Novel g-csf conjugates
OwnerFrancesco Veronese, Manuela Bernainventors
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Abstract
The present application relates to novel PEG-G-CSF conjugates in which a PEG molecule is linked to the cyteine residue in position 17 of native G-CSF primary sequence or to the cysteine residue of the corresponding position of a G-CSF analogue. The present application also describes a process for the manufacture of such conjugates, such process comprising the following steps: (i) subjecting the G-CSF protein to conditions inducing reversible denaturation of the protein, (ii) conjugation of the denatured protein obtained is step i with a thiol-reactivc PEG under denaturing conditions, (iii) subjecting the conjugates obtained in step ii to conditions promoting renaturation of the conjugate yielding biologically active G-CSF-PEG conjugate.
Claims16
A conjugate of PEG and human G-CSF or an analogue of G-CSF, characterized in that the cysteine thiol in position 17 of the human G-CSF or in the corresponding position of the G-CSF analogue is conjugated to a PEG molecule.
The conjugates of any of the preceding claims where the PEG has MW ranging between 800 to 80.000 Da and preferably 5.000 to 40.000 Da.
Pharmaceutical composition comprising a conjugate according to any of the previous claims.
Use of a conjugate according to any of the previous claims for the manufacturing of a pharmaceutical formulation for the treatment of neutropenia (chronic, chemotherapy induced, HIV induced, bone marrow transplantation).
A process for the manufacture of a conjugate of a polymer and a polypeptide with a sterically hindered cysteine group comprising the following steps:
- (i) subjecting the polypeptide to conditions inducing reversible denaturation of the polypeptide,
- (ii) conjugation of the denatured polypeptide obtained in step (i) with a thiol reactive polymer, under denaturing conditions,
- (iii) subjecting the conjugate obtained in step (ii) to conditions which promote renaturation of the conjugate and afford the desired conjugate.
The process according to any of the preceding claims, characterized by being carried out in an aqueous buffered solution at pH of 5 to 11.
Dependencies are inferred from the claim text.