U.S. patent application number 09/799906 was filed with the patent office on 2002-01-03 for method for large scale plasmid purification.
This patent application is currently assigned to Merck & Co., Inc.. Invention is credited to Lee, Ann L., Sagar, Sangeetha.
Application Number | 20020001829 09/799906 |
Document ID | / |
Family ID | 27402739 |
Filed Date | 2002-01-03 |
United States Patent
Application |
20020001829 |
Kind Code |
A1 |
Lee, Ann L. ; et
al. |
January 3, 2002 |
Method for large scale plasmid purification
Abstract
A process is disclosed for the large scale isolation and
purification of plasmid DNA from large scale microbial
fermentations. All three forms of plasmid DNA; supercoil (form I),
nicked or relaxed circle (form II), and linearized (form III), are
individually isolatable using the disclosed process. Highly
purified DNA suitable for inclusion in a pharmaceutical composition
is provided by the disclosed process.
Inventors: |
Lee, Ann L.; (Lansdale,
PA) ; Sagar, Sangeetha; (Lansdale, PA) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Assignee: |
Merck & Co., Inc.
Rahway
NJ
07065
|
Family ID: |
27402739 |
Appl. No.: |
09/799906 |
Filed: |
March 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09799906 |
Mar 6, 2001 |
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08952428 |
Nov 7, 1997 |
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6197553 |
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08952428 |
Nov 7, 1997 |
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PCT/US96/07083 |
May 15, 1996 |
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PCT/US96/07083 |
May 15, 1996 |
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08446118 |
May 19, 1995 |
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08446118 |
May 19, 1995 |
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08275571 |
Jul 15, 1994 |
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Current U.S.
Class: |
435/91.1 ;
424/184.1; 514/44R |
Current CPC
Class: |
C12N 1/06 20130101; C12N
15/1006 20130101; C12N 15/10 20130101; C12N 15/1017 20130101; C12N
15/101 20130101 |
Class at
Publication: |
435/91.1 ;
514/44; 424/184.1 |
International
Class: |
C12P 019/34; A01N
043/04; A61K 031/70; A61K 039/00 |
Claims
What is claimed is:
1. A process for large scale isolation and purification of plasmid
DNA from large scale microbial cell fermentations, comprising: a)
harvesting microbial cells from a large scale fermentation; b)
adding to the harvested microbial cells a sufficient amount of a
lysis solution; c) heating the microbial cells of step b) to a
temperature between 70.degree. C. and 100.degree. C. in a
flow-through heat exchanger to form a crude lysate; d) centrifuging
the crude lysate; e) filtering and diafiltering the supernatant of
step d) providing a filtrate; f) contacting the filtrate of step e)
with an anion exchange matrix; g) eluting and collecting plasmid
DNA from the anion exchange matrix; h) contacting the plasmid DNA
from step g) with a reversed phase high performance liquid
chromatography matrix; i) eluting and collecting the plasmid from
the reversed phase high performance liquid chromatography matrix of
step h); j) optionally concentrating and/or diafiltering the
product of step i) into a pharmaceutically acceptable carrier; and
k) optionally sterilizing the DNA product.
2. The process of claim 1 wherein the lysis solution of step b) is
modified STET buffer.
3. The process of claim 1 wherein the heating of step c) is to a
temperature between 70.degree. C. and 77.degree. C.
4. The process of claim 1 wherein the lysis solution of step b)
contains a sub-microgram concentration of lysozyme.
5. The process of claim 1 optionally including RNase treatment at
any step following step a).
6. An isolated and purified plasmid DNA obtained by the process of
claim 1.
7. The plasmid DNA of claim 6 wherein said plasmid is suitable for
administration to humans.
8. The plasmid DNA of claim 6 wherein said plasmid is suitable for
administration to non-human animals.
9. The plasmid DNA of claim 6 wherein said plasmid is a
polynucleotide vaccine.
10. An isolated and purified plasmid DNA suitable for
administration to humans.
11. The plasmid DNA of claim 10 wherein said plasmid DNA is a
polynucleotide vaccine.
Description
RELATED APPLICATION
[0001] This is a continuation-in-part application of U.S. Ser. No.
08/446,118 filed May 19, 1995.
