Gaetano Orsini

Protein purification in multicompartment electrolyzers with isoelectric membranes

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher pI. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.

A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization

We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH(2)-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8°C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen

BackgroundFilgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics.MethodsCell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence.ResultsThe results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®.ConclusionsBK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients.Trial registrationTrial registration number (TRN): NCT01933971. Date of registration: Sept 6th 2013.

Efficient bacterial expression of fusion proteins and their selective processing by a recombinant Kex-1 protease

A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

Enzymatic techniques for PEGylation of biopharmaceuticals

Modification of therapeutic proteins and peptides by polyethylene glycol conjugation is a well known method to improve the pharmacological properties of such drugs.

Enzymatic mono-pegylation of glucagon-like peptide 1 towards long lasting treatment of type 2 diabetes

Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates. The compound GLP-1-(7-36)-amide-Q23-PEG 20 kDa monopegylated on the single glutamine residue naturally present in position 23 maintained the ability to activate the GLP-1 receptor expressed in the rat β-cell line RIN-m5F with nanomolar potency along with an increased in vitro resistance to DDP IV and a circulating half-life of about 12 h after subcutaneous administration in rats. These properties enabled GLP-(7-36)-amide-Q23-PEG 20 kDa to exert a glucose-stabilizing effect for a period as long as 8 h, as demonstrated by a single subcutaneous injection to diabetic mice concomitantly challenged with an oral glucose load. The results reported in this work indicate that GLP-(7-36)-amide-Q23-PEG 20 kDa could be a lead compound for the development of long-lasting anti-diabetic agents useful in the treatment of type 2 diabetes affected patients.