High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter.

Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters.

Recombinant filgrastim (BK0023) pharmacodynamics and pharmacokinetics after single and multiple escalating doses in an equivalence study in healthy men.

Background And Objectives: The new filgrastim formulation, BK0023, whose synthesis method is patented, was tested in a phase I clinical study that was aimed at investigating the pharmacodynamic and pharmacokinetic equivalence and the safety of BK0023 in healthy male subjects.

Methods: Single and multiple escalating doses were administered to healthy male volunteers according to a double-blind, randomised, two-way crossover design. Thirty-two subjects received subcutaneous filgrastim 2.

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

Background: Filgrastim 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.

Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation.

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation.

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.

Self-assembling nanocomposites for protein delivery: supramolecular interactions between PEG-cholane and rh-G-CSF.

PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5β-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5 ± 1.

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.

Supramolecular association of recombinant human growth hormone with hydrophobized polyhydroxyethylaspartamides.

The protein delivery properties of polymer supramolecular assemblies were investigated by using recombinant human growth hormone (rh-GH) and two polyhydroxyethylaspartamide (PHEA) derivatives: (a) PHEA-C16 obtained by PHEA random grafting with hexadecylalkylamine; (b) PHEA-PEG 5000-C16 obtained by PHEA random co-grafting with hexadecylalkylamine and 5 kDa poly(ethylene glycol). The two polymers possessed similar self-assembling properties: critical micelle concentration (CMC) and particle size. The protein loading (protein/polymer, w/w, %) was 12.