BACKGROUND OF THE INVENTION
[0002] The classical techniques for isolating plasmid DNA from
microbial fermentations are suitable for small or laboratory scale
plasmid preparations. One such procedure involves the alkaline
lysis of microbial host cells containing the plasmid, followed by
acetate neutralization causing the precipitation of host cell
genomic DNA and proteins which are then removed by, for example,
centrifugation. The liquid phase contains the plasmid DNA which is
alcohol precipitated and then subjected to isopycnic centrifugation
using CsCl in the presence of ethidium bromide. The ethidium
bromide is required in order to separate the total plasmid DNA into
the three different forms, supercoiled (form I), nicked circle
(form II), and linearized (form III), and the desired plasmid form
is collected. Further extraction with butanol is required to remove
residual ethidium bromide followed by DNA precipitation using
alcohol. Additional purification steps follow to remove host cell
proteins. The removal of host proteins is performed by repeated
extractions using phenol or a mixture of phenol and chloroform. The
plasmid DNA is alcohol precipitated and residual phenol is removed
by repeated isoamyl/chloroform extractions. The final alcohol
precipitated plasmid DNA is dissolved in water or a suitable buffer
solution.
[0003] There are numerous drawbacks and limitations to this process
including:
[0004] a) this process requires the use of expensive and hazardous
chemicals (CsCl and EtBr, which are used within the density
gradient centrifugation; EtBr is a known mutagen and must be
removed from products; also it is an intercalating agent which can
nick the plasmid);
[0005] b) the density centrifugation step is not easily
scaleable;
[0006] c) there is a need for organic solvent extraction to remove
residual EtBr;
[0007] d) phenol extraction is used to remove residual proteins and
DNase, a process that would require a centrifuge to break
phenol/water emulsion;
[0008] e) highly repetitive steps making it laborious and time
consuming (isolation requires several days);
[0009] f) scalability of the chemical lysis step is an obstacle
i.e., lysozyme/alkaline/KOAc treatment step is efficient in lysing
cells on a small scale, however, the increase in viscosity makes
large scale processing very difficult; and
[0010] g) use of large quantities of lysozyme to enzymatically
weaken the microbial cell wall prior to lysis.
[0011] The mixture is then neutralized by addition of acid which
results in precipitation of the high molecular weight chromosomal
DNA. The high molecular weight RNA and protein-SDS complexes
precipitate with the addition of high concentration of KOAc salt.
The plasmid product remains in the clarified supernatant following
centrifugation. Limitations here include the need to process
quickly and on ice in order to retard the activity of nucleases
which are not removed until phenol extraction. The main contaminant
remaining in the supernatant with the product is RNA.
[0012] Another commonly utilized method for isolating and purifying
plasmid DNA from bacteria provides a rapid process suitable for
only very small scale preparations.
[0013] Holmes and Quigley (1981, Analytical Biochem., 114, pp
193-197) reported a simple and rapid method for preparing plasmids
where the bacteria are treated with lysozyme, then boiled at about
100.degree. C. in an appropriate buffer (STET) for 20-40 seconds
forming an insoluble clot of genomic DNA, protein and debris
leaving the plasmid in solution with RNA as the main contaminant.
Lysozyme is apparently a requirement for this technique to work,
and as such adds a treatment step which is less desirable for large
scale manufacture of DNA for human or veterinary use. However, the
addition of lysozyme may enhance plasmid release during lysis. An
advantage is that heat treatment of the cells would also denature
the DNase. However, this technique is not suitable for scale up to
a high volume of microbial fermentations and is meant for
fermentations less than five liters.
[0014] Alternatives to isopycnic centrifugation using CsCl for
plasmid purification have been published. These alternatives are
suitable only for laboratory scale plasmid isolation and
include:
[0015] a) size exclusion chromatography, which is inherently
limited in throughput;
[0016] b) hydroxyapatite chromatography, which has the disadvantage
of requiring high concentrations of urea for efficiency;
[0017] c) reversed phase chromatography; and
[0018] d) ion exchange chromatography.
[0019] Large scale isolation and purification of plasmid DNA from
large volume microbial fermentations, therefore, requires the
development of an improved plasmid preparation process. An
isolation and purification process for large scale plasmid DNA
production is necessitated by recent developments in many areas of
molecular biology. In particular, recent advances in the field of
polynucleotide-based vaccines for human use, and potentially human
gene therapy, requires the ability to produce large quantities of
the polynucleotide vaccine in a highly purified form.
[0020] Unprecedented technology is required for
developing/implementing a large scale commercially viable process
for fermentation, isolation, purification and characterization of
DNA as a biopharmaceutical.
SUMMARY OF THE INVENTION
[0021] The current laboratory method used to isolate and purify
plasmid DNA consists of a series of classical laboratory techniques
that are not suitable for a manufacturing process. For example,
density gradient centrifugations are not scaleable; the
purification procedure necessitates the use of hazardous and
expensive chemicals/solvents such as ethydium bromide, a known
mutagen, and is labor intensive and time consuming. Therefore, a
scaleable alternative process was developed, and is disclosed
herein. In addition, an HPLC assay was established to track the
plasmid product through the process steps and to distinguish
between the plasmid forms. The microbial cells harboring the
plasmid are suspended and optionally incubated with lysozyme in a
buffer containing detergent, heated using a flow-through heat
exchanger to lyse the cells, followed by centrifugation. After
centrifugation the clarified lysate, which contains predominately
RNA and the plasmid product, is filtered through a 0.45 micron
filter and then diafiltered, prior to loading on the anion exchange
column. The plasmid product may optionally be treated with RNase
before or after filtration, or at an earlier or later step. The
anion exchange product fraction containing the plasmid is loaded
onto the reversed phase column, and is eluted with an appropriate
buffer, providing highly pure plasmid DNA suitable for human
use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. A schematic of a suitable heat exchanger apparatus
is shown.
[0023] FIG. 2. The relationship between the outlet temperature and
the flow rate is shown, graphically.
[0024] FIG. 3. Comparative chromatograms of total plasmid in
clarified supernatant with 50 mMEDTA and 100 mMEDTA are shown.
[0025] FIG. 4. The yield of supercoiled plasmid as a function of
exit temperature is shown.
[0026] FIG. 5. The elution profiles of anion exchange columns run
with RNase treated (bold line) and untreated (thin line) clarified
lysates are shown.
[0027] FIG. 6. The elution profiles of anion exchange
chromatography with clarified lysate that was diafiltered before
the column or not diafiltered before the column are shown.
[0028] FIG. 7. An elution profile of plasmid DNA from cell lysate
is shown.
[0029] FIG. 8. An agarose gel electrophoresis analysis of the DNA
product obtained at various intermediate steps of purification is
shown.
[0030] FIG. 9. A tracing of the anion exchange HPLC analysis of the
DNA product demonstrating the purity of the product is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0031] We have identified a novel, scaleable, alternative
lysis/debris removal process for large scale plasmid isolation and
purification which exploits a rapid heating method to induce cell
lysis and precipitate genomic DNA, proteins and other debris while
keeping the plasmid in solution. The utility of this process is the
large scale isolation and purification of plasmid DNA. We have
found that suspending the microbial cells in modified STET buffer
(described below) and then heating the suspension to about
70-100.degree. C. in a flow-through heat exchanger results in
excellent lysis. Continuous flow or batch-wise centrifugation of
the lysate effects a pellet that contains the cell debris, protein
and most of the genomic DNA while the plasmid remains in the
supernatant. This invention offers a number of advantages including
higher product recovery than by chemical lyses, inactivation of
DNases, operational simplicity and scaleability.
[0032] The present invention is drawn to a process for the large
scale isolation and purification of plasmid DNA from microbial
fermentations. Large scale microbial cell fermentations as used
herein are considered to be total cell fermentation volumes of
greater than about 5 liters, or the cells harvested from a
fermentation volume greater than about 5 liters.
[0033] The present invention is also drawn to providing plasmid DNA
in a highly purified form suitable for human use. DNA for human use
includes, but is not limited to, polynucleotide vaccines and DNA
for human gene therapy. Polynucleotide vaccines are intended for
direct injections into humans [Montgomery, D. L. et al., 1993, Cell
Biol., 169, pp. 244-247; Ulmer, J. B. et al., 1993, Science, 259,
pp. 1745-1749].
[0034] The present invention is also drawn to an in-line monitoring
process for the tracking of the various forms of plasmid DNA
through the isolation and purification steps. The various forms of
plasmid DNA referred to above which can be individually isolated by
the process of the present invention are form I (supercoiled
plasmid), form II (nicked or relaxed plasmid), and form III
(linearized plasmid).
