U.S. patent application number 13/925189 was filed with the patent office on 2013-12-12 for methods and devices for producing biomolecules.
This patent application is currently assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH. The applicant listed for this patent is Christine ASCHER, Roman NECINA, Jochen URTHALER, Helga WOEHRER. Invention is credited to Christine ASCHER, Roman NECINA, Jochen URTHALER, Helga WOEHRER.
Application Number | 20130331560 13/925189 |
Document ID | / |
Family ID | 34108324 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130331560 |
Kind Code |
A1 |
URTHALER; Jochen ; et
al. |
December 12, 2013 |
METHODS AND DEVICES FOR PRODUCING BIOMOLECULES
Abstract
A scalable process and device for producing a biomolecule, in
particular pharmaceutical grade plasmid DNA. The process includes
the steps of alkaline lysis and a neutralization. For separating
the lysate and the precipitate, the mixture is allowed to gently
flow downward through a clarification reactor that is partially
filled, in its lower part, with retention material like glass
beads, whereby the precipitate is retained on top of and within the
retention. In a preferred embodiment of the lysis step, cell
suspension and alkaline lysis solution flow through a lysis reactor
that is filled with particulate material like glass beads. The
process can be run continuously and fully automated.
Inventors: |
URTHALER; Jochen; (Maria
Enzersdorf, AT) ; NECINA; Roman; (Vienna, AT)
; ASCHER; Christine; (Wein, AT) ; WOEHRER;
Helga; (Modling, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
URTHALER; Jochen
NECINA; Roman
ASCHER; Christine
WOEHRER; Helga |
Maria Enzersdorf
Vienna
Wein
Modling |
|
AT
AT
AT
AT |
|
|
Assignee: |
BOEHRINGER INGELHEIM INTERNATIONAL
GMBH
Ingelheim am Rhein
DE
|
Family ID: |
34108324 |
Appl. No.: |
13/925189 |
Filed: |
June 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10806346 |
Mar 23, 2004 |
8501402 |
|
|
13925189 |
|
|
|
|
60472729 |
May 23, 2003 |
|
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Current U.S.
Class: |
536/25.41 |
Current CPC
Class: |
C12N 15/1006
20130101 |
Class at
Publication: |
536/25.41 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 2003 |
EP |
03006568.4 |
Claims
1-39. (canceled)
40. A method of purifying a polynucleotide of interest from host
cells comprising the polynucleotide of interest using a device,
wherein the device comprises a lysis reactor, a neutralization
reactor and a clarification reactor fluidly connected to one
another, the method comprising: (a) providing a cell suspension of
the host cells that have been cultivated to produce the
polynucleotide of interest, wherein the cell suspension is a
fermentation broth within which the host cells were cultivated or a
re-suspension of the cultivated host cells that were harvested from
the fermentation broth; (b) introducing a flow of the cell
suspension and a flow of a lysis solution into the lysis reactor,
the lysis reactor containing filling elements made of glass,
plastic, stainless steel or fibrous material, such that the flow of
the cell suspension and the flow of the lysis solution through the
lysis reactor filling elements provides homogenous mixing of the
flows in the absence of shear forces and whereby the cultivated
host cells of the flowed suspension are substantially disintegrated
by alkaline lysis alone to produce a lysed solution; (c)
neutralizing the cell solution via the neutralization reactor,
wherein the lysed cell solution is mixed with a neutralization
solution to produce a mixture comprising a lysate and a precipitate
comprising cellular debris and impurities, and wherein the lysate
contains the polynucleotide of interest; (d) introducing the
mixture comprising the precipitate and the lysate into the
clarification reactor and mixing slowly with a stirrer or
introducing gas through a distributor from the top or from an inlet
in the bottom of the clarification reactor; (e) purifying the
polynucleotide of interest, where the polynucleotide of interest is
purified from the lysate that flows out of the clarification
reactor, wherein said method is operated on a manufacturing
scale.
41. The method of claim 40, wherein in step d), the mixing is
carried out by introducing air through a distributor from the top
or from an inlet in the bottom of the reactor.
42. The method of claim 41, wherein in step d), the mixing is
carried out by introducing air through an inlet in the bottom of
the reactor.
43. The method of claim 40, wherein the device is an automated or
semi-automated system.
44. A method of purifying a polynucleotide of interest from host
cells comprising the polynucleotide of interest using device,
wherein the device comprises a lysis reactor and a clarification
reactor fluidly connected to one another, the method comprising:
(a) providing a cell suspension of the host cells that have been
cultivated to produce the polynucleotide of interest, wherein the
cell suspension is a fermentation broth within which the host cells
were cultivated or a re-suspension of the cultivated host cells
that were harvested from the fermentation broth; (b) introducing a
flow of the cell suspension and a flow of a lysis solution into the
lysis reactor, the lysis reactor containing filling elements made
of glass, plastic, stainless steel or fibrous material, such that
the flow of the cell suspension and the flow of the lysis solution
through the lysis reactor filling elements provides homogenous
mixing of the flows in the absence of shear forces and whereby the
cultivated host cells of the flowed suspension are substantially
disintegrated by alkaline lysis alone to produce a lysed solution;
(c) introducing the lysed cell solution into the clarification
reactor; (d) neutralizing the cell solution, wherein the lysed cell
solution is mixed with a neutralization solution to produce a
mixture comprising a lysate and a precipitate comprising cellular
debris and impurities, and wherein the lysate contains the
polynucleotide of interest; (e) mixing slowly with a stirrer or
introducing gas through a distributor from the top or from an inlet
in the bottom of the clarification reactor; (f) purifying the
polynucleotide of interest, where the polynucleotide of interest is
purified from the lysate that flows out of the clarification
reactor, wherein said method is operated on a manufacturing
scale.
45. The method of claim 44, wherein in step e), the mixing is
carried out by introducing air through a distributor from the top
or from an inlet in the bottom of the reactor.
46. The method of claim 44, wherein in step e), the mixing is
carried out by introducing air through an inlet in the bottom of
the reactor.
47. The method of claim 44, wherein the device is an automated or
semi-automated system.
48. The method of claim 44, wherein the lysis reactor and the
neutralization reactor are combined to form a two-step automated or
semi-automated system.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority benefit of U.S.
Provisional Application No. 60/472,729, filed May 23, 2003, herein
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of producing
biomolecules, in particular polynucleotides like plasmid DNA. In
particular, the present invention relates to a method on a
manufacturing scale that includes cell lysis under alkaline
conditions followed by neutralization and subsequent clarification
of the cell lysate.
[0004] 2. Related Art
[0005] The advances in molecular and cell biology in the last
quarter of the 20.sup.th century have led to new technologies for
the production of biomolecules (biopolymers). This group of
naturally occurring macromolecules includes proteins, nucleic acids
and polysaccharides. They are increasingly used in human health
care, in the areas of diagnostics, prevention and treatment of
diseases.
[0006] Recently some of the most revolutionary advances have been
made with polynucleotides in the field of diagnostics, gene therapy
and nucleic acid vaccines. Common to these applications is the
introduction of DNA or RNA into cells with the aim of a diagnostic,
therapeutic or prophylactic effect.
[0007] Polynucleotides are a heterogeneous group of molecules in
terms of size, shape and biological function. Common to all of them
are their building blocks (nucleotides as Adenine (A), Guanine (G),
Cytosine (C), Thymine (T), Uracil (U)) and their high negative
charge under physiological conditions. Representative members of
polynucleotides are RNA (messenger RNA, transfer RNA, ribosomal
RNA), genomic DNA (gDNA) or chromosomal DNA (cDNA), and plasmid DNA
(pDNA). These macromolecules can be single- or double-stranded.
Similar to proteins, they are able to build three-dimensional
structures and aggregates under distinct conditions.
Polynucleotides are sensitive to enzymatic degradation (DNases and
RNases) and shear forces, depending on their size and shape.
Especially chromosomal DNA, in its denatured and entangled form, is
highly sensitive to mechanical stress, resulting in fragments with
similar properties to pDNA. This becomes more and more critical
with the duration of the shear force exposure (Ciccolini L A S,
Shamlou P A, Titchener-Hooker N, Ward J M, Dunnill P (1998)
Biotechnol Bioeng 60:768; Ciccolini L A S, Shamlou P A,
Titchener-Hooker N (2002) Biotechnol Bioeng 77:796).
[0008] Plasmids (pDNA) are double stranded extrachromosomal
circular polynucleotides. A typical plasmid contains between 1 and
20 kilo base pairs which corresponds to
3.times.10.sup.6-13.times.10.sup.6 Da and several thousand .ANG..
Different topological forms of pDNA can be distinguished. The
supercoiled (sc) or covalently closed circular (ccc) form is
considered as most stable for therapeutic application and is
therefore the desired form. The other topological pDNA forms are
derived from the ccc form by either single strand nick (open
circular or oc) or double strand nick (linear). Breakage of the
strands can be caused by physical, chemical or enzymatic activity.
For therapeutic use the percentage of ccc form is the
main-parameter for assessing the quality of the pDNA
preparation.
[0009] Therapeutic treatment based on pDNA is considered to be an
alternative to treatment with classical chemical drugs or
recombinant proteins. Due to the increasing amounts of pDNA
required for preclinical and clinical trials, there is a demand for
processes that can be performed on a manufacturing scale. These
production processes must fulfill regulatory requirements (FDA,
EMEA) and should be economically feasible.
[0010] In the past, the majority of biotechnological production
processes have been developed for manufacturing of purified
recombinant proteins. Due to the differences in the
physico-chemical properties between polynucleotides and proteins,
these methods cannot easily be adapted for the production of
polynucleotides. Thus, there is a need for methods that are
applicable to polynucleotides, in particular for production of
plasmid DNA on a manufacturing scale.
[0011] In brief, a process for producing recombinant biomolecules,
which are not secreted by the host, in particular DNA and large
proteins, follows the steps of:
[0012] a) Fermentation (cultivation of cells that carry the
biomolecule of interest and optionally harvesting the cells from
the fermentation broth),
[0013] b) Disintegration of the cells (release of the biomolecule
of interest from the cells),
[0014] c) Isolation and purification (separation of the biomolecule
of interest from impurities).
[0015] These steps are more specifically characterized for the
production of polynucleotides, in particular for the production of
pDNA, as follows:
[0016] Currently, E. coli is the most commonly used host for pDNA
production. Other bacterial, yeasts, mammalian and insect cells may
also be used as host cells in the fermentation step. Selection of a
suitable host strain is of major importance for the pDNA quality. A
high cell density and plasmid copy number and its stable
maintenance during the fermentation are crucial for a robust
economic process. For this purpose, a well-defined culture medium
is needed. The end point of fermentation and the conditions during
cell harvest, which usually follows fermentation, contribute to the
quality of the polynucleotide (Werner R G, Urthaler J, Kollmann F,
Huber H, Necina R, Konopitzky K (2002) Contract Services Europe, a
supplement to Pharm. Technol. Eur. p. 34).
