide, displaying high antibacterial activity against human pathogens. In this work, we have identified and characterized the entire biosynthetic gene cluster responsible for lysobactin assembly. Sequential analysis of the Lysobacter sp. ATCC 53042 genome revealed the lysobactin gene cluster to encode two multimodular nonribosomal peptide synthetases. As the number of modules found within the synthetases LybA and LybB directly correlates with the primary sequence of lysobactin, a linear logic of lysobactin biosynthesis is proposed. Investigation of adenylation domain specificities in vitro confirmed the direct association between the synthetases and lysobactin biosynthesis. Furthermore, an unusual tandem thioesterase architecture of the LybB termination module was identified. Biochemical characterization of the individual thioesterases in vitro provides evidence that solely penultimate thioesterase domain mediates the cyclization and simultaneous release of lysobactin.

Biosynthesis of the siderophore rhodochelin requires the coordinated expression of three independent gene clusters in Rhodococcus jostii RHA1.

In this work we report the isolation, structural characterization, and the genetic analysis of the biosynthetic origin of rhodochelin, a unique mixed-type catecholate-hydroxamate siderophore isolated from Rhodococcus jostii RHA1. Rhodochelin structural elucidation was accomplished via MS(n)- and NMR-analysis and revealed the tetrapeptide to contain an unusual ester bond between an L-δ-N-formyl-δ-N-hydroxyornithine moiety and the side chain of a threonine residue. Gene deletions within three putative biosynthetic gene clusters abolish rhodochelin production, proving that the ORFs responsible for rhodochelin biosynthesis are located in different chromosomal loci. These results demonstrate the efficient cross-talk between distantly located secondary metabolite gene clusters and outline new insights into the comprehension of natural product biosynthesis.

Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery.

Daptomycin (Cubicin) is a branched cyclic lipopeptide antibiotic of nonribosomal origin and the prototype of the acidic lipopeptide family. It was approved in 2003 for the nontopical treatment of skin structure infections caused by gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), and in 2006 for the treatment of bacteremia. Understanding the ribosome-independent biosynthesis of daptomycin assembly will provide opportunities for the generation of daptomycin derivatives with an altered pharmaceutical spectrum to address upcoming daptomycin-resistant pathogens. Herein, the structural properties of daptomycin, its biosynthesis, recent efforts for the generation of structural diversity, and its proposed mode of action are discussed.

Erythrochelin--a hydroxamate-type siderophore predicted from the genome of Saccharopolyspora erythraea.

The class of nonribosomally assembled siderophores encompasses a multitude of structurally diverse natural products. The genome of the erythromycin-producing strain Saccharopolyspora erythraea contains 25 secondary metabolite gene clusters that are mostly considered to be orphan, including two that are responsible for siderophore assembly. In the present study, we report the isolation and structural elucidation of the hydroxamate-type tetrapeptide siderophore erythrochelin, the first nonribosomal peptide synthetase-derived natural product of S. erythraea. In an attempt to substitute the traditional activity assay-guided isolation of novel secondary metabolites, we have employed a dedicated radio-LC-MS methodology to identify nonribosomal peptides of cryptic gene clusters in the industrially relevant strain. This methodology was based on transcriptome data and adenylation domain specificity prediction and resulted in the detection of a radiolabeled ornithine-inheriting hydroxamate-type siderophore. The improvement of siderophore production enabled the elucidation of the overall structure via NMR and MS(n) analysis and hydrolysate-derivatization for the determination of the amino acid configuration. The sequence of the tetrapeptide siderophore erythrochelin was determined to be D-alpha-N-acetyl-delta-N-acetyl-delta-N-hydroxyornithine-D-serine-cyclo(L-delta-N-hydroxyornithine-L-delta-N-acetyl-delta-N-hydroxyornithine). The results derived from the structural and functional characterization of erythrochelin enabled the proposal of a biosynthetic pathway. In this model, the tetrapeptide is assembled by the nonribosomal peptide synthetase EtcD, involving unusual initiation- and cyclorelease-mechanisms.

TioS T-TE--a prototypical thioesterase responsible for cyclodimerization of the quinoline- and quinoxaline-type class of chromodepsipeptides.

The family of chromodepsipeptides constitutes a class of structurally related pseudosymmetrical peptidolactones and peptidothiolactones synthesized by nonribosomal peptide synthetases. The chromodepsipeptides, which are analogous to the extensively characterized echinomycin, attain their DNA-bisintercalating properties from chromophore moieties attached to the N-termini of the oligopeptide chain. Thiocoraline, a quinoline-substituted DNA-bisintercalator isolated from marine actinomycetes, is a two-fold symmetric octathiodepsipeptide currently undergoing preclinical trials phase II. In the present study, the excised peptide cyclase TioS T-TE (thiolation-thioesterase bidomain) was employed as a general catalyst for the in vitro generation of thiocoraline analogs. TioS T-TE is capable of catalyzing ligation and the subsequent cyclization of tetrapeptidyl-thioester substrates, circumventing the demanding synthesis of octapeptidyl substrates. The general importance of several amino acid residues within the tetrapeptide was evaluated and revealed new insights with respect to the iterative mechanism utilized by the thioesterase. Additionally, substrate tolerance towards the cyclizing nucleophile allows the formation of macrolactones instead of the native macrothiolactones. Several thiocoraline analogs were isolated and investigated for DNA-bisintercalation activity. Relaxed substrate specificity regarding the chromophore moiety enables the chemoenzymatic synthesis of the quinoxaline- and quinoline-type class of chromodepsipeptides. TioS T-TE is the first nonribosomal peptide synthetase-derived thioesterase, capable of macrothiolactonization and macrolactonization, working in an iterative manner.

Insights into the biosynthesis and stability of the lasso peptide capistruin.

Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Gly1, Arg11, Val12, and Ile13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations.