Glycopegylated granulocyte colony stimulating factor
A colony-stimulating factor, granulocyte technology, applied in the direction of colony-stimulating factor, cytokines/lymphokines/interferons, chemical instruments and methods, etc., can solve the problems of reducing peptide biological or enzyme activity, lack of uniformity of finished products, etc.
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[0298] Particular embodiments according to the invention include:
[0299]
[0300] and carbonates and active esters of these substances, such as:
[0301]
[0302] Other reactive groups or leaving groups suitable for activating linear PEGs used to prepare the compounds described herein include, but are not limited to, the following:
[0303]
[0304] PEG molecules activated with these and other substances and methods of making activated PEGs are listed in WO04 / 083259.
[0305] Those skilled in the art will recognize that one or more of the m-PEG arms of a branched polymer may be replaced by a PEG moiety having a different end, e.g., OH, COOH, NH 2 、C 2 -C 10 - Alkyl etc. In addition, the above structures are easily modified by inserting an alkyl linker between the α-carbon atom and the side chain functional group or removing a carbon atom. Thus, "homo" derivatives and higher and lower homologues are within the core scope of the branched PEGs used in the present ...
Embodiment 1
[0541] GlycoPEGylation of G-CSF produced in CHO cells
[0542] a. Preparation of asialo (Asialo) granulocyte colony-stimulating factor (G-CSF)
[0543] Dissolve G-CSF produced in CHO cells at 2.5 mg / mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl 2 medium and concentrated to 500 μL in a Centricon Plus 20 centrifugal filter. The solution was incubated with 300 mU / mL neuraminidase II (Vibrio cholerae) at 32°C for 16 hours. To monitor the reaction, a small sample of the reaction was diluted with the appropriate buffer and subjected to an IEF gel. The reaction mixture was then added to prewashed N-(p-aminophenyl)oxamate-agarose conjugate (800 μL / mL reaction volume) and the washed beads were gently rotated at 4°C for 24 hours. The mixture was centrifuged at 10,000 rpm, and the supernatant was collected. The beads were washed 3 times with Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and once with 0.2 mL Tris-EDTA buffer, and all supernatants were pooled. I...
Embodiment 2
[0552] Recombinant GCSF - expression, refolding and purification
[0553] ●Collect cells by centrifugation and discard supernatant. Growth results on various media are shown in Figure 9 middle.
[0554] • Resuspend the cell pellet in 10 mM Tris pH 7.4, 75 mM NaCl, 5 mM EDTA using 10 ml / g (lysis buffer).
[0555] • Microlluidize cells (and French pressure processing).
[0556] ● Centrifuge at 5,000 RPM for 30 minutes at 4°C, -discard the supernatant.
[0557] • Resuspend pellet in lysis buffer and centrifuge as above.
[0558] • Wash IB's in 25mM Tris pH 8, 100mM NaCl, 1% TX-100, 1% NaDOC, 5mM EDTA. Resuspend the pellet by pipetting and vortexing. Centrifuge at 5,000 RPM for 15 minutes at 4°C. This step was repeated again (two washes in total).
[0559] • Wash the pellet twice in 25mM Tris pH 8, 100mM NaCl, 5mM EDTA to remove detergent and centrifuge as above.
[0560] ●Resuspend the pellet in dH 2 Aliquot in O and centrifuge as above. The pellet was fr...
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