[0017] After fermentation, the cells are usually harvested, mostly
by means of centrifugation. The harvested wet biomass is
resuspended in an appropriate buffer. Before final isolation (by
e.g. column chromatography, ultradiafiltration, extraction or
precipitation) of the polynucleotide of interest from proteins,
gDNA, RNA and other host related impurities, the cells need to be
processed, either directly or after freezing and thawing.
Alternatively to harvesting and resuspending the cells before
further processing, the fermentation broth per se may be subject to
further processing (WO 97/29190).
[0018] Processing starts with disintegration of the cells and ends
with the first isolation step of the polynucleotide of interest,
which is also termed "capture step".
[0019] Disintegration of the cells can be achieved by physical,
chemical or enzymatic methods. Most of currently available
procedures were developed for the release of proteins from the
cells and can not be used for polynucleotides without certain
adaptations. Limitations of the established techniques are due to
the differences of the physico-chemical properties between proteins
and polynucleotides. High-pressure homogenization, the most common
technology for the recovery of proteins, cannot be used for
polynucleotides due to their size-depending shear force sensitivity
and possible destruction of gDNA. (Carlson A, Signs M, Liermann L,
et al. (1995) Biotechnol Bioeng 48:303). Chemical (Foster D (1992)
Biotechn 10, (12):1539) and enzymatic (Asenjo J A, Andrews B A
(1990) Bioprocess Technol 9: 143) methods cause minimal mechanical
stress and minimal irreversible deterioration of the plasmid. Since
it is the gentlest method, enzymatic disintegration utilizing
lysozyme is the method of choice on laboratory scale. Typically,
lysozyme is animal-derived (most commonly from chicken egg white)
and therefore its use is a potential health risk (prions) and is
considered as problematic by regulatory authorities like FDA or
EMEA. Using recombinant lysozyme involves high raw material costs
and analytical efforts. Thermal treatment of the cells is another
option for a disintegration technique that avoids shear forces, as
described in WO 02/057446 A2 and WO 96/36706. The suspension of
microorganisms processed by short time exposure (30 seconds to some
minutes) to 80.degree. C. in a sink heater or in a filter (with
filtering aids). This method is usually carried out in combination
with a detergent (e.g. Triton.RTM.) and lysozyme.
[0020] Usually, disintegration and release of plasmid DNA from
bacterial cells is performed by alkaline lysis (a chemical method)
as described by Birnboim and Doly (Birnboim H C, Doly J (1979) Nucl
Acids Res 7: 1513).
[0021] The disintegration/release process disclosed therein can be
divided into two steps, the first one being the intrinsic cell
disintegration or lysis step and the second one being the
neutralization step.
[0022] During alkaline lysis, cells are subjected to an alkaline
solution (preferably NaOH) in combination with a detergent
(preferably SDS). In this environment, the cell wall structures are
destroyed thereby releasing the polynucleotide of interest and
other cell related compounds. Finally, the solution is neutralized
by addition of a solution of an acidic salt, preferably an acetate,
in particular potassium acetate (KAc) or sodium acetate (NaAc). The
alkaline conditions lead to denaturation of pDNA by unwinding the
supercoiled structure. Up to a pH-value of 12 to 12.5 the complete
separation of the complementary strands is prevented. This enables
entire renaturation of the plasmid molecule, when the pH is
decreased again. If the pH-value exceeds the renaturation limit,
the unseparated regions are lost and the pDNA is irreversibly
denatured. At this stage the polynucleotide contains large domains
of single stranded material (with a large exposure of hydrophobic
bases) (Diogo M M, Queiroz J A, Monteiro G A, Prazeres D M F (1999)
Analytical Biochemistry 275:122). The exact pH-value for
irreversible denaturation of the plasmid is strongly influenced by
the base pair composition, the resulting hydrogen bonds and its
size (WO 97/29190). In parallel, genomic DNA and proteins are
denatured, too. Denaturation of DNA leads to entanglement and
formation of long single pair strands with low mechanical
stability. Impact of mechanical stress may cause breakage of DNA,
especially of the large gDNA molecules. The resulting fragments
have properties comparable to those of pDNA. Since precipitation
during the subsequent neutralization step is a size dependent
process, these fragments may remain soluble and thus behave
similarly to pDNA (Marquet M, Horn N A, Meek J A (1995) BioPharm
September: 26). Therefore they would interfere during the isolation
process. The incubation time at high pH value is critical for the
renaturation of the target polynucleotide, the degree of cell
disintegration and the genomic DNA content in the preparation.
Therefore the main parameter for quality and quantity of the
polynucleotide preparation is the contact time with the alkaline
lysis solution. Usually RNAse is added to the suspension to digest
RNA into small pieces not to interfere the isolation process
(Sambrook J, Fritsch E F, Maniatis T, (1989) Molecular Cloning: A
Laboratory Handbook, CSH Press, Cold Spring Harbor, USA). After
addition of NaOH and SDS, the solution becomes highly viscous.
Local pH extremes, which irreversibly denature the plasmid (Rush M
G, Warner R C (1970) J Biol Chem 245:2704) have to be avoided. Fast
and efficient mixing has to be guaranteed in order to achieve a
homogenous solution. Usually small containers like glass bottles
containing the viscous solution are mixed very gently by hand
(Qiagen.RTM. Plasmid-Handbuch 01/2001, Qiagen GmbH, Germany). This
procedure can only be performed in a batchwise mode with a maximum
of about 5 l lysate per bottle. It is mainly operator dependent,
providing low reproducibility and is therefore not suited for a
manufacturing scale. For large scale conventional stirrers are not
suited because they may cause damage to pDNA and gDNA. Some
processes use optimized tanks and stirrers or a combination of
different mixers in order to overcome these problems (Prazeres D M
F, Ferreira G N M, Monteiro G A, Cooney C L, Cabral J M S (1999)
Trends Biotechnol 17:169; WO 02/26966).
[0023] In the subsequent neutralization step, cell debris, proteins
as well as genomic DNA are co-precipitated with SDS by formation of
a complex floccose precipitate (Levy M S, Collins I J, Yim S S, et
al. (1999) Bioprocess Eng 20:7). Again gentle, but homogeneous
blending (homogeneous neutralization) is essential for complete
precipitation and for maintenance of pDNA quality. Vigorous mixing
causes destruction of the plasmid and the flocks, resulting in
redissolution of the impurities precipitated before (Levy M S,
Ciccolini L A S, Yim S S, et al. (1999) Chem Eng Sci 54:3171;
Marquet M, Horn N A, Meek J A (1995) BioPharm (September):26). This
burdens the subsequent chromatographic separations (by e.g. loss of
capacity for pDNA orthe negative impact on the separation of RNA
and gDNA, which have similar binding properties).
[0024] In the next step that follows alkaline lysis and
neutralization, the precipitate has to be separated from the
plasmid containing solution (this step is, in the meaning of the
present invention, termed "clarification step"). In view of further
purification by means of a resin, it is often necessary to adjust
the parameters of the solution (like salt composition,
conductivity, pH-value) to guarantee binding of the desired
polynucleotide on the resin (this step is, in the meaning of the
present invention, termed "conditioning step"). Subsequently, the
solution is subjected to the first chromatographic step (capture
step).
[0025] Centrifugation on fixed angle rotors (is the most frequently
used method employed as the clarification step on laboratory and
pre-preparative scales (Ferreira G N M, Cabral J M S, Prazeres D M
F (1999) Biotechnol Prog 15:725). For lysate amounts usually
handled in bottles the clear liquid phase separating from floating
flocks and descending precipitate is sucked off and filtered.
Otherwise the big flock-volume would shortly block the used filter.
Since the fluid between the flocks contains residual plasmid DNA
(Theodossiou I, Collins I C, Ward J M, Thomas O R T, Dunnhill P
(1997) Bioprocess Engineering 16:175), high losses have to be taken
into account. As further problem strong adsorption of nucleotides
and pDNA to many filter-media has to be mentioned (Theodossiou I,
Collins I J, Ward J M, Thomas O R T, Dunnhill P (1997) Bioprocess
Eng 16:175; Theodossiou I, Thomas O R T, Dunnhill P (1999)
Bioprocess Eng 20: 147). In many cases, bulk filter materials or
bag filters are used for clarification of the lysate. Since these
materials are either not certified or not scalable, they are not
applicable for the production of pharmaceutical-grade plasmids on a
manufacturing scale. More recent technologies utilize expanded bed
adsorption (EBA), which allows removal of precipitated material
while capturing the desired product (Chase H A (1994) Trends
Biotechnol 12: 296). For capturing plasmid DNA direct after lysis
by this chromatographic technique, it has to be taken into account
that due to the large diameter of the (during neutralization built)
aggregates of flocks pre-clarification prior to EBA is essential
(Ferreira G N M, Cabral I M S, Prazeres D M F (2000) Bioseparation
9:1; Varley D L, Hitchkock A G, Weiss A M E, et al. (1998)
Bioseparation 8:209).
[0026] There have been several attempts to develop improved
technologies for each of the above-described steps. These attempts
were mostly based on the following considerations:
[0027] Resuspension of the cells has to be carried out as fast as
possible (especially when the cells have been frozen before), while
avoiding high shear forces. Several commercially available types of
stirrers are available for mixing the cell paste with the
resuspension buffer in a batchwise mode in a vessel until
homogeneity is achieved, the most commonly used device being a
magnetic or impeller stirrer. Another method is described in US
2001 0034435 A1. Here the cell paste is diluted with a resuspension
buffer and the cell/buffer mixture is circulated through a static
mixer in a pump-around mode. It has also been suggested to directly
dilute the fermentation broth with the resuspension buffer in a
static mixer prior to lysis (WO 97/23601 A1).
[0028] For disintegration (lysis) of the cells in view of obtaining
polynucleotides, several different methods have been suggested,
e.g. methods that use thermal or chemical treatment. For the
thermal lysis, a process using a flow-through heat exchanger
(70-100.degree. C.), in which the cells are continuously
disintegrated after incubation of the resuspended cells in presence
of a detergent and optionally lysozyme, is described (WO 96/02658
A1). Another physical method, which works in a temperature range of
70-90.degree. C., is shown in WO 02/057446 A2: In a first step, the
harvested cells are filtered utilizing filter aids and the
resulting mixture is thermally lysed in a second step.