[0035] The process of the present invention is suitable for use
with microbial fermentations in general. It is readily apparent to
those skilled in the art that a wide variety of microbial cells are
suitable for use in the process of the present invention, including
but not limited to, fungal cells including yeast, and bacterial
cells. A preferred microbial fermentation is a bacterial
fermentation of cells containing the plasmid to be isolated and
purified. A preferred bacterial fermentation is a fermentation of
E. coli containing the plasmid to be isolated and purified. It is
readily apparent to those skilled in the art that bacterial
fermentations other than E. coli fermentations are suitable for use
in the present invention. The microbial fermentation may be grown
in any liquid medium which is suitable for growth of the bacteria
being utilized.
[0036] The plasmid to be isolated and purified by the process of
the present invention can be any extrachromosomal DNA molecule. The
plasmids can be high copy number per cell or low copy number per
cell. The plasmids can also be of virtually any size. It is readily
apparent to those skilled in the art that virtually any plasmid in
the microbial cells can be isolated by the process of the present
invention.
[0037] Microbial cells containing the plasmid are harvested from
the fermentation medium to provide a cell paste, or slurry. Any
conventional means to harvest cells from a liquid medium is
suitable, including, but not limited to centrifugation or
microfiltration.
[0038] Isolation of the plasmid DNA from harvested microbial cells
using the current lab scale procedures consist mainly of enzymatic
treatment of microbial cells to weaken the cell wall followed by
cell lysis. The purification steps include repetitive CsCl/EtBr
centrifugations followed by organic solvent extractions and
precipitation to remove tRNA, residual proteins, EtBr and other
host contaminants. These steps are not scaleable and therefore not
suitable for use in large-scale processing. In contrast,
preparative scale chromatography is a powerful purification tool
that provides high resolution, operational ease and increased
productivity for purifying DNA plasmid products. Two different
modes of chromatography, reversed phase and anion exchange, show
suitability in purifying DNA plasmid to the stringent levels
required for human use. Separations based on reversed phase are
governed by hydrophobic interactions while those for anion exchange
are based on electrostatic interaction. These two orthogonal
chromatography steps achieve separations between various forms of
plasmid (supercoiled, open relaxed, linear and concatemers) and
remove host contaminants like LPS (endotoxin), RNA, DNA and
residual proteins.
[0039] In the process of the present invention, harvested microbial
cells are resuspended in modified STET buffer which is comprised of
about 50 mM TRIS, about 50-100 mM EDTA, about 8% Sucrose, about 2%
TRITON X-100, and optionally sub-microgram concentrations of
lysozyme, at a pH in the range of 6.0-10.0. The concentration of
lysozyme optionally used in the process of the present invention is
substantially less than the concentration of lysozyme used in the
procedures known in the art. It is readily apparent to those
skilled in the art that modifications of this basic buffer formula
can be made and are suitable for use in the present invention.
Modifications to this basic buffer formula that do not
substantially affect or alter the outcome of the present process
are intended to be within the scope of the process of the present
invention. The pH range may be adjusted according to the best
results provided for the particular strain of bacteria being used.
The preferred pH range is about 8.0-8.5. The suspension is then
heated to about 70-100.degree. C., with about 70-77.degree. C.
preferred, in a flow-through heat exchanger. The lysate is
centrifuged to pellet large cell debris, protein and most genomic
DNA.
[0040] A prototype heat exchanger was built to demonstrate the
feasibility of flow-through heat lysis of microbial cells
containing plasmid. The particular heat exchanger consisted of a 10
ft..times.0.25 inch O.D. stainless steel tube shaped into a coil.
The coil was completely immersed into a constant high temperature
water bath. The hold-up volume of the coil was about 50 mL.
Thermocouples and a thermometer were used to measure the inlet and
exit temperatures, and the water bath temperature, respectively.
The product stream was pumped into the heating coil using a
Masterflex peristaltic pump with silicone tubing. Cell lysate
exited the coil and was then centrifuged in a Beckman J-21 batch
centrifuge for clarification. FIG. 1 provides a schematic of this
particular apparatus, however other types of heat exchanger
construction are suitable for use in the present invention,
including but not limited to a shell and tube construction, which
is preferrable.
