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Purification process for piasmid DNA

一种质粒、溶胞产物的技术，应用在DNA制备、重组DNA技术、生物化学设备和方法等方向，能够解决可能性没被到认识到等问题

Active Publication Date: 2008-11-19
MERCK SHARP & DOHME BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Positively charged polymeric particles have also been used for flocculation of E. coli cell debris (Kim, C.W., et al., "Removal of Cell and Cell Debris by Electrostatic Adsorption of Positively Charged Polymeric Particles," Flocculation in Biotechnology and Separation Systems, Ed. Y. A. Attia, Amsterdam: Elsevier, 1987, 429-439), however, the possibility of designing an adjustable process using polymeric flocculants to clarify the lysate was not recognized

Method used

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  • Purification process for piasmid DNA
  • Purification process for piasmid DNA
  • Purification process for piasmid DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0099] Flocculation of host cell debris with PEG

[0100] Analytical Methods - Analytical methods used included: (i) anion-exchange ("AEX") HPLC assay; (ii) gel electrophoresis with E-gel (Invitrogen; 0.8% agarose with ethidium bromide) and (iii) qPCR using Taq man PCR assay. The E-gel was run at 60 volts for 50 minutes with a loading volume of 20 [mu]L per well. Samples were pretreated with ethanol precipitation (2 equal volumes) and centrifuged for 5 minutes in an Eppendorf microcentrifuge before performing the E-gel assay. The supernatant was decanted and the pellet was allowed to dry for 5 min before resuspending in 10 mM Tris buffer pH 8.0. The pretreated samples were then diluted in IX TAE buffer before loading onto the E-gel. The same pretreatment was performed for the samples used for qPCR analysis.

[0101] Lysis with Lysozyme - An initial E. coli lysis study was performed to assess the effect of lysozyme, base / acid treatment (addition of base to pH 12-13 followed...

Embodiment 2

[0111] Plasmid DNA purification after lysozyme incubation, alkali / acid treatment and PEG flocculation

[0112] Analytical Methods - See Example 1 for methods.

[0113] 30 L Lysis: Frozen cells containing supercoiled DNA collected from a 2,000 L E. coli fermentation were thawed in a warm (approximately 35 °C) water bath and processed in a 50 L Lee tank with a 5-inch A310 impeller using STET buffer ( 50mM Tris HCl, 100mM EDTA, 2% v / v X-100, 8% w / v sucrose, pH 8.2) diluted to OD 600 70. A resuspension volume of 30 L was obtained. Add Ready-Lyse TM Lysozyme (500 U / mL, Epicentre), and the resulting cell slurry was incubated at 37°C for ca. 2 hours. The cell slurry was then cooled to 20°C and 5M NaOH was slowly added subsurface over 60 minutes to raise the pH of the lysozyme lysate to ca. 12. After a 30-minute rest period, then over 60 minutes 2.5M acetic acid was added to lower the pH to 8-9. PEG 3000 (50% w / v) in RCM 6 (saline, 150 mM NaCl) was then slowly added to a final...

Embodiment 3

[0125] Final DNA polishing treatments: PEG-precipitation and microfiltration of PEG precipitate

[0126] One prior DNA purification method described in U.S. Patent Application No. 09 / 875,379 (U.S. Publication No. US2002 / 0012990) utilized an ultrafiltration ("UF") step at the end of the method to separate calcium silicate (e.g., LRA ) filtrate was concentrated to ~7+ mg / mL DNA, followed by 10X diafiltration for buffer exchange into formulation buffer, PBS (see Figure 6 A), said patent application number is incorporated herein by reference. This final finishing step is satisfactory for volumes of about 100 L or less, but larger production scale purification processes show pump sizes and flow rates starting to reach the limits of the technology. To avoid these scale-up problems, the final UF step of the plasmid DNA method was replaced with a PEG precipitation step, followed by two separate filtration / diafiltration steps (see Figure 6 B). The first filtration / diafiltration st...

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Abstract

Methods of isolating clinical-grade plasmid DNA from manufacturing processes, including large-scale fermentation regimes, are disclosed which encompass alternatives to two core unit operations common to plasmid DNA purification processes. The novel upstream and downstream purification processes disclosed herein provide for reduced production costs and increase process robustness. Either or both of the purification processes disclosed herein may be used in combination with additional purification steps known in the art that are associated with DNA plasmid purification technology.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application 60 / 648,670, filed January 31, 2005, which is incorporated herein by reference. field of invention [0003] The invention relates to a method for isolating clinical-grade plasmid DNA, including a large-scale fermentation method from a production process, including two optional core unit operations for purifying plasmid DNA. The novel upstream and downstream purification processes disclosed here serve to reduce product cost and increase process stability. The novel upstream purification process described in this invention partly involves two steps of lysis / lysate clarification: first generating host cell lysate containing plasmid DNA, followed by flocculation of host cell debris for clarification. As described in another part of the present invention, a novel downstream purification process involves the precipitation of plasmid DNA from host cell lysate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12N15/1017C12N15/1003C12N1/06
Inventor D·B·博伊德A·J·克里斯托佩特R·J·兰德J·C·墨菲M·A·温特斯
Owner MERCK SHARP & DOHME BV
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