Alternatively, disintegration can be carried out by pumping hot
lysis buffer through the filter cake or by a flow through heat
exchanger. Chemical lysis methods are operated at an alkaline
pH-value, they are therefore referred to as "alkaline lysis". A
commonly used composition of the intrinsic lysis solution is
described by Bimboim and Doly, but there are exist many variants of
this solution. As the detergent that is part of lysis solution
usually SDS is used, but other (e.g. non-ionic) detergents like
Tween.RTM. or Triton.RTM. are also suitable (e.g. WO 95/21250 A2).
According to EP 0376080 A1, SDS is replaced by desoxycholate (DOC),
while the three phase extraction method of U.S. Pat. No. 5,637,687
uses a novel composition for the cell-solubilization (benzyl
alcohol+sodium iodide+guanidinium thiocyanate and/or guanidinium
chloride). Most methods for alkaline lysis are operated in a
batchwise mode. By way of example, the alkaline treatment can be
carried out directly by adding a NaOH/SDS solution to a bacterial
cell culture during exponential growth (in this case, no harvest of
the cells is performed) or after resuspension of the cells in a
proper buffer. Thereby, an alkaline solution is added until a pH
value is reached that is 0.2 units lower than the pH value at which
the pDNA-molecules are completely denatured, a pH value that is
empirically determined and different for each single plasmid (WO
97/29190 A1). Another method utilizes a column comprising a carrier
on a membrane filter that is capable of retaining a solution and
permeating it by aspiration. When adsorbed onto the carrier, a
certain amount of cells can be lysed in this column by means of
lysozyme and further processed (EP 0814156 A2). A similar device
that consists of a hollow body (tube) with a built-in
filtration-layer is disclosed in EP 0616638 B1, EP 0875271 A2, and
WO 93/11218 A1 Alkaline lysis is carried out in the part of the
tube above the filtration section. The cell suspension and the used
solutions are distributed and mixed in a non-continuous way.
[0029] The above-described methods are operated in non-continuous
open systems that bear the risk of possible contamination. Handling
and mixing is not automated and therefore user-dependent. The only
way to handle larger pDNA-amounts, is multiplication of the
devices, e.g. running them in parallel. These methods and devices
are not suitable for production of pharmaceutical grade
polynucleotides on a manufacturing scale. To achieve contacting and
mixing of the cells with the lysis solution, it has also be
suggested to use static mixers or simple tubings. This approach has
been described for a cell lysis method, which is based on simply
connecting the streams containing the pumped cell suspension and
the lysing agent at a defined meeting point. The contact time is
defined by the tubing volume (diameter and length of the tube)
behind the meeting point and by the pump-velocity of the connected
streams through the tubing. To facilitate rapid homogenization, the
inner diameter of the tubing has to be reduced (2-8 mm) (WO
99/37750 A1). For connecting the two pumped streams at the meeting
point, "Y"-connectors are proposed (WO 00/09680 A1). To enhance
homogenous mixing of the cells with the lysis solution, especially
designed static mixers are suggested. These devices are
commercially available continuous flow-through supports. The
contact time of the cells with the lysis solution is defined by the
mixer dimensions and the flux (WO 97/23601 A1, WO 00/05358 A1).
These online-contacting devices can also be combined with a
subsequent stirred tank reactor. In this stirred tank reactor the
neutralization step may also take place. (WO 02/26966 A2). Another
process describes the combination of a static mixer, a so called
"lysis coil" and an impeller (US 2001/0034435 A1).
[0030] The above-described continuous methods either work with
simple connections of the flow stream or, in the case of using
static mixers, with various fixtures, (e.g. helical structures.
[0031] Among the above-described methods, those using a simple
tubing do not guarantee homogenous mixing, while the variant with
the reduced tubing diameter (<1 cm) was designed for small-scale
applications. The methods using static mixers (or reduced tubing
diameters) may cause high shear forces to the polynucleotides.
[0032] In the neutralization step, normally an acidic solution
containing potassium acetate is used. For concurrent precipitation
of RNA, compositions that contain, in addition, sodium chloride,
potassium chloride or ammonium acetate (up to 7 M) have been
suggested (US 2001/0034435 A1). It was also shown that a solution
containing divalent alkaline earth metal ions like CaCl.sub.2 that
is added to the mixture after neutralization results in the
precipitation of RNA and chromosomal DNA (U.S. Pat. No. 6,410,274
B1).
[0033] Neutralization of the lysed cell solution is often carried
out as one single step in a batch mode. In EP 0814156 A2, WO
93/11218 A1, EP 0616638 B1, and EP 0875271 A2 the lysed cell
solution is contacted with the neutralization/precipitation
solution in the same device (column or tube with an in-line
filter-material) like used before for the lysis step (already
described above). Again, these techniques would be subject to
several major limitations when transferred to the manufacturing
scale production of pharmaceutical-grade polynucleotides, the
problems also being possible contamination due to the
non-continuous open system, user-dependence, and lacking
scalability.
[0034] For the neutralization step, a stirred tank reactor that
already contains the lysed cell solution, has been suggested, into
which the neutralization solution is filled under continuous mixing
with the stirrer at a speed of 500 rpm (WO 02/26966). A similar
method is claimed in US 2001/0034435 A1, according to which
neutralization is achieved by mixing the solutions with an impeller
in a chilled jacketed holding tank or before in an in-line static
mixer. Two very simple continuous contacting techniques are
disclosed in WO 99/37750 A1 and WO 00/09680 A1. Both methods use
the same setup as already described above for the lysis step
connecting the two pumped streams at a meeting point with a reduced
inner diameter of the resulting tubing (WO 99/37750) or a simple
"Y" connector and tubing (WO 00/09680). For both methods, static
mixers may be used in the neutralization step (WO 97/23601 A1, WO
00/05358 A1). These mixers are utilized in the same manner already
described above for the lysis step.
[0035] The contact time of the pDNA-with the lysis solution has a
major impact on its quality and depends on the time point and
effectiveness of the neutralization step. Therefore, mixing of the
lysed cell solution with the neutralization solution has to be fast
and homogenous. This requirement can not be met by the techniques
utilizing stirred tank reactors. Fast mixing with an impeller may
cause rupture of the precipitated flocks and re-dissolution of
impurities. The methods using a simple tubing do not guarantee
homogenous mixing, while the variant with the reduced tubing
diameter (<1 cm) may also cause undesired destruction of the
flocks and is not suitable for larger scales. Although static
mixers are expected to achieve homogenous mixing, they may get
blocked due to the large volume of the flocks. Another disadvantage
is that genomic DNA may be sheared by the internal structure of the
mixer to a size, which will cause problems in the subsequent
purification steps, let alone the possible negative impact of the
mechanical stress to the desired polynucleotide.
[0036] To obtain a cleared lysate; the precipitated material has to
be separated from the polynucleotide containing solution.
Conventionally this clarification step is carried out in a
batchwise mode using techniques known in the art like filtration or
centrifugation (e.g. US 2001/0034435 A1, WO 02/04027 A1). Most
commonly, the filters are depth filters (WO 00/09680). Other filter
means for macrofiltration are macroporous diaphragms consisting of
e.g. compressed gauze or an equivalent filter material (EP 0376080
A1). According to some protocols, filtration is carried out in
presence of a filter aid (WO 95/21250 A2, WO 02/057446 A2, US
2002/0012990 A1). WO 96/21729 A1 discloses a method that contains a
filtration step using diatomaceous earth after a centrifugation
step, thereby achieving the additional effect of reducing the RNA
content. Furthermore, combinations of a membrane filter with a
loose matrix (glass, silica-gel, anion exchange resin or
diatomaceous earth), which concurrently act as carrier for DNA,
have been described (EP 0814156 A2). According to WO 96/08500 A1,
WO 93/11218 A1, EP 0616638 B1 and EP 0875271 A2, clarification is
achieved by a device that has been described above for the lysis
and for the neutralization step, whose filtration part may consist
of different materials (e.g. glass, silica-gel, aluminum oxide . .
. ) in the form of loose particles, layers or filter plates
(especially with an asymmetric pore size distribution). The flux
through the filter is accomplished by gravitation, vacuum, pressure
or centrifugation. As a continuous clarification method,
centrifugation (e.g. disc stack centrifuge or decanting centrifuge)
are mentioned (WO 99/37750 A1, WO 96/02658 A1). Also combinations
of centrifugation followed by filtration are described for the
clarification purpose (WO 02/26966 A2, WO 96/02658 A1).
[0037] The above-described clarification methods are usually
carried after the material has been incubated with the
neutralization buffer for a certain period of time. This does not
allow continuous connection with the foregoing steps and is only
suitable for the laboratory scale. Apart from this, filtration
techniques are usually carried out in open devices with the risk of
possible contamination. Since any material that is used in a cGMP
process must be validated, additional filter aids that might
improve performance of the filtering process, are usually
avoided.
[0038] In general, conventional filters have a limited capacity and
are soon blocked by the large amount of voluminous flocks. In
addition, a constant flux over the precipitate that is retained by
the material may result in destruction of the flocks and
re-dissolution of impurities, which would again have a negative
impact on the following steps. For larger amounts of pDNA it has
been suggested for some devices to multiply them (e.g. run them in
parallel), which is insufficient for operating on a manufacturing
scale. Centrifugation could be applicable continuously, but due to
the sensitivity of polynucleotides to shear forces this treatment
may also cause degradation of plasmid DNA and genomic DNA and
detachment of precipitated impurities by rupture of the flocks.
[0039] In the subsequent conditioning step, the salt composition
and/or the conductivity and/or the pH-value of the cleared lysate
is adjusted to a value (to be determined empirically) that ensures
binding to the resin in the subsequent capture step. Several
conditioning methods have been described, e.g. in WO 97/29190 A1,
WO 02/04027 A1 and WO 98/11208 A1. In the methods described in EP
0814156 A2, WO 93/11218 A1, EP 0616638 B1 and EP 0875271 A2 the
conditioning step is carried out as a washing and eluting step in
the same device in which the previous steps took place.
[0040] Furthermore, as a pretreatment before the final
purification, addition of an "Endotoxin Removal (ER) Buffer"
(Quiagen.RTM.) (WO 00/09680 A1) or Triton X.RTM.-114 (WO 99/63076
A1) has been suggested.
[0041] Common to all of the described methods is their
non-continuous and non-automated mode of operation that does not
connect the operational steps.