[0041] After centrifugation, the clarified lysate can optionally be
treated with RNase, and the plasmid product can be filtered to
further remove small debris. A wide variety of filtration means are
suitable for use in this process, including but not limited to
filtration through a membrane having a small pore size. A preferred
filtration method is filtration through a 0.45 micron filter.
[0042] To further remove contaminants from the DNA product, the
material can be diafiltered. Standard, commercially available
diafiltration materials are suitable for use in this process,
according to standard techniques known in the art. A preferred
diafiltration method is diafiltration using an ultrafilter membrane
having a molecular weight cutoff in the range of 30,000 to 500,000,
depending on the plasmid size. The DNA preparation described above
is diafiltered using an ultrafiltration membrane (about 100,000
molecular weight cutoff) against column buffer prior to loading on
the anion exchange column. Diafiltration prior to the anion
exchange column is preferred, and it greatly increases the amount
of lysate that can be loaded onto the column.
[0043] A wide variety of commercially available anion exchange
matrices are suitable for use in the present invention, including
but not limited to those available from POROS Anion Exchange
Resins, Qiagen, Toso Haas, Sterogene, Spherodex, Nucleopac, and
Pharmacia. The column (Poros II PI/M, 4.5 mm.times.100) is
initially equilibrated with 20 mM Bis/TRIS Propane at pH 7.5 and
0.7 M NaCl. The sample is loaded and washed with the same initial
buffer. An elution gradient of 0.5 M to 0.85 M NaCl in about 25
column volumes is then applied and fractions are collected. Anion
exchange chromatography is an ideal first polishing step because it
provides excellent clearance of RNA, genomic DNA and protein. FIG.
5 (bold) shows a sample elution profile of filtered clarified cell
lysate from the anion exchange column. Agarose gel analysis
revealed that the second peak which appears after the flow-through
is composed of the plasmid product. The earlier large peak is due
to RNA. This is confirmed by incubating the clarified cell lysate
with ribonuclease prior to loading on the column, which showed that
the large peak disappears and is replaced by several smaller more
rapidly eluting peaks, due to the degradation products of
ribonuclease digestion.
[0044] The anion exchange product fraction is loaded onto a
reversed phase column. A wide variety of commercially available
matrices are suitable for use in the present invention, including
but not limited to those available from POROS, Polymer Labs, Toso
Haas, Pharmacia, PQ Corp., Zorbax, BioSepra resins, BioSepra Hyper
D resins, BioSepra Q-Hyper D resins and Amicon. The matrices can
also be polymer based or silica based. The reversed phase column
(Poros R/H), is equilibrated with about 100 mM Ammonium Bicarbonate
at pH 8.5. A gradient of 0-11% isopropanol is then used to elute
bound material. The three forms of plasmids, forms I, II and III
described above, can be separated by this method.
[0045] The eluted plasmid DNA can then be concentrated and/or
diafiltered to reduce the volume or to change the buffer. For DNA
intended for human use it may be useful to diafilter the DNA
product into a pharmaceutically acceptable carrier or buffer
solution. Pharmaceutically acceptable carriers or buffer solutions
are known in the art and include those described in a variety of
texts such as Remington's Pharmaceutical Sciences. Any method
suitable for concentrating a DNA sample is suitable for use in the
present invention. Such methods includes diafiltration, alcohol
precipitation, lyophilyzation and the like, with diafiltration
being preferred. Following diafiltration the final plasmid DNA
product may then be sterilized. Any method of sterilization which
does not affect the utility of the DNA product is suitable, such as
sterilization by passage through a membrane having a sufficiently
small pore size, for example 0.2 microns and smaller.
[0046] The following examples are provided to illustrate the
process of the present invention without, however, limiting the
same thereto.
