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Polyethylene glycol modified human serum albumin and preparation method thereof

A human albumin and polyethylene glycol technology, applied in the preparation method of peptides, serum albumin, albumin peptides, etc., can solve problems such as prolonging the dosing cycle, reduce the frequency of dosing, prolong the half-life, and reduce the source of remission nervous effect

Active Publication Date: 2010-05-19
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the research and application of using PEG and its derivatives to modify human albumin to prepare human albumin conjugates to prolong the administration cycle and achieve long-acting administration of albumin.

Method used

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  • Polyethylene glycol modified human serum albumin and preparation method thereof
  • Polyethylene glycol modified human serum albumin and preparation method thereof
  • Polyethylene glycol modified human serum albumin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Site-directed Single Modification of Human Serum Albumin by Modifier mPEG-MAL

[0040]mPEG-MAL (Mr=20kD) and human serum albumin (based on protein content) were dissolved in pH 6.5, 50mmol / L phosphate buffer (containing 10mmol / L EDTA) at a molar ratio of 2:1, shaken in a water bath at 30°C Bed, shake gently for 20h. Take 3 μL of the modified reaction solution for SDS-PAGE detection, see the staining results figure 1 . figure 1 It shows that compared with HSA, a new band appears in the modified reaction solution lane (mixtures lane), and the molecular weight is about 90kD. The molecular weight of the PEG modifier used in the modification reaction is 20kD, and the molecular weight of albumin is 66.5kD, so it can be inferred that the newly appeared band is PEG-cys 34 -HSA, and one HSA molecule is connected with one PEG chain.

Embodiment 2

[0042] mPEG site-directed single-modification product of cysteine ​​34 of albumin (PEG-cys 34 - Separation and purification of HSA)

[0043] The modified product was separated by DEAE Sepharose FF chromatography medium. The modified reaction solution was diluted 5 times with pH 6.5, 10mmol / L phosphate buffer solution, and then loaded. The flow rate is 5.0ml / min, and the detection wavelength is 280nm. The elution curve shows two elution peaks, which are collected separately, desalted and concentrated by ultrafiltration, and the purity of the sample is detected by SDS-PAGE electrophoresis. The electrophoresis film is stained with iodine first and then stained with iodine. Both PEG and protein-bound PEG can be stained; Coomassie brilliant blue staining can stain proteins, and the bands that can be stained by both dyes are PEG-cys 34 -HSA. DEAE chromatograms and SDS-PAGE spectra are shown in figure 2 and image 3 . image 3 Display: DEAE peak 1 (swimming lane 4) in the iod...

Embodiment 3

[0046] PEG-cys 34 -HSA and HSA secondary structure determination

[0047] Determination of PEG-cys by Circular Dichroism (CD) 34 -HSA and the secondary structure of HSA. Weigh 10mgHSA and 9mg PEG-cys respectively 34 -HSA, dissolved in pH 7.4, 50mmol / L phosphate buffer, so that the final protein concentration is 5μmol / L. Use a 1mm colorimetric cell to measure HSA and PEG-cys at 25°C 34 - CD spectrum of HSA in the wavelength range 190-260 nm. The secondary structure of HSA and PEG-cys34-HSA was calculated by random special software. HSA and PEG-cys 34 Circular Dichroism (CD) of -HSA see Figure 4 . Depend on Figure 4 It can be seen that HSA and PEG-cys 34 - The CD spectrum of HSA has a similar peak shape, and the CD spectrum of the two is almost completely overlapped, which shows that the secondary structure of the two is almost completely consistent, and the secondary structure calculated by the random software has further confirmed this (see Table 1 ), suggesting t...

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Abstract

The invention relates to the biomedical technical field, in particular to polyethylene glycol modified human serum albumin and a preparation method thereof. The polyethylene glycol modified human serum albumin comprises sulfydryl of 34-bit cysteine residues on albumin molecules and N-terminal amino or free amino group combined polyethylene glycol. Modifiers adopted by the fixed-point single modification are respectively polyethylene glycol-maleimide and polyethylene glycol-propionaldehyde; modifiers adopted by the free amino group random modification are respectively polyethylene glycol-succinimide active ester or methoxy-polyethylene glycol activated by cyanuric chloride; and the modification rate of the free amino group on the albumin molecules combined with the polyethylene glycol is 3%-7%. Compared with the natural albumin, the polyethylene glycol modified human serum albumin has the excellent characteristics of prolonged half-life period, slow plasma elimination, and the like and is beneficial to reducing the medication frequency and releasing the rare albumin source.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to polyethylene glycol-modified human serum albumin and a preparation method thereof. Background technique [0002] Human serum albumin (human serum albumin, HSA) is a single-chain aglycosylated protein containing 585 amino acid residues. It is the most abundant protein in human plasma, accounting for about 60% of the total plasma protein. [0003] Human albumin is a natural blood product commonly used clinically. It is separated and extracted from the plasma of healthy people. Increased intracranial pressure due to injury, and edema and ascites due to hypoalbuminemia and cirrhosis or nephropathy. Human serum albumin has a wide range of indications and a large dosage (each patient consumes several grams of human serum albumin per day), and has a huge market demand. [0004] In recent years, the supply of human serum albumin is very tight due to the shortage of blood sources an...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K1/18C07K1/107
Inventor 王凤山庞广礼赵婷谭海宁仲立军刘纯慧马山刘欣晏
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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