[0042] For capturing the polynucleotide of interest, several
techniques are known in the art, e.g. tangential flow filtration
(WO 01/07599 A1), size exclusion chromatography (WO 96/21729 A1, WO
98/11208), anion exchange chromatography (WO 00/09680 A1, U.S. Pat.
No. 6,410,274 B1, WO 99/16869), hydrophobic interaction
chromatography (WO 02/04027 A1).
[0043] It has already been suggested combining some of the steps
described above, e.g. for the processes described in EP 0814156 A2,
WO 93/11218 A1, EP 0616638 B1 and EP 0875271, according to which
cell lysis, neutralization, clarification, washing, optionally
conditioning and capturing are carried out in the same apparatus.
Typically, these methods are open systems that are operated in a
non-automated/non-continuous mode including several holding steps.
The devices are only suitable for the laboratory scale and cannot
be transferred into manufacturing scale. The techniques also lack
of reproducibility and suitability for cGMP large-scale
production.
[0044] Alternatively, combinations utilizing different devices have
been described, in which the individual steps are directly
connected with each other.
[0045] The continuous combination of two ore more steps has been
described in several patent documents: WO 96/02658 A1 describes the
combination of thermal lysis and clarification by means of a
centrifuge, WO 00/09680 A1 and WO 02/26966 A2 suggest combining
alkaline lysis and neutralization. The methods described in US
2001/0034435 A1 and WO 97/23601 A1 combine the three steps
resuspension of the cells, alkaline lysis and neutralization; WO
00/05358 A1 and WO 99/37750 A1 describe the combination of alkaline
lysis, neutralization and clarification by centrifugation.
[0046] None of these processes combines more than three steps of
the isolation procedure, the first step being the resuspension step
and last one being the capture step. The devices used in these
methods for contacting the solutions during lysis and
neutralization do either not guarantee homogenous mixing or may
apply disadvantageous shear forces to the solutes.
SUMMARY OF THE INVENTION
[0047] It was an object of the invention to provide a method for
isolating a biomolecule, in particular a polynucleotide, of
interest from a cell culture that overcomes the limitations of the
known methods. Such method should be suitable for the production of
therapeutically applicable polynucleotides. Thus, such process
should neither require the use of enzymes like RNase and lysozyme
nor the use of detergents apart from SDS.
[0048] In particular, it was an object of the invention to provide
a automatable and scalable process for isolating a polynucleotide
of interest, in particular plasmid DNA, on a manufacturing scale
that includes, as a cell disintegration step, an improved alkaline
lysis method. In addition to an alkaline lysis step, the process
should include a neutralization step, a clarification step, and
optionally a conditioning step and/or a concentration step.
[0049] To solve the problem underlying the invention, the following
steps were taken:
[0050] Since clarification of the lysate was considered to be the
limiting step for operating a process for isolating a biomolecule
of interest in a continuous and automated way, in a series of
experiments, several different methods were investigated to address
the issue of clarification. It was surprisingly found that a tank
that is filled with glass beads to a certain level and has an
outlet at the bottom, provides excellent clarification results and
allows automation of the entire process.
[0051] Furthermore, it was sought to provide an improved method for
achieving disintegration of the bacterial cells by alkaline lysis
that may be combined with the improved clarification step. To this
end, several mixing techniques were tested in preliminary
experiments using differently colored test solutions. It was
surprisingly found that a tube filled with glass beads leads to
sufficient mixing and contacting of two solutions when brought
together by pumping through this tube. This finding was confirmed
when using, as the two solutions, the resuspended cell suspension
and the lysis solution.
[0052] A further unexpected result was obtained when procedures for
mixing the lysed cell solution with the neutralization solution
were tested. It was found that after connecting the streams of the
pumped lysed cell solution with the stream of the pumped
neutralization solution by a conventional T-connector, an
especially oriented tubing results in satisfactory mixing of the
solutions and formation of compact voluminous flocks, which are not
influenced by strong shear forces.
[0053] These findings were developed further by combining the
single steps to a system that can be operated in a continuous mode
and automated.
[0054] The present invention relates to a process for producing a
biomolecule that is not secreted by the host cell, comprising the
steps of
[0055] a) cultivating cells to produce the biomolecule of interest
and optionally harvesting and resuspending the cells,
[0056] b) disintegrating the cells by alkaline lysis,
[0057] c) precipitating the cell debris and impurities by
neutralizing the lysate,
[0058] d) separating the lysate from the precipitate obtained in
step c),
[0059] e) purifying the biomolecule of interest,
wherein in step d) the mixture comprising the precipitate and the
lysate is allowed to gently flow downward through a clarification
reactor that is partially filled in its lower part with retention
material, whereby the precipitate is retained on top of and within
the layer of retention material and the cleared lysate leaves the
reactor through an outlet in the bottom of the reactor.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0060] FIG. 1: Flowchart of a combined continuous three step
process comprising alkaline lysis, neutralization and
clarification
[0061] FIG. 2: Flowchart of the combined continuous three-step
process of FIG. 1, extended by a continuous conditioning step
[0062] FIG. 3: Flowchart including an additional capture step
[0063] FIG. 4: Flowchart of the combined continuous system
including an on-line filtration step between conditioning and
capture step
[0064] FIG. 5: Flowchart of the combined continuous process (FIG.
4) extended by a concentration step before conditioning
[0065] FIG. 6: Scheme for the continuous combination of alkaline
lysis, the neutralization and the clarification reactor
[0066] FIG. 7: Device comprising a combination of lysis reactor,
neutralization reactor and clarification reactor (pilot apparatus
suitable for up to 1 kg wet cells)
[0067] FIG. 8: Up-scaled version of the device of FIG. 7 (pilot
apparatus suitable for up to 6 kg wet cells)
[0068] FIG. 9: cGMP device comprising a combination of lysis
reactor, neutralization reactor and clarification reactor (suitable
for up to 20 kg wet cells)
[0069] FIG. 10: Analytical HPLC chromatogram of a lysate obtained
by the continuous method of the invention including the steps
lysis, neutralization and clarification in the pilot device
[0070] FIG. 11: Analytical HPLC chromatogram of a reference lysate
obtained by a conventional method on the laboratory scale
[0071] FIG. 12: Analytical HPLC chromatogram of a pool from the
capture step obtained by the extended continuous method of the
invention including the steps lysis, neutralization, clarification,
conditioning, filtration and capturing
[0072] FIG. 13: Analytical HPLC chromatogram of a lysate obtained
by the continuous method of the invention including the steps
lysis, neutralization and clarification in the up-scaled
device.
[0073] FIG. 14: Analytical HPLC chromatogram of a pool of the
capture step. Lysate obtained by the continuous method of the
invention including the steps lysis, neutralization and
clarification in the up-scaled device.
[0074] FIG. 15: Analytical HPLC chromatogram of a lysate obtained
by the continuous method of the invention including the steps
lysis, neutralization and clarification, which was concentrated by
ultrafiltration.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Steps a) to c) and step e) may be performed according to
known methods, preferably according to methods that can be run
continuously and automated.
a) Fermenting/Cultivating:
[0076] In the method of the invention, preferably E. coli is used
as host, in particular when the biomolecule of interest is
pDNA.
[0077] In one embodiment of the invention continuously operated
devices, e.g. tube centrifuges or separators, are used for
separating the cells from the cultivation medium. If the cells (the
biomass) are frozen prior to further processing, the cells can be
frozen directly after harvesting or after resuspension of the cells
in a suitable buffer, typically a buffer containing 0.05 M Tris,
0.01 M EDTA at pH 8. In this case no resuspension buffer has to be
added prior to alkaline lysis or it is required in lower
amounts.
[0078] Biomass that is obtained in a fermentation, may be, before
being further processed (resuspended, lysed, etc.), frozen, in
particular cryo-pelleted. Cryo pelletation is an advantageous
method to prepare cells for storage. Since this method guarantees
fast freezing of the cells undesired temperature gradients, within
the biomass, can be avoided. Slow freezing in a conventional
freezer may lead to inhomogeneous freezing and the building of ice
crystals, which may damage the cells and reduce their shelf life
and the quality of the polynucleotide of interest. The same may be
observed when the biomass is thawn again.
[0079] In a preferred embodiment, the biomass obtained in step a)
is cryo-pelletized. With a cryo-pelletation system the fermentation
broth can be directly frozen or after harvest and resuspension in a
suitable ratio of resuspension buffer. Cryo-pelletation devices
normally work with fluid gases in which the (stirred) material to
freeze is applied dropwise and the resulting pellets continuously
brought out of the system or/and avoided to agglomerate by slowly
stirring inside. These devices are commercially available and have
been used so far in the food industry in the production of ice
cream or in the pharmaceutical industry for the storage of final
products. The size of the pellets depends on the nozzle used for
distribution of the material into the gas and the velocity of
application. For the harvested biomass an average pellet size
between 0.5 and 2 cm was found to be suitable. After
cryo-pelletation the pellets may be stored in a conventional
freezer at -20-100.degree. C. The method results in homogenous
pellets that can be easily divided into several aliquots, which is
another advantage compared to the conventional technique. For the
processing of the biomass the aliquots can be thawn faster, due to
the larger surface of the pellets compared to larger blocks. The
thawing can be further accelerated when resuspension buffer at room
temperature is added and this suspension is stirred with
conventional stirrers.
[0080] In an embodiment of the invention, harvesting and
resuspending the cells may be omitted, in this case the
fermentation broth can be directly further processed in the lysis
step b) without separation of cells and cultivation
supernatant.
b) Disintegrating by Alkaline Lysis:
[0081] The harvested cells of step a) are either directly processed
or thawn, if frozen before. Common to both procedures is that the
harvested cells are resuspended in the resuspension buffer
described in a) prior to the intrinsic cell disintegration step
b).
[0082] Alternatively the fermentation broth obtained in step a) is
directly further processed without harvest and resuspension of the
cells. In this case, the cells may be disintegrated by directly
conducting alkaline lysis (and optionally subsequent
neutralization) in the fermentor or by introducing the fermentation
broth into the lysis reactor.
[0083] (In the following, with respect to cell disintegration in
step b), the term "cell suspension" is used for both the
resuspended cells after harvest and the fermentation broth.)
[0084] In principle, step b) can be performed according to methods
known per se, preferably according to methods that are gentle and
can be run in a continuous and automated mode.
[0085] In a preferred embodiment, in step b) the cell suspension
and the alkaline lysis solution are allowed to flow through a lysis
reactor that is essentially completely filled with particulate
material, thereby contacting and mixing the cell suspension with
the alkaline lysis solution.
