Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim.

We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 µg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim. The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2-8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia.

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V).

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.