EXAMPLE 1
Growth of Microbial Cells, Cell Lysis and Clarification
[0047] One liter of frozen E. coli cell slurry was used to make 8
liters of cell suspension in STET buffer (8% sucrose, 0.5% TRITON,
50 mM TRIS buffer, pH 8.5 and 50 mM EDTA). The absorbance of the
cell suspension at 600 nm was about O.D. 30. The suspension was
stirred continuously to ensure homogeneity. The viscosity of the
cell suspension was measured to be about 1.94 cp at room
temperature (24.degree. C.). The cell suspension was pumped through
the heat exchanger at 81 mL/min which corresponded to a residence
time of the cell solution in the heat exchanger of about 35
seconds. The bath temperature was maintained at 92.degree. C. The
inlet and outlet temperatures of the cell solution were measured to
be about 24.degree. C. and about 89.degree. C. (average),
respectively. About 1 liter of sample was run through the heat
exchanger. No visible clogging of the tube was observed although
the lysate was somewhat thicker than the starting material. The
lysate was cooled to room temperature and its viscosity was
measured to be about 40 cp. The cell lysate was clarified by batch
centrifugation at 9000 RPM for 50 minutes using the Beckman J-21.
Analysis of the supernatant confirmed effective cell lysis and
product recovery. The product yield produced by flow-through heat
lysis was at least comparable to that made by the Quigley &
Holmes boiling method. The latter method; however, must be carried
out at the laboratory scale in batch mode and is therefore
unsuitable for large-scale (5 liters or greater) processing. Since
the heat exchanger process is flow-through, there is no maximum
limit to the volume of cell suspension that can be processed. This
process can therefore accomodate very large scale fermentations of
bacteria to produce large quantities of highly purified plasmid
DNA.
[0048] The clarified lysate was then filtered through a membrane
having a pore size of 0.45 microns to remove finer debris. The
filtrate was then diafiltered using a membrane having a molecular
weight cutoff of about 100,000.
EXAMPLE 2
Control and Reproducibility of Cell Lysis with the Heat
Exchanger
[0049] Adjusting the flowrate (i.e., residence time) at which the
cell slurry is pumped through the heat exchanger permits tight
control of the temperature of lysis, i.e., the outlet temperature.
A cell slurry solution was prepared as described in Example 1 and
pumped through the heat exchanger at flow rates ranging from 160 to
850 mL/min. The corresponding outlet temperatures ranged between
93.degree. C. and 65.degree. C., respectively. FIG. 2 illustrates
the relationship between flow rate and temperature. The initial
temperature of the cell slurry was 24.degree. C. and the bath
temperature was kept constant at 96.degree. C. In addition, a
number of runs were performed where an outlet temperature of
80.degree. C. was targeted. Yields of 24 mg of circular DNA per L
of clarified supernatant were consistently obtained demonstrating
the reproducibility of the process.
EXAMPLE 3
Purification of Plasmid DNA
[0050] Microbial cells and lysates were prepared as described in
Examples 1 and 2, and the following analyses were performed.
[0051] To illustrate that the addition of 100 mM EDTA vs 50 mM EDTA
increased the percentage of supercoiled DNA, and to determine an
acceptable range of outlet temperatures (i.e., lysis temperature)
with respect to recovery of supercoiled DNA, the following analyses
were performed. The supercoiled form of plasmid DNA is desirable
since it is more stable than the relaxed circle form. One way that
supercoiled DNA can be converted to open circle is by nicking with
DNase. We found that the addition of 100 mM EDTA vs 50 mM in the
STET buffer minimized the formation of open circle plasmid. FIG. 3
shows comparative chromatograms of the total plasmid in the
clarified supernatant with 50 mM EDTA vs 100 mM EDTA. The cell
suspension was prepared as described in Example 1. The operating
flow rate for these runs was approximately 186 ml/min. The
temperatures of the inlet, outlet and bath are 24.degree. C.,
92.degree. C. and 96.degree. C. respectively.
[0052] An acceptable range of lysis temperatures was determined by
measuring the percentage of supercoiled plasmid generated for each
run. FIG. 4 illustrates the concentration of supercoiled plasmid as
a function of exit temperature. An acceptable range of lysis
temperatures is between 75.degree. C. and 92.degree. C. At
temperatures below 75.degree. C., more relaxed circle plasmid was
generated, most likely due to increased DNase activity. Above
93.degree. C., the levels of supercoiled plasmid appear to
diminish, possibly due to heat denaturation.
