Chemical synthesis method of single-chain antibody fragment and product obtained by chemical synthesis method

Abstract

The invention discloses a novel method for complete chemical synthesis of protein of a single-chain antibody fragment (scFv). The obtained product and a biological counterpart thereof are similar in structure and equivalent in function, but have the advantages that the product does not contain impurities, and the properties are more repeatable. The method comprises the general steps of: (a) respectively synthesizing subsequences (peptide fragments); (b) sequentially assembling the subsequences obtained in the step (a) according to the appearing sequence of the subsequences in the target scFv amino acid sequence; and (c) subjecting the assembled intact peptide molecules obtained in step (b) to oxidative folding and dialyzing the resulting product in a buffer solution.

Claims

1. A method for synthesizing a single-chain variable antibody fragment (scFv) whose amino acid sequence (target sequence) comprises n cysteine ​​residues separated by a subsequence of m amino acids, the process comprising the following steps: a. synthesizing all of the subsequences separately, including the cysteine ​​residue preceding it, if any; b. assembling the subsequences obtained in step a. in the order in which the subsequences appear in the target amino acid sequence; c. The assembled structure obtained in step b. is subjected to oxidative folding, and the resulting scFv is dialyzed in a buffer solution.

2. The method according to claim 1, wherein n is 1-10, preferably 2-6, and m is 0-100, preferably 5-80.

3. The method according to claims 1-2, wherein the target sequence comprises at most 350 amino acids, more preferably 100 to 300 amino acids, even more preferably 200 to 300 amino acids.

4. The method according to claims 1-3, wherein step a. can be performed by manual or automated peptide synthesis.

5. The method according to claim 4, wherein the manual peptide synthesis comprises condensation on a resin, and the automated peptide synthesis is peptide chain elongation performed on a peptide synthesizer.

6. The method according to claim 1, wherein: In step a.: (a) the preceding cysteine ​​residue is present as its N-protected precursor; and (b) the final carboxyl group of the resulting subsequence is further converted to a thioester form. 7 . The method according to claim 6 , wherein the thioester is a phenylthioester, and the N-protected cysteine ​​precursor is an N-protected cyclized cysteine.

8. The method according to claims 1 to 7, wherein in step b., the assembly is performed by native chemical ligation followed by restoration of the original cysteine ​​residue if it is present in its N-protected precursor.

9. The method of claims 1-8, wherein the native chemical ligation comprises: (a) dissolving the subsequences to be ligated in a ligation buffer; (b) stirring overnight; (c) terminating the reaction; (d) adjusting the pH of the reaction mixture to a range of 3-5; and (e) purifying the resulting mixture.

10. The method according to claim 9, wherein: In (a), the ligation buffer is a phosphate buffer at pH 7 to which a mixture of mercaptophenylacetic acid, guanidine hydrochloride, tris(2-carboxyethyl)phosphine hydrochloride and ascorbic acid or its salts is added; in (c), the termination is carried out by adding mercaptoalkanesulfonate, optionally followed by hydroxylamine; step (d) is carried out by adding HCl.

11. The method according to claims 1 to 10, wherein the oxidative folding is performed by adding the peptide to be folded into a folding buffer, followed by stirring at room temperature for 1 day.

12. The method according to claim 11, wherein the folding buffer is a Tris buffer added with trehalose and guanidine hydrochloride, with a final pH range of 7-9.

13. The method according to claims 1-12, wherein in step c., the dialysis is performed in a buffer having a pH of 4.6 to 8.0, preferably a pH of 7.4±0.

5. The method according to claim 13 , wherein the buffer is phosphate buffer.

15. The method according to claims 1-14, wherein step c. comprises further dialysis in water.

16. The process according to claims 1-15, wherein the final product obtained from step c. is purified by HPLC.

17. The method of claims 1-16, wherein the target sequence is SEQ.ID.NO: 1 (scFV D2B), SEQ.ID.NO: 2 (scFV trastuzumab), or SEQ.ID.NO: 3 (scFV adalimumab).

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