Pharmaceutical composition for treating colorectal cancer and application thereof
A composition and drug technology, applied in the field of chemical medicine, can solve problems such as unsatisfactory curative effect, and achieve the effect of good therapeutic effect
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Embodiment 1
[0093] Example 1: Preparation of PEGylation reaction mixture
[0094] Take IL-2 protein (provided by Beijing Jiankai Technology Co., Ltd.), exchange it into 5mM PB pH 7.0 buffer, and add PEG modifier (provided by Beijing Jiankai Technology Co., Ltd.) according to a certain reaction molar ratio, Mix well, shake the reaction at room temperature for 2 hours, and check the preparation of the reaction mixture by HPLC after the reaction. The result is as figure 1 and shown in Table 1.
[0095] The structure of the PEG modifier used is shown below, with a molecular weight of 20,000 Dalton:
[0096]
[0097] Table 1 Component analysis of the reaction mixture (UV-0217-0002)
[0098]
[0099] The results showed that 2PEG-IL2 accounted for about 37%.
Embodiment 2
[0100] Example 2: Separation of 2PEG-IL2 by CM chromatography
[0101] The reaction mixture obtained in Example 1 was diluted with water while the pH was adjusted to 3.0 and the conductivity was 1.5 mS / cm, and the samples were loaded into the equilibrated CM column through the AKTA chromatography system, with 10 mM pH 3.0 sodium acetate buffer as A Phase, taking 10mM sodium acetate buffer containing 1M NaCl pH 3.0 as phase B, adopting phase B phase gradient elution, collect the elution fraction when the concentration of phase B is 8%, and analyze the purity by HPLC, that is, 2 PEG-IL2 samples.
[0102] The separated samples of 2PEG-IL2 were exchanged into 5mM PB pH 7.0 buffer using a G25 desalting column for use.
[0103] CM chromatographic separation pattern such as figure 2 shown. Using CM ion-exchange column chromatography resin, the samples coupled with different PEGs were separated by elution with increasing salt concentration. The peaks from left to right were 3PEG-I...
Embodiment 3
[0104] Example 3: alpha affinity chromatography
[0105] Specific steps are as follows:
[0106] (1) Equilibrate the filler with 5mM PB pH7.0 buffer;
[0107] (2) loading the sample obtained in Example 2 into the affinity filler;
[0108] (3) Elution is carried out with the configured eluent, and the elution samples are collected;
[0109] (4) Using the G25 desalting column to exchange the collected elution samples into 5mM PB pH7.0 buffer;
[0110] (5) Concentrate the sample obtained in step (4), filter and sterilize it through a 0.22 μm filter, and check the purity by HPLC.
[0111] 2PEG-IL2 sample purity analysis map such as image 3 As shown, the final desired 2PEG-IL2 was obtained.
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