[0053] Following continuous heat lysis and centrifugation, 1 mL of
clarified lysate was either incubated with 5 .mu.g RNase for 2
hours, or was used untreated. The RNase treated and untreated
samples were then loaded onto an anion exchange column (Poros Q/M
4.6.times.100) that had been previously equilibrated with a 50-50
mixture of solvents A and B [HPLC solvent A: 20 mM Tris/Bis
Propane, pH 8.0; and solvent B: 1 M NaCl in 20 mM Tris/Bis Propane,
pH 8.0]. The column was eluted using a gradient of 50% to 85% B run
over 100 column volumes. Open circle plasmid elutes at
approximately 68% B and supercoiled elutes at 72% B.
[0054] A comparison of the anion exchange column eluate from
clarified lysate treated with RNase (thin line) and untreated
(thick line) is shown in FIG. 5. The peak at about 10 minutes is
plasmid DNA, and is followed by a large peak in the untreated
sample which is RNA. In the RNase treated sample the large RNA peak
has been eliminated and a greater separation of the plasmid peak
from contaminant peaks is produced.
[0055] As described above, diafiltration prior to anion exchange
chromatography greatly increases the amount of lysate that can be
loaded onto the column. This is demonstrated in FIG. 6 which shows
a comparison of clarified lysate which was diafiltered and
clarified lysate which was not diafiltered prior to anion exchange
chromatography. Samples were prepared as described above except
that one sample was diafiltered before loading onto the anion
exchange column, and the other sample was not diafiltered. The
column was run and eluted as described above. FIG. 6 shows that the
amount of contaminant material eluted from the column is vastly
greater in the sample that was not diafiltered. The large amount of
contaminating material which binds the anion exchange column matrix
can overwhelm the maximunm capacity of the column causing loss of
DNA product because of the unavailability of the matrix to bind any
more material. Therefore, diafiltration removes contaminants and
allows more of the DNA product to bind the anion exchange matrix,
and in turn allows a greater volume of clarified lysate to be
loaded onto the column.
[0056] The plasmid DNA eluted from the anion exchange column was
separated into the individual forms by reversed phase HPLC
analysis. The separation of supercoiled plasmid (form 1) from
nicked circle (form 2) is shown in FIG. 7. The two forms were
easily separated and allowed the isolation of individual forms of
the plasmid.
EXAMPLE 4
Highly Purified Plasmid DNA From a Chromatography-based Process
[0057] A fermentation cell paste was resuspended in modified STET
buffer and then thermally lysed in a batchwise manner.
Alternatively a fermentation cell paste is resuspended in modified
STET buffer and then thermally lysed in the flow-through process
described above. The lysate was centrifuged as described above.
Twenty ml of the supernatant were filtered as described above and
loaded onto an anion exchange column (Poros Q/M 4.6.times.100) that
was previously equilibrated with a 50-50 mixture of buffers A and B
described above. A gradient of 50% to 85% B was run over 50 column
volumes with a flow rate of 10 ml/minute. Fractions of 2.5 ml each
were collected from the column. The supercoiled plasmid DNA eluted
from the column at 72% B.
[0058] The anion exchange product was then loaded onto a reversed
phase chromatography column (Poros R/H) which had been previously
equilibrated with 100 mM ammonium bicarbonate at pH 8.0, and a
gradient of 0% to 80% methanol was used to elute the bound
material. The highly purified supercoiled plasmid DNA eluted at 22%
methanol.
[0059] An agarose gel of the product fractions from each of the
major steps of the purification processis shown in FIG. 8. Based on
the agarose gels and the colorimetric and HPLC assays described in
Example 3, the final product, shown in FIG. 9, is highly pure. The
product consists of greater than 90% supercioled and less than 10%
open circle plasmid. RNA was below the limits of detection of the
assay used. Genomic DNA and protein contaminant levels were also
below the limits of detection in the assays used. The overall
supercoiled plasmid yield at the end of the process was
approximately 60% of the supercoiled plasmid in the clarified
lysate. EXAMPLE 5
Multi-Gram Scale Purification of Plasmid DNA
[0060] 4.5 L of frozen E. coli cell slurry was used to make 33.7 L
of cell suspension in STET buffer (8% sucrose, 2% Triton, 50 mM
Tris buffer, 50 mM EDTA, pH 8.5) with 2500 units/ml of lysozyme.