[0086] Preferably, the cell suspension and the alkaline lysis
solution are combined, without further mixing, before entering the
lysis reactor, thus forming a single flow that is thoroughly mixed
when flowing through the particulate material in the lysis reactor
and achieving very gentle lysis. By avoiding disadvantageous shear
forces, plasmid DNA quality is maintained at a very high level.
Furthermore, the yield of supercoiled plasmid DNA is higher as
compared to conventional methods. This is due to two reasons:
Firstly, degradation, which can occur when using harsher mixing
conditions and devices, is reduced. Secondly, due to the homogenous
mixing, the cells are completely disintegrated (releasing the whole
pDNA-amount), avoiding local pH-extremes, which may also result in
degradation of the target plasmid DNA molecule.
[0087] Alternatively, the cell suspension is introduced into the
reactor simultaneously with the lysis solution, preferably through
inlets that are as close as possible to each other.
[0088] Preferably, in both embodiments, the cell suspension and the
lysis solution are transported, e.g. by pumps or pressurized gas,
at a defined ratio of flow rates, thereby achieving a constant
ratio of cell suspension and lysis solution volumes.
[0089] In terms of the lysis reaction per se, step b) in the
preferred embodiment of the present invention, is performed
according to methods known in the art, using an alkaline lysis
solution that contains a detergent. A typical lysing solution
consists of NaOH (0.2 M) and SDS (1%), but also other alkaline
solutions and other detergents can be used (see e.g. WO
97/29190).
[0090] The lysis reactor, which is also a subject of the present
invention, is a flow-through container that is filled with the
particulate material, preferably a hollow body formed as a cylinder
or tube, in particular a glass or stainless steel tube. The tube
may also be made of plastic or any other material acceptable for
biopharmaceutical production. The particles, in particular beads,
which are preferably made of glass, but can also consist of
stainless steel, plastic or other materials, are packed into the
reactor in a random way, so that it is completely or essentially
completely filled, with free space of random size and shape between
the particles. Due to this, functionality of the lysis reactor is
independent of whether and in which way the flows of the two
solutions have been connected before they enter the lysis reactor.
It is even possible that the solutions are combined directly in the
device. The beads can be of equal or different diameter, their size
depending on the scale on which the process is operated, generally
ranging from ca. 1 to ca. 100 mm. In a preferred embodiment the
diameter of the beads is 5 mm. Instead of beads, other filling
elements that provide efficient mixing can be used, e.g. rods,
fibrous material like fiberglass, grids in shifted layers, nets, or
particles of irregular shape. Mixing of the solutions is not
limited by the direction of the flow through the device, it may be
performed in any direction, e.g. vertically upwards or downwards,
horizontally or in any angle.
[0091] The parameters of step b) and the dimensions of the device
used therein are advantageously designed such that homogenous
mixing is guaranteed and contact time is kept in a certain range
from 5 seconds to 5 minutes or more, preferably at 1 to 3 minutes,
in order to avoid denaturation of the desired polynucleotide. These
parameters can be adjusted by the dimension of the device, the free
volume between the packed beads and the flow rate. The contact time
for adequate alkaline lysis of cells depends on the host strain and
is independent of the biomolecule of interest, in the case of pDNA
it is independent of plasmid size or the plasmid copy number
(PCN).
c) Neutralizing/Precipitating:
[0092] Also this step may, in principle, be performed according to
methods known per se, preferably according to methods that are
gentle and can be run in a continuous and automated mode.
[0093] In a preferred embodiment, in the neutralization step c) the
lysed cell solution is mixed with the neutralizing solution in a
continuous, preferably automated manner. This is accomplished by
combining the lysed cell solution and the neutralizing solution, at
a constant ratio of the flow rates (e.g. by means of a T connector
or Y connector) and ensuring mixing and neutralizing/precipitating
during transportation of the reaction mixture between the lysis
reactor and the subsequent clarification reactor.
[0094] For this purpose, a novel neutralization reactor, which is
also subject of the invention, is used. This reactor consists of a
connector means and a tubing system, which is designed such that
homogenous mixing of lysed cell solution and neutralization
solution is guaranteed and the newly formed flocks of the
precipitate are not destroyed by shear forces. The tubing system
may be rigid or flexible, preferably it is in the form of a coil,
the dimension (diameter and overall length of the tubing and
diameter of the coil) depending on the scale of the process. The
tube can be made of any material acceptable for biopharmaceutical
production, preferably plastic or stainless steel.
[0095] According to a preferred embodiment, the flow paths of the
lysed cell solution and the neutralization solution are combined by
a conventional connector, e.g. a T connector or a Y connector. Once
the lysed cell solution is contacted with the neutralization
solution, the formation of the flocks starts. The resulting mixture
is then transported, preferably by pumps or pressurized gas,
through the tubing system. Depending on the scale of the process,
the inner diameter of the tubing is in the range of ca. 3 to ca.
100 mm, preferably greater than 8 mm in order to avoid shear of the
flocks at the tubing wall. The orientation of the flow may be
upwards, downwards, horizontally or in any other direction,
preferably in the form of a spiral. A mixing distance of 30 cm to
several meters allows gentle and complete mixing of the solutions
and thus precipitating the cell-derived impurities. The mixing
distance, the inner diameter of the tube as well as the retention
time in the mixing device effect the quality of mixing and the
formation of the precipitate.
[0096] Typically a buffered solution with acidic pH and high salt
concentration is used for neutralization. Preferable this solution
consists of 3 M potassium acetate (KAc) at pH 5.5. But also other
neutralizing salts can be used or added.
d) Separating/Clarifying and Optionally Washing:
[0097] In step d) the mixture obtained in step c) comprising the
precipitate and the lysate (which in the case of pDNA and usually
in the case of proteins contains the biomolecule of interest) is
allowed to gently flow downward through a clarification reactor
that contains, in its lower part, a retention layer, whereby the
precipitate is retained on top of and within the retention layer
and the cleared lysate leaves the reactor through an outlet in the
bottom of the reactor. If the aeration valve on the top of the
clarification reactor is, which is usually the case, completely
closed, the free volume in the clarification reactor decreases in
the course of the process due to the increasing level of the
flock/lysate mixture. Therefore, the pressure in the reactor
increases constantly over time. This results in a constant outflow
that is obtained without further handling.
[0098] In a preferred embodiment, in step d) the retention layer in
the clarification reactor is composed of a particulate
material.
[0099] In another embodiment, to accelerate the process, increasing
pressure is applied to the mixture in the clarification reactor,
e.g. by applying pressurized gas, in particular air, from the top
of the reactor. Normally, application of pressure is not required
at the beginning of the process but when the process further
proceeds. Usually, the pressure is increased stepwise, e.g. in the
range of 0.2 bar, the intervals being defined by the points of time
when predetermined aliquots of the precipitate/lysate-mixture have
entered the reactor. Alternatively, the pressure may be increased
continuously.
[0100] The clarification reactor, in which the mixture containing
the flocks (which are, in the case of pDNA, a co-precipitate of
gDNA, proteins, cell debris and SDS) is further processed and which
is also subject of the invention, can be made of glass, stainless
steel, plastic or any other material that is acceptable for
pharmaceutical production. A preferred shape is cylindrical, but in
principle every other hollow body is possible. Step d) in the
method of the invention is independent of the shape of the
reactor.
[0101] The reactor has an inlet at the top or at any other position
above the retention layer and has an outlet at the bottom,
underneath the retention layer. Inside the reactor, preferably in
the center, there is a distribution means that reaches to the
surface of the retention layer and evenly and gently, without
destroying the flocks, distributes the mixture into the
clarification reactor. This distributor is connected with the
supply means that transports the mixture through the inlet, or it
represents an extension of the supply means. The distributor may be
in the form of a tube or coil with apertures like slots, which may
be in any direction, e.g. vertically or horizontally, or
perforations or other apertures, or in the form of a chute, it may
be a simple rod or a combination of two or more identical or
different of such distributing devices, that are preferably
arranged vertically or slightly inclined. The distributor has
apertures over at least 10% of its total length, the apertured
section being located above the retention layer. Preferably, the
distributor carries apertures over its entire length.
[0102] In a preferred embodiment, the distributor is a perforated
tube that reaches to the surface of the retention layer and has a
rod in its center. In case the retention layer consists of
particulate filling material, this may be of regular (e.g
spherical, cylindric, in form of plates) or irregular (sandy,
gritty . . . ) shape, preferably, in the form of beads.
[0103] The beads may be of identical or different diameter, ranging
from 0.1 to 10 mm. In a preferred embodiment the diameter of the
beads is 1 mm.
[0104] If the retention elements are beads, they are preferably
made of glass, but they may also be made of stainless steel,
plastic or other materials that are acceptable for
biopharmaceutical production. The particles are packed into the
reactor in a random way up to a certain height, providing
sufficient clarification
[0105] The volume that the retention material occupies is not
critical as long as it ensures that the residual reactor volume is
sufficiently large to collect the flock volume to be processed. By
way of example, independent of the reactor base, the height of the
retention material should be in the range of 1-15 cm, in particular
2-5 cm, for a total reactor height of 40-100 cm. The height of the
filling material in the reactor depends on the specific size and
shape of the filling material itself and its capacity to retain the
flocks. The optimum filling height has to be determined empirically
for the selected retention material: Due to their larger retention
capacity, a thinner layer is necessary for particles or retention
material with smaller pores as compared to larger particles or
material with larger pores. Preferably, the filling material takes
approximately at least 5% to a maximum of 30% of the total reactor
volume.
[0106] In case of particulate retention material, the particles are
held back by a device in the outlet, e.g. a frit. Naturally, this
frit must have pores smaller than the particles used in the
reactor. The frit may be made of polypropylene or any other
suitable material with an average pore size of 10 to 200 .mu.m,
preferably 30-100 .mu.m.
[0107] The outlet of the clarification reactor may be extended by a
tubing. In this case, the frit may be situated distant from the
outlet inside the tubing; thus the tubing above the frit is filled
with the retention material.
[0108] Instead of particulate retention elements, the bottom of the
clarification reactor may be filled with rigid retention material,
e.g. sinter plates, preferably made of glass and having a pore size
from apx. 100 .mu.m to apx. 500 .mu.m. In a specific embodiment, a
sinter plate with larger pores can be placed on top of one with
smaller pores.
[0109] In the course of the separation process, the flocks float in
the reactor whereas the clear lysate runs through the retention
layer. Flocks that are not floating are retained by the retention
layer.
[0110] In a preferred embodiment, connections for supply of
compressed gas, e.g. air, are located in the top of the
clarification reactor. In this case, the clarification reactor has
to be pressure-resistant (since the pressure in the reactor
increases even if no compressed gas is supplied, when the aeration
valve on the top is closed, the reactor should be pressure proof up
to 6 bar). By applying compressed gas, the clarification process
can be accelerated, which is a very gentle method of increasing the
outlet flow and at the same time avoiding shear forces that might
damage the biomolecule.
