Factor VII conjugates
A technology of conjugates and factors, applied in the direction of drug combination, biochemical equipment and methods, extracellular fluid diseases, etc., can solve problems such as attachment of half-life extension parts that are not suitable for highly functionalized
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Embodiment 1
[0284] Example 1: Synthesis of HEP-maleimide and HEP-aldehyde polymers
[0285] Maleimide- and aldehyde-functionalized HEP polymers of defined sizes were prepared by enzymatic (PmHS1) polymerization using two sugar nucleotides, UDP-GlcNAc and UDP-GlcUA. Use trigger trisaccharide (GlcUA-GlcNAc-GlcUA) NH 2 The reaction was initiated and the polymerization was run until the sugar nucleotide building blocks were depleted. The terminal amine (derived from the primer) is then functionalized with a suitable reactive group, in this case a maleic acid designed for conjugation to free cysteine and thio-GSC derivatives. Imide functionality, or benzaldehyde functionality designed for reductive amination chemistry with GSC. The size of HEP polymers can be predetermined by variations in sugar nucleotide:primer stoichiometry. This technique is described in detail in US 2010 / 0036001.
[0286] The trisaccharide primer was synthesized as follows:
[0287] Step 1: Synthesis of (2-Fmoc-ami...
Embodiment 2
[0313] Example 2: Selective reduction of FVIIa407C
[0314] FVIIa407C was reduced using a glutathione-based redox buffer system as described in US 20090041744. 50 mM Hepes, 100 mM NaCl, 10 mM CaCl in a total volume of 41 ml containing 0.5 mM GSH, 15 uM GSSG, 25 mM p-aminobenzamidine and 3 μM Grx2 2 , pH 7.0, unreduced FVIIa 407C (15.5 mg) was incubated at room temperature for 17 h. The reaction mixture was then cooled on ice and added to 8.3 ml of 100 mM EDTA solution while maintaining the pH at 7.0. The entire contents were then loaded onto a 5 ml HiTrap Q FF column (Amersham Biosciences, GE Healthcare) equilibrated in Buffer A (50 mM Hepes, 100 mM NaCl, 1 mM EDTA, pH 7.0) to capture FVIIa407C. After washing with buffer A to remove unbound glutathione buffer and Grx2, wash with buffer B (50mM Hepes, 100mM NaCl, 10mM CaCl 2 , pH 7.0) to elute FVIIa 407C in one step. The concentration of FVIIa407C in the eluate was determined by HPLC. In 50mM Hepes, 100M NaCl, 10mM CaCl 2...
Embodiment 3
[0315] Example 3: Synthesis of 38.8kDa HEP-[C]-FVIIa407C
[0316] Synthesis of 38.8k HEP-[C]-FVIIa 407C: Monocysteine-reduced FVIIa 407C (25 mg) was mixed with 38.8K HEP-maleimide (26.8 mg) in 50 mM Hepes, 100 mM NaCl at 5°C , 10mM CaCl2, pH 7.0 buffer solution (8.5ml) for 22 hours. The reaction mixture was then loaded onto a FVIIa-specific affinity column (CV=64ml) modified with a Gla-domain-specific antibody, and first washed with 2 column volumes of buffer A (50mM Hepes, 100mM NaCl, 10mM CaCl 2 , pH 7.4), and then stepwise eluted with two column volumes of buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4). The method essentially follows the principles described by Thim, L et al. Biochemistry (1988) 27, 7785-779. The product with unfolded Gla-domain was collected and applied directly onto a 3x5ml HiTrap Q FF ion exchange column (Amersham Biosciences, GE Healthcare, CV=15ml) pre-equilibrated with 10mM His, 100mM NaCl, pH 7.5. The column was washed with 4 column volum...
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