The absorbance of the suspension at 600 nm was about O.D. 30. The
suspension was stirred at room temperature for 15 minutes to ensure
proper mixing and then was incubated for 45 minutes with continuous
stirring at 37.degree. C. Following incubation, mixing was
continued at room temperature and the cell suspension was pumped
through the heat exchanger at a flowrate of 500 ml/min. The batch
temperature was maintained at 100.degree. C. and the inlet and
outlet temperatures of the cell suspension were measured to be
about 24.degree. C. and between 70-77.degree. C., respectively. The
cell lysate exiting the heat exchanger was collected in Beckman
centrifuge bottles (500 mls each) and the material was centrifuged
immediately in Beckman J-21 centrifuges for 50 minutes at 9000 RPM.
Following centrifugation, the supernatant was found to contain 4-5
times more plasmid product than in the case without lysozyme
incubation. The supernatant product of the centrifugation was
immediately diafiltered against 3 volumes of TE buffer (25 mM
Tris-EDTA at pH 8.0) and then incubated with 20.times.10.sup.5
units of E. coli RNase for 2-4 hours at room temperature. After
completion of the incubation, the product solution was then
diafiltered an additional 6 volumes with TE buffer using a 100 kD
MWCO membrane and then filtered through a 0.45 micron filter to
remove residual debris. The filtered lysate was diluted to 0.7 M
NaCl with a 20 mM Bis/Tris Propane-NaCl buffer at pH 7.5, which
prepares the diluted filtrate for loading onto the anion exchange
column. The anion exchange column (3.6 L of POROS PI/M) was
previously equilibrated with 20 mM Bis/Tris Propane and 0.7M NaCl.
The filtered lysate was loaded to column capacity. In this case 5
grams of supercoiled plasmid was loaded onto the anion exchange
column. After loading, the column was washed with 2-4 column
volumes of 20 mM Bis/Tris Propane and 0.7 M NaCl. A 10 column
volume gradient from 0.7 M NaCl to 2.0 M NaCl in 20 mM Bis/Tris
Propane was performed to clear most of the E. coli protein. RNA and
some endotoxin. The supercoiled plasmid fraction eluted between 1.4
M and 2.0 M NaCl. The supercoiled fraction from the anion exchange
column, which contained 4 grams of supercoiled plasmid was then
diluted 2-3 times with pyrogen free water, adjusted to 1.2% IPA and
pH adjusted to 8.5 with 1 N NaOH. The diluted anion exchange
supercoiled fraction was then loaded onto a 7 L reversed phase
column (POROS R2/M) which had been previously equilibrated with 100
mM Ammonium Bicarbonate containing 1.2% IPA. In this case, 3.2
grams of supercoiled plasmid were loaded onto the reversed phase
column and then the column was washed with 6-10 column volumes of
1.2% IPA in 100 mM Ammonium Bicarbonate. This extensive wash was
performed to clear impurities. Next, a gradient of 1.2% IPA to
11.2% IPA in 5 column volumes was performed. The supercoiled
plasmid fraction elutes at about 4% IPA. The supercoiled product
fraction from the reversed phase column was then concentrated and
diafiltered into normal saline using a 30 kD MWCO membrane. The
final product bulk was filtered through a 0.22 micron filter. Table
1 provides a purification table describing clearance of impurities
and yields across each of the major process steps. The overall
product yield of the process was more than 50% of the supercoiled
plasmid in the clarified cell lysate as indicated by the anion
exchange HPLC assay described in EXAMPLE 3. The purity of the
product was very high with less than 1% E. coli RNA and protein,
and less than 2.9% E. coli genomic DNA.
1TABLE 1 MULTI-GRAM PURIFICATION AND RECOVERY SUMMARY Plasmid
genomic Product % Step DNA Protein RNA LAL STEP (mg) Yield (mg/mg)
(mg/mg) (mg/mg) Eu/mg clarified lysate 6750 100 0.52 7.6 196 1.1
.times. 10.sup.7 concentration/RNase/ 6500 93 0.50 1.6 2.21 3.4
.times. 10.sup.6 diafiltration/dead-end filtration anion exchange
4000 of 80 0.41 0.3 0.1 1.2 .times. 10.sup.4 5000 reversed phase
2300 of 77 0.029 <0.01.dagger-dbl. <0.01.dagger-dbl. 62 3200
concentration 2110 100 0.029 <0.01.dagger-dbl.
<0.01.dagger-dbl. 2.8 diafiltration into final buffer final
process yield 54 .dagger-dbl.below detection limits of assay
method
* * * * *