[0111] The pressure has to be in a range such that the flocks are
not pressed through the retention material, especially at the end
of the procedure. Preferably, the applied pressure is in the range
of 0.1 to 3 bar, most preferred up to 2 bar. The resulting
neutralized lysate is visually clear and can directly be further
captured and processed, usually by chromatographic techniques.
[0112] At the end of separation/clarifying step d), the residual
fluid between the flocks, which are then present on top of and
possibly also within the retention layer, in particular when using
larger particulate material or rigid retention material with larger
pores, may be recovered by applying pressure. This leads to
drainage of the flocks.
[0113] This provides an advantage in that the residual fluid
between the flocks that contains the biomolecule of interest, e.g.
plasmid DNA, and that can normally not, or only insufficiently be
recovered, is obtained at maximum yield. Thus, practically the
entire lysate is obtained as a clear solution.
[0114] In addition, one or more wash steps may be inserted between
steps d) and e). In this case, at the end of step d), the flocks
are washed with a suitable buffer that does not re-dissolve the
flocks, e.g. 3 M potassium acetate at pH 5.5, or a mixture of the
solutions used in the resuspension, lysis (without SDS) and
neutralization step, e.g. at a ratio of 1:1:1, by pumping the
solution successively or simultaneously in either of the two or in
both directions through the flocks, i.e. from the inlet and/or the
outlet of the reactor. If pumping is done from the inlet, the wash
step can be continuous or batchwise. If it is done from the outlet,
which is preferred, the wash buffer may, but does not need to, be
pumped into the tank up to the inlet. Then the solution is
recovered at the outlet, applying the same method as described
above (compressed air).
e) Purifying:
[0115] A process following steps a) to d) of the invention
facilitates isolating (capturing) and purifying of the biomolecule
of interest in the subsequent chromatographic steps.
[0116] Before capturing/purification by means of a resin, it may be
necessary to adjust the parameters of the solution (like salt
composition, conductivity, pH-value) to ensure binding of the
desired biomolecule to the chromatographic support, usually a resin
(this step is, in the meaning of the present invention, termed
"conditioning step"). The simplest conditioning procedure is
dilution of the cleared lysate with water or low salt buffer,
especially in case the chromatographic resin in the subsequent
capture step is achieved by anion exchange chromatography (WO
97/29190 A1). Furthermore, in particular when hydrophobic
interaction chromatography is used as first purification step, a
high concentration salt solution may be added and the possibly
resulting precipitate (which is present if a certain salt
concentration in the solution is exceeded) separated by filtration
or centrifugation (WO 02/04027 A1). In the case ammonium sulfate is
used in high concentrations, this treatment reduces the RNA content
(WO 98/11208 A1).
[0117] For capturing and purification several steps are applied to
obtain a highly purified biomolecule which meets the requirements
for pharmaceuticals. As for the previous steps, enzymes, detergents
and organic solvents should be avoided. Isolation and purification
are performed according to methods known in the art, in particular
by a combination of different chromatographic techniques (anion
exchange chromatography AIEC, hydrophobic interaction
chromatography HIC, size exclusion chromatography (SEC),
ultra(dia)filtration, filtration or precipitation and extraction. A
method that may advantageously be used, in particular for obtaining
pDNA for therapeutic applications, comprises a combination of two
steps that are based on different chromatographic principles, in
which either of the two steps is selected from hydrophobic
interaction chromatography (HIC), polar interaction chromatography
(PIC) and anion exchange chromatography (AIEC) and in which at
least in one of the two steps, preferably in both steps, the
chromatographic support is a porous monolithic bed, preferably a
rigid methacrylate-based monolith in the form of a monolithic
column. Suitable monolithic columns are commercially available
under the trademark CIM.RTM. from BIA Separations). This
purification process may advantageously be performed with a
chromatographic support in the form of a single monolithic bed
comprising a tube-in-a-tube system, the outer and inner tube
carrying different functional moieties. In such a system one of the
monolithic tubes represents the support for the chromatographic
principle of one step and the other tube represents the support for
the chromatographic principle of the other step. Preferably, the
capturing/purification step can be operated in a batchwise mode or
in a quasi-continuous or continuous mode, employing technologies
such as annular chromatography, carousel chromatography or a
simulated moving bed process.
[0118] The process of the invention is suited for, but not limited
to, biomolecules that are sensitive to shear forces, preferably to
polynucleotides, in particular plasmid DNA, and large proteins,
e.g. antibodies.
[0119] The process of the invention can be used for any biomolecule
of interest. For the production of proteins, it may be designed
such that the specific needs of the protein of interest are met.
The method of the invention is independent of the fermentation
process and of the source of the protein (e.g. bacteria,
yeast).
[0120] The choice of specific methods suitable for cell
disintegration and the following processing steps is strongly
influenced by the protein's state in the cells after
fermentation:
[0121] If the protein is overexpressed, it may be present in the
form of so-called "inclusion bodies". In this case, the treatment
with e.g. strong alkali in combination with a reducing agent (e.g.
DTT) during lysis results in a resolubilization of the protein,
which is, at this stage, in its denatured form. To reconstitute the
protein's native structure, refolding can be achieved by
neutralization (e.g. by addition of phosphoric acid) in the
neutralization reactor or in a second reactor similar to the lysis
reactor. Insoluble components are separated from the
protein-containing solution in the clarification reactor.
[0122] In the case the protein of interest is soluble in the cell,
the cells are disintegrated in the lysis reactor in a similar
manner as described above.
[0123] In the lysis reactor, the conditions (contact time,
concentration of the lysis solution) may be chosen in a way that
the protein stays soluble or, alternatively, the parameters are set
to specifically denature and precipitate the protein. In the first
case, the solution is further processed in the neutralization
reactor (which, in terms of construction, is similar to the lysis
reactor or the neutralization reactor used for polynucleotides) and
the clarification reactor, as described for solubilized inclusion
bodies. If the protein is in its denatured state, precipitation can
either already take place in the lysis reactor or afterwards in the
neutralization reactor (by addition of a neutralizing and/or
precipitating agent). In both cases, the conditions for the
precipitation are preferably chosen to specifically precipitate the
protein of interest (while undesired impurities like e.g. RNA,
endotoxins, and DNA stay soluble). The precipitate is subsequently
separated from the solution in the clarification reactor.
Afterwards, the precipitate is either removed from the
clarification reactor (e.g. by sucking off or flushing out with an
appropriate buffer) or directly further processed in this device.
After it has been removed from the reactor, the precipitate is
resolubilized in a separate container using a suitable buffer,
which is empirically determined on a case-by-case basis. In the
case the precipitate remains in the clarification reactor,
resolubilization is done there (by addition of a suitable buffer
and optionally mixing). As soon as the precipitate (especially the
protein of interest) is resolubilized, it can easily be removed
from the clarification reactor through the outlet in the
bottom.
[0124] Common to all variations of the method of the invention in
the production of proteins are the options for further processing
the resulting protein solution. Beside additional refolding steps,
the same steps as described for processing of polynucleotide
solutions (continuous or non continuous concentration,
conditioning, filtration, capturing) may take place.
[0125] The process of the invention meets all regulatory
requirements for the production of therapeutic biomolecules. When
applied to polynucleotides, the method of the invention
yields--provided the fermentation step has been optimized to
provide high quality raw material--high proportion of plasmid DNA
in the ccc form and a low proportion of proteins and chromosomal
DNA. The process neither requires the use of enzymes like RNase and
lysozyme nor the use of detergents except in lysis step b).
[0126] The process of the invention is scalable for processing
large amounts of polynucleotide containing cells, it may be
operated on a "manufacturing scale", to typically process more than
100 grams wet cells, and yielding amounts from 0.1 g to several 100
g up to kg of the polynucleotide of interest that meet the demands
for clinical trials as well as for market supply.
[0127] The applicability of the process is not limited or
restricted with regard to size, sequence or the function of the
biomolecule of interest. A polynucleotide of interest may be a DNA
or RNA molecule with a size ranging from 0.1 to approximately 100
kb or higher. Preferably, the polynucleotide of interest is
circular DNA, i.e. plasmid DNA with a size of preferably 1 to 20
kbp.
[0128] The process and the devices of the invention are not limited
with regard to the cell source from which a biomolecule of interest
is to be obtained.
[0129] The process can be easily implemented and is flexible with
regard to automatization and desired scale; adjustment of the flows
and the reaction times can be achieved by commercially available
pump and pressure systems that ensure steady flows and a low impact
of mechanical stress.
[0130] Another advantage of the present invention is that the
devices are sanitizeable, depyrogenysable and allow cleaning in
place (CIP) and steaming in place (SIP).
[0131] The method and apparatus employed therein provides a
controllable and consistent performance in a closed system,
allowing direct further processing of the continuously produced
lysate obtained after clarification, e.g. loading it to. a
chromatography column or allowing online conditioning of the lysate
prior to column loading. After clarification, there may be an
intermediate concentration step before conditioning or loading onto
the chromatographic column.
[0132] In the process of the present invention, irrespective of
whether steps a) is performed batchwise or in a continuous mode,
each subsequent step may be run in a continuous and automated mode.
Preferably, at least a combination of two steps selected from steps
b) to e) is run continuously connecting the individual steps.
[0133] In the case the lysis step b) is the automated step, it is
independent of how the cell suspension has been obtained (batchwise
or continuous operation, direct use of fermentation broth or
harvest and resuspension, optionally after freezing). It is also
independent of the host from which the lysate has been
obtained.
[0134] In the case the neutralization step c) is the automated
step, the application is independent of how the processed alkaline
lysed cell solution has been prepared (e.g. batchwise or
continuous). In a preferred embodiment the collector tank is
designed in the same way as described for the clarification
step.
[0135] In the case the clarification step d) is the automated step,
the application is independent of how the processed neutralized
lysed cell solution containing flocks has been prepared (e.g.
batchwise or continuous). It is also independent of how the
resulting clarified lysate is further processed.
[0136] In a preferred embodiment, the outflow of the clarification
reactor is combined with the flow of the solution necessary for the
next processing step (conditioning solution) by means of a
connector, e.g. a T- or Y-connector or directly in a mixing device.
The two solutions may be pumped by conventional pumps.
