Method of preparation of pharmaceutically grade plasmid DNA

A plasmid-level technology, applied in the production and separation of pharmaceutical-grade plasmid DNA, can solve the problems of difficulty in plasmid DNA and inability to obtain nucleic acids in large quantities

Inactive Publication Date: 2006-12-20
AVENTIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] However, using the conventional method, there is a problem that a sufficiently high-purity nucleic acid, particularly plasmid DNA, cannot be obtained in large quantities

Method used

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  • Method of preparation of pharmaceutically grade plasmid DNA
  • Method of preparation of pharmaceutically grade plasmid DNA
  • Method of preparation of pharmaceutically grade plasmid DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] The adjustment of diameter to flow rate used in the coils of the continuous lysis system is based on the calculation of Reynolds numbers. Because the following analysis assumes Newtonian behavior of the fluid, the numbers reported below are only fully valid in B1a and to some extent in B2.

[0209] The value of the Reynolds number allows one skilled in the art to determine the type of behavior encountered. Here, we will only focus on fluid flow in pipes (hydraulic engineering).

[0210] 1) Non-Newtonian fluid

[0211] The two most commonly encountered non-Newtonian fluids in industry are Bingham and Ostwald de Waele.

[0212] In this case, the Reynolds number (Re) is calculated as follows:

[0213] ReN is the generalized Reynolds number

[0214] ReN=(1 / (2 n-3 ))×(n / 3n+1) n ×((ρ×D n ×w 2-n ) / m) (1)

[0215] D: Inner diameter of the cross section (m)

[0216] ρ: volume mass of fluid (volum nass) (kg / m 3 )

[0217] w: space velocity of the fluid (m / s)

[0218]...

Embodiment 2

[0258] We can decompose the CL system into 5 steps: In a specific implementation, the structure is as follows:

[0259] 1) Mixing: cells (in solution 1) + solution 2 (M1 + 3m of 6mm tubing). Begin cell lysis by SDS, there is no risk of fragmentation as long as the DNA is not denatured.

[0260] 2) End of lysis and denaturation of gDNA (13m, 16mm tubing).

[0261] 3) Mixing: lysate + solution 3 (M2 + 3m, 6mm tubing).

[0262] 4) Harvest the neutralized lysate at 4°C

[0263] 5) Sediment flocs and large gDNA fragments overnight at 4°C

[0264] The following conditions can be used to perform sequential lysis:

[0265] - Solution 1: EDTA 10 mM, Glucose (Glc) 9 g / l and Tris HCl 25 mM, pH 7.2.

[0266] - Solution 2: SDS 1% and NaOH 0.2N.

[0267] - Solution 3: acetic acid 2M and potassium acetate 3M.

[0268] - Flow rate 60 l / h: solution 1 and solution 2

[0269] - Flow rate 90 l / h: solution 3.

[0270] - The cells were adjusted to 38.5 g / l with solution 1.

[0271] Cells ...

Embodiment 3

[0287] The column used was a 1 ml HiTrap column, activated with NHS (N-hydroxysuccinimide, Pharmacia), connected to a screw-type compression pump (output < 1 ml / min. The specific oligonucleotide used had NH2 at the 5' end The group, the sequence of which is as follows: 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID NO: 1)

[0288] The buffers used in this example are as follows:

[0289] Coupling buffer: 0.2M NaHCO3, 0.5M NaCl, pH 8.3.

[0290] Buffer A: 0.5M ethanolamine, 0.5M NaCl, pH 8.3.

[0291] Buffer B: 0.1M acetic acid, 0.5M NaCl, pH 4.

[0292] The column was washed with 6 ml of 1 mM HCl, then the oligonucleotide diluted in coupling buffer (50 nmol in 1 ml) was applied to the column for 30 minutes at room temperature. The column was washed three times with 6 ml buffer A followed by 6 ml buffer B. The oligonucleotides are thus covalently bound to the column via CONH linkages. The column is stored at 4°C in PBS, 0.1% NaN3 and can be used at least 4 times.

[0293] The fo...

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Abstract

This invention provides a process for the continuous alkaline lysis of a bacterial suspension in order to harvest pDNA. It further provides for optional additional purification steps, including lysate filtration, anion exchange chromatography, triplex affinity chromatography, and hydrophobic interaction chromatography. These optional purification steps can be combined with the continuous lysis in order to produce a highly purified pDNA product substantially free of gDNA, RNA, protein, endotoxin, and other contaminants.

Description

field of invention [0001] The present invention relates to the preparation of highly purified plasmid DNA (pDNA), in particular to the production and isolation of pharmaceutical grade plasmid DNA for use in plasmid-based therapies. Background of the invention [0002] Developments in molecular biology have clearly demonstrated that plasmid-based therapies, particularly in the areas of vaccines and human gene therapy, are effective avenues for treating disease. However, a significant obstacle to this technique is finding efficient ways to deliver the gene of interest into cells. A promising method to safely and efficiently deliver normal genes into human cells is through plasmid DNA. Plasmid DNA is a closed, circular piece of bacterial DNA into which a DNA sequence of interest can be inserted. Once delivered to a human cell, the pDNA begins to replicate and produces a copy of the inserted DNA sequence. Thus, researchers see plasmid DNA as a promising vehicle for delivering...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/06C12N15/10A61K48/00
CPCA61K48/0091C12N1/06C12N15/1003C12M47/06
Inventor 弗朗西斯·布兰奇米歇尔·库德尼古拉斯·梅斯特拉利戴维·盖拉克蒂里·吉尔明
Owner AVENTIS PHARMA INC
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