Development of long-acting fibroblast growth factor-23 antagonist
A fibroblast and growth factor technology, applied in the protein field, can solve the problems of unpredictability, reduced protein activity, inactivation, etc., and achieve the effects of good activity retention, good uniformity, and prolonged in vivo half-life
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Embodiment 1
[0038] Example 1 Preparation of PEG-FGF-23 variant peptide
[0039] 1. Modification reaction: Mix 10 mg of humanized FGF-23 variant peptide dissolved in 10 mL Pipes buffer (25 mM, PH=6.5) with mPEG-maleimide (PEG molecular weight: 5000 Da) at a molar ratio of 1:5, and place in 4 ℃, protected from light for 12 hours. Add 1 mL of 1M glycine to terminate the modification reaction.
[0040] , Separation and purification of modified products: Pass the reaction solution in step 1 through a Sephedax G-25 column (20cm×1.5cm) to desalt: use 25mM Pipes buffer (pH=6.5) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1mL / min, and use 25mM Pipes buffer The solution was eluted and the first eluted peak was collected. The first elution peak corresponds to the sample on the Resource S cation exchange column and is eluted with a gradient of Pipes buffer containing 0-1.0 M NaCl at a flow rate of 5mL / min. Collect each elution peak and use 12% SDS-...
Embodiment 2
[0042] Example 2 Preparation of Polyethylene Glycol-FGF-23 Variant Peptide
[0043] 1. Modification reaction: Mix 10mg of humanized FGF-23 variant peptide dissolved in 10mL Pipes buffer (25mM, PH=6.5) with mPEG-maleimide (PEG molecular weight: 10000Da) at a molar ratio of 1:10, and place in 4 ℃, protected from light for 12 hours. Add 1 mL of 1M glycine to terminate the modification reaction.
[0044] , Separation and purification of modified products: Pass the reaction solution in step 1 through a Sephedax G-25 column (20cm×1.5cm) to desalt: use 25mM Pipes buffer (pH=6.5) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1mL / min, and use 25mM Pipes buffer The solution was eluted and the eluted peaks were collected. Put the elution peak corresponding to the sample on the Resource S cation exchange column and use the Pipes buffer gradient elution containing 0-1.0 M NaCl, the flow rate is 5mL / min, collect each elution peak, and use 1...
Embodiment 10
[0074] Example 10 Preparation of Polyethylene Glycol-FGF-23 Variant Peptide
[0075] 1. Modification reaction: Mix 10 mg murine FGF-23 variant peptide dissolved in 10 mL Pipes buffer (25 mM, PH=6.5) with mPEG-butyraldehyde (PEG molecular weight: 20000 Da) at a molar ratio of 1:5, and place it at 4 °C, avoiding Light reaction for 24 hours. Add 1 mL of 1M glycine to terminate the modification reaction.
[0076] , Separation and purification of modified products: Pass the reaction solution in step 1 through a Sephadex G-25 column (20cm×1.5cm) to desalt: use 25mM Pipes buffer (pH=6.0) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1mL / min, and use 25mM Pipes buffer The solution was eluted and the eluted peaks were collected. Put the elution peak corresponding to the sample on the Resource S cation exchange column and use the Pipes buffer gradient elution containing 0-1.0 M NaCl, the flow rate is 3mL / min, collect each elution peak, ...
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