[0137] In another embodiment only the flow rate of the second
solution is adjusted to the flow-rate of the lysate leaving the
clarification reactor. The mixing device for this purpose may be a
device filled with beads like the one described for the automated
lysis step or a tubing system like the one described for the
neutralization step. Such a setup may be used if conditioning of
the lysate for the first chromatographic step is necessary. For
example, a solution of ammonium sulfate (or simply water) can be
added in this way.
[0138] In another embodiment, the process also contains an
intermediate concentration step: as soon as a sufficient volume of
the lysate leaving the clarification reactor is present, the lysate
is concentrated, e.g. by means of ultrafiltration, prior to
conditioning and/or loading onto the chromatography column.
Concentration may be done in one or more passages. In the latter
case, the concentration step as such may be in a continuous or
batchwise mode. If only one passage takes place, the retentate
(e.g. containing the pDNA) may subsequently be directly conditioned
or loaded to a chromatography column. In the case of several
passages, the retentate is recycled until the desired final
volume/concentration is reached, and subsequently further
processed. For this concentration step, conventional devices can be
used, e.g. membranes in form of cassettes or hollow fibres. The
cut-off of suitable membranes depends on the size of the
biomolecule processed. For pDNA, usually membranes with a cut-off
between 10 and 300 kDa are used.
[0139] In a preferred embodiment, the lysis reactor and the
neutralization reactor are combined to form a two-step automated
system. In this case, the outflow of the lysis reactor is connected
and mixed with the flow of the neutralization solution in the
manner described for the automated neutralization step. By this,
the flow rate of the pumped neutralization solution is adjusted to
the flowrate of the outflow of the lysis reactor.
[0140] In another preferred embodiment the neutralization reactor
and the clarification reactor are combined to form a two-step
automated system. In this case, the outflow of the neutralization
reactor is connected with the automated clarification reactor of
the invention. In this case, the pressure of the compressed air has
to be adjusted such that the outflow of the reactor is kept
constant. This may be achieved by measuring the fluid level by
means of an integrated floater or similarly by measuring the flow
at the outlet. Also other systems like light barriers are
applicable. By means of an electronic connection to the pressure
gauge the pressure can be adjusted steplessly according to the
fluid level or the outlet flow.
[0141] In another embodiment the lysis step and the clarification
step are connected by directly connecting the two reactors without
an intermediate distinct neutralization step. Neutralization may in
this case be carried out in the clarification reactor. In this
embodiment, the outlet of the reactor is closed at first and the
lysed cell solution is mixed with a certain volume of
neutralization solution by mixing slowly with a stirrer or
introducing air through the distributor from the top or from an
inlet in the bottom of the reactor. At the end of neutralization,
automated clarification takes place in the same manner as described
above.
[0142] In an even more preferred embodiment, the whole system is
fully automated by employing at least all steps b) to d) and
optionally, in addition, step a) and/or e) in a continuous system.
In this embodiment, the outflow of the lysis reactor is directly
connected with the neutralization device and the outflow of the
neutralization device is directly connected with the clarification
reactor. The design for the individual connections and devices is
the same as described above for the two-step automated systems.
[0143] In a most preferred embodiment, the fully automated system
is connected to an optional automated conditioning step (and
device). This embodiment allows continuous mixing of the clarified
lysate that leaves the clarification reactor with a conditioning
solution (e.g. an ammonium sulphate solution). As described above,
such conditioning step may be necessary to prepare the
polynucleotide containing lysate for the subsequent
(chromatographic) purification steps (e.g. hydrophobic interaction
chromatography).
[0144] Adding such a conditioning step results in an extension of
the automated three-step system to a continuous four-step system.
In this embodiment, a conditioning solution can be continuously
mixed with the clarified lysate using a device, which is preferably
of the same type as the lysis reactor. This device was found to be
most gentle for continuous mixing of solutions containing
polynucleotides that are sensitive to shear forces. Yet also other
devices (e.g. as described for the neutralization step) can be
utilized for this purpose, e.g. conventional static mixers. The
flow rate of the pump that pumps the conditioning solution can be
adjusted to the flow rate of the outflow of the clarification
reactor by installing a flow measurement unit. The pump can be
connected with this unit and thus regulated, keeping the ratio of
the flow rates of the two mixed solutions constant.
[0145] Between conditioning and capture step, an on-line filtration
step may be inserted.
[0146] In yet another embodiment of the invention, an
ultrafiltration step is added. By such an extension of the
automated three-step system, the process represents a continuous
four-step system. In this embodiment the resulting lysate of the
previous steps is concentrated by ultrafiltration. While the
permeate is discarded, the retentate is either directly further
processed by the conditioning step and/or by the loading step
(which means an extension of the continuous system by one or two
additional steps) or recycled until a desired final
concentration/volume is reached. In the latter case, the resulting
concentrate is further processed (conditioning and/or loading)
after concentration is finished.
[0147] In another embodiment, the lysate flowing out of the
clarification reactor may be directly loaded onto a chromatographic
column, or it may be loaded onto the column after conditioning
(with or without subsequent on-line filtration).
[0148] In all described embodiments utilizing the automated
clarification step the obtained cleared lysate may either be
collected in a suitable tank or directly further processed (e.g. by
connecting the outflow of the clarification reactor with a
chromatographic column). If a conditioning step is employed in this
automated process, the conditioned lysate can either be collected
in a suitable tank or directly further processed.
[0149] The method and devices of the invention are independent of
the pumps used for pumping the solutions. In a special embodiment,
the flow of the several suspensions and solutions is accomplished
by air pressure in pressurized vessels instead of pumps.
[0150] Due to these advantages, the process and devices of the
invention are suitable for cGMP (Current Good Manufacturing
Practice) production of pharmaceutical grade pDNA. The process can
be adapted to any source of pDNA, e.g. to any bacterial cell
source. In particular due to the properties of the system, the
process of the invention allows fast processing of large volumes,
which is of major importance for processing cell lysates. Since the
lysates contain various pDNA-degrading substances such as DNAses,
process time is a key to high product quality and yield.
[0151] The process and device of the invention are suited for
production of pDNA for use in humans and animals, e.g. for
vaccination and gene therapy applications. Due to its high
productivity, the process can be used for production of preclinical
and clinical material as well as for market supply of a registered
product.
Example 1
Production of pDNA-Containing E. Coli Cells
[0152] The pDNA containing E. coli biomass for the pilot scale runs
was produced at 20 l or 200 l fermentation scale according to the
following procedure (this description relates to the 20 l
fermentation):
Pre-Culture
[0153] The working cell bank of a production strain of the plasmid
pRZ-hMCP1 (Escherichia coli K12 JM108; ATTC no. 47107; plasmid
size: 4892 kbp) was maintained in cryo vials (glycerol stocks) at
-70.degree. C. A cryo vial of the working cell bank was thawn at
room temperature for 15 min and a 200 .mu.l aliquot thereof was
inoculated in a 1000 ml Erlenmeyer shake flask containing 200 ml
autoclaved preculture medium (composition in gL-1: Vegetable
Peptone/Oxoid 13.5; Bacto Yeast Extract/Difco 7.0; glycerol 15.0;
NaCl 1.25; MgSO.sub.4*7H.sub.2O 0.25; K.sub.2HPO.sub.4 2.3;
KH.sub.2PO.sub.4 1.5). The preculture was incubated at
37.degree.+0.5.degree. C. and 150 rpm up to an optical density (OD
550) of 1-1.5.
Fermenter Preparation
[0154] A fermenter of a total volume of 30 l (continuous stirred
tank reactor) was used for fermentation. Three of the medium
components (in gL-1 final culture medium: Vegetable Peptone/Oxoid
13.5; Bacto Yeast Extract/Difco 7; glycerol 15) was heat sterilized
inside the fermenter at 121.degree. C. for 20 min. After cooling
down the fermenter content to <40.degree. C., a macro element
solution (in gL-1 final culture medium: tri-Sodium citrate
dihydrate 0.5; KH.sub.2PO.sub.4 1.2; (NH.sub.4).sub.2SO.sub.4 5.0;
MgSO.sub.4*7H.sub.2O 8.8; Na.sub.2HPO.sub.4*12H.sub.2O 2.2;
CaCl.sub.2*2H.sub.2O 0.26; NH.sub.4Cl 4.0) was sterile-filtered
into the fermenter. By sterile filtration with a syringe, 5 ml of a
1% m/v thiamine solution and 1.5 ml of a trace element solution was
transferred into the fermenter. The trace element solution consists
of (in gL-1 solution): CoCl.sub.2*6H.sub.2O 0.9;
CuSO.sub.4*5H.sub.2O 1.23; FeSO.sub.4*7H.sub.2O 38.17;
MnSO.sub.4*H.sub.2O 1.82; Na.sub.2MoO.sub.4*2H.sub.2O 0.48;
NiSO.sub.4*6H.sub.2O 0.12, ZnSO.sub.4*7H.sub.2O 5.14. The
fermentation medium was filled up with sterile deionized water to
the final working volume of 20 L.
Fermentation and Harvest of Cell Paste:
[0155] The total pre-culture volume of 200 ml was transferred into
the fermenter under sterile conditions. The cultivation conditions
were set as follows: aeration rate 20 l min-1=1 vvm; agitation rate
400 rpm, 37+0.5.degree. C.; 0.5 bar; pH 7.0+0.2). The pH value was
automatically controlled with 5 M NaOH and 25% m/v H.sub.2SO.sub.4.
The concentration of dissolved oxygen (DO, pO.sub.2) was maintained
at >20% of saturation by automatic control of agitation rate
(400-700 rpm).
[0156] Cultivation was terminated 12 h after inoculation of the
fermenter. After cooling down the culture broth to <10.degree.
C., the cells were harvested by separating in an ice water-cooled
tube centrifuge. The obtained cell paste was packaged and stored at
-70.degree. C.
[0157] The experiments were performed with pDNA containing E. coli
biomass of different 20 l batch and fed-batch fermentations as well
as of 200 L batch fermentations (two different hosts, four
different plasmids).
Example 2
Setting Up a Pilot Scale System for Continuous Alkaline Lysis,
Neutralization and Clarification
[0158] The setup of the pilot scale system for the continuous
combination of alkaline lysis, neutralization and clarification on
which the experiment of Example 3 is based, is shown in FIG. 7.
FIG. 6 shows a schematic image of the components and the basic
construction of the continuous three step combination. This Figure
also relates in principle to the lab scale model (up to 100 g wet
cell weight; used for preliminary experiments), to the up-scale
variant (capable for handling up to 6 kg biomass) of the system and
to the cGMP production system (up to 20 kg wet cell weight).
[0159] In FIG. 6, {circle around (1)} are three similar pumps,
which transport the cell suspension (I), the lysis solution (II)
and the neutralization solution (III). {circle around (2)} is the
first meeting point constructed as a T-connection. {circle around
(3)} shows the lysis reactor (inner diameter: 6 cm, height 45 cm)
filled with glass beads of 5 mm diameter. {circle around (4)}
indicates the second meeting point, again constructed as T
connection. {circle around (5)} shows the neutralization reactor
(coiled polypropylene tubing of 12.5 mm inner diameter and 3.5 m
length). {circle around (6)} shows the clarification reactor
constructed as a glass cylinder of 180 mm inner diameter and a
height of 500 mm. In the center, a slotted stainless steel tube is
built in to distribute the entering solution. On the top of the
clarification reactor, a connection for pressurized gas and a
pressure gauge is located. In the bottom of the clarification
reactor the retention material (glass beads; diameter 0.75-1 mm) is
filled in up to a height of 4-5 cm. Next to the retention layer
{circle around (7)} of the system is the outlet of the
clarification reactor. {circle around (8)} is the collector tank,
which collects the cleared lysate that leaves the clarification
reactor. {circle around (9)} are conventional three-way valves to
change the flow-paths to carry out degassing of the system and
washing of the flocks in the clarification reactor.
Example 3
Utilization of the Pilot Scale System for Continuous Alkaline
Lysis, Neutralization and Clarification
[0160] 990 g wet biomass, prepared according to the above mentioned
procedure (Example 1), was resuspended in 10 l of a buffer
containing 0.05 M Tris-HCl, 0.01 M EDTA at pH 8, by mixing at room
temperature (impeller stirrer) in a glass container for one
hour.
[0161] Before the subsequent process was started, the lysis reactor
and the neutralization reactor were degassed by pumping the
suspensions and solutions with the 3 pumps.
[0162] Afterwards, all three pumps (pump I for the resuspended
biomass, pump II for the lysis solution and pump III for the
neutralization solution) were started simultaneously and adjusted
to the same flow-rate (150 ml/min) providing the desired contact
time of the cells with the lysis solution and of the lysed cell
solution with the neutralization solution in the respective
reactors (lysis reactor, neutralization reactor).
[0163] Thereby the resuspended cells came into contact with the
lysis solution (0.2 M NaOH, 1% SDS) at the first meeting point. The
resulting stream was subsequently mixed homogeneously and contacted
(1.5-2 min) in the lysis reactor by passing the glass beads.
Directly after leaving the neutralization reactor, the now lysed
cell solution was brought into contact with the neutralization
solution (3 M potassium acetate at pH 5.5) at a second meeting
point (T-connector). Both streams were mixed homogeneously in the
following neutralization reactor and contacted (1-1.5 min). The
mixture of the pDNA containing lysate and the precipitated
impurities (flocks) were then transported into the clarification
reactor by gently flowing down the special designed device. In this
way, the mixture reaches the retention material in the bottom part
of the reactor. Later the mixture is distributed on the surface of
the lysate/flock-mixture that is already present in the reactor,
with the majority of flocks floating. When the clarification
reactor is filled up to 10 cm (above the retention material), the
outlet of the reactor (which was closed so far) was opened to
recover the cleared lysate at the outlet. Thereby the flocks are
retained by the retention material. To accelerate the process, the
pressure was increased stepwise (0.25 bar/2 l lysate) by
introducing pressurized air to ensure constant outflow from the
reactor. At the end, the system was washed with 1 l of the
respective solutions (without cells) in order to recover the
residual cells in the system in the form of lysed cells. The
residual mixture in the clarification reactor was exposed to a
maximum of 2 bar pressure and the lysate recovered to the largest
possible extent, while the flocks stayed in the reactor. To also
recover the pDNA containing lysate between the flocks, a gentle
washing procedure was applied. Neutralization solution was pumped
from the outlet of the clarification reactor into the device and
through the precipitate. After this, the flocks were drained by
exposing the mixture of flocks and wash-solution to an overpressure
of up to 2 bar. The cleared lysate including the wash fraction was
further processed by several subsequent chromatographic steps (HIC,
AIEC using an 80 ml CIM.RTM. tube and SEC). Analysis was carried
out by HPLC. As a reference sample, an aliquot of the resuspended
cells equal to 1 g wet biomass was lysed and neutralized in a small
tube according to the conventional lab-scale procedure,
clarification being carried out by centrifugation (12.000 g). This
sample was used to calculate the yield of the pilot-scale process
and to compare homogeneity (criterion for smoothness and quality).
In addition the purity of the pDNA-solution could be approximated
(HPLC).
[0164] The comparison of the reference lysate and the lysate
obtained from the continuous system (Table 1) shows that the
results of the lab-scale lysis, that is known to be very gentle,
and of the novel pilot-scale-system were comparable.
TABLE-US-00001 TABLE 1 mg pDNA/ Homogeneity g WCP Purity oc ccc
unid. Reference lysate 1.667 mg 4.0% 2.1% 89.9% 8.0% Lysate of
1.694 mg 5.0% 2.3% 90.0% 7.7% continuous system
Example 4
Setting Up an Up-Scaled System and a cGMP Production System for
Continuous Alkaline Lysis, Neutralization and Clarification
[0165] The principle construction of the up-scaled system and of
the system for cGMP production is similar to the pilot scale system
described in Example 2. FIG. 8 shows the up-scaled system, while
FIG. 9 displays the cGMP-production system. The dimensions of the
systems were adapted such that key parameters like linear velocity,
contact time and process time are kept in a range comparable to the
pilot system when larger amounts of resuspended cells need to be
processed.
[0166] The stainless steel lysis reactor of the up-scaled system
has an inner diameter of 10 cm and a height of 70 cm. The
neutralization reactor is a polypropylene tubing of 19 mm inner
diameter and 900 cm length, while the clarification reactor is
constructed as a glass cylinder of 45 cm inner diameter and 50 cm
height.
[0167] The lysis reactor of the production system was constructed
as a stainless steel cylinder of 11 cm inner diameter and 84 cm
height. The neutralization coil is made of a polypropylene-tubing
of 25.4 mm inner diameter and 900 cm length. In this setup the
clarification reactor consists also of stainless steel. A cylinder
of 60 cm inner diameter and 65 cm height was constructed as a
CIPable (CIP=cleaning in place) version.
[0168] Both systems are equipped with a flushing device in the
clarification reactor to remove the majority of flocks of the
reactor before the reactor is cleaned. In addition for safety
reasons the systems are equipped with burst disks. The production
system is especially designed for hygienic use and cleaning. All
parts are CIPable and SIPable (SIP=steaming in place).
[0169] As retention material glass beads of 0.75-1 mm or 0.42-0.84
mm were used.
Example 5
Utilization of the Pilot System for Continuous Alkaline Lysis,
Neutralization and Clarification and Further Continuous
Conditioning, Filtration and Capturing
[0170] As an option to extend the system of Example 2 and 3, the
direct continuous connection of the subsequent steps conditioning,
filtration and capturing was tested. Since hydrophobic interaction
chromatography (HIC) was the first step of the chromatographic
purification sequence, the lysate had to be conditioned by addition
of ammonium sulfate to obtain binding of pDNA to the resin.
[0171] Therefore a lysate (of about 250 g wet cell paste, produced
according to Example 1) obtained by the method described in Example
3 and by the device described in Example 2 was collected in a
collection vessel. As soon as a sufficient volume of clarified
lysate was present in this container, the automated
conditioning-filtration-capturing procedure (according to FIG. 4)
was started.
[0172] Two additional piston pumps were used in this extended
setup. One piston pump was used to transport the cleared lysate at
a flow rate of 28 mL/min while the other one, pumping a 4 M
ammonium sulfate solution was adjusted to the double velocity (56
ml/min). Both streams were connected by a conventional Y-connector.
The combined stream was entered to a mixing device similar to the
lysis reactor, allowing sufficient homogenous mixing and
contacting. As mixing device, a tube of 2.6 cm inner diameter and
100 cm length, filled with glass beads of 5 mm inner diameter was
used. The contact time (here: about 2.5 minutes) was defined by the
flow-rate through this conditioning reactor and by the free volume
inside the reactor. During this conditioning procedure,
precipitation of RNA and other impurities (e.g. endotoxins) took
place. In order to load a solution free of particles to the
chromatography column, a filter (4.5 .mu.m pore size) was connected
with the outlet of the conditioning reactor, thus providing an
on-line filtration. The clear solution (containing the pDNA)
leaving the filter was directly and continuously loaded onto the
chromatography column (inner diameter 7 cm, bed height 25 cm)
filled with Toyopearl Butyl 650 M. Under these conditions, pDNA was
binding onto the resin and was separated from the majority of
impurities during elution (performed after the entire conditioned
lysate was loaded and a subsequent "wash"-step with an appropriate
buffer). The result is displayed as HPLC-chromatogram of the
resulting HIC-pool in FIG. 12. Impurities could be decreased to
about 45% and that the oc pDNA (before in the range of 10%) was
separated mostly.
Example 6
Utilization of the Up-Scaled System for Continuous Alkaline Lysis,
Neutralization and Clarification
[0173] To show scalability of the continuous three-step-system, the
up-scaled system (described in Example 4) capable for up to 6 kg of
wet cell paste was used to prepare a clarified lysate processing
5.4 kg wet cell paste produced according to Example 1 in a 200 L
fermentation. After resuspension of the previously frozen biomass
in 54.4 l resuspension buffer and degassing the system lysis,
neutralization and clarification were carried out methodically as
described in Example 3. The pumps were adjusted to 0.5 l/min
providing a contact/mixing time of 1-1.5 minutes in the lysis and
neutralization reactor. The resulting flock lysate mixture was
separated in the clarification reactor, where the flocks were
floating and retained by the retention material (0.42-0.84 mm). At
the end of the process the retained flocks in the clarification
reactor were washed from both sides with a buffer containing 0.017
M Tris-HCl, 0.003 M EDTA, 0.067 M NaOH and 1M potassium acetate at
a flow rate of 1 l/min. Finally the flocks were drained by applying
2.3 bar over pressure (pressurized air). The result is shown as
analytical HPLC-chromatogram in FIG. 13. The obtained clarified
lysate was further (stepwise) processed by the conditioning step
(including filtration) and capturing (HIC). FIG. 14 shows the
analytical HPLC chromatogram of the pool from the first
chromatography step. The homogeneity of the lysate was about 93.5%
and the approximated purity (roughly estimated by HPLC) about 10%
while the HIC-pool showed a homogeneity of about 94% and an
approximated purity of about 92%.
Example 7
Utilization of the Lab Scale System for Continuous Alkaline Lysis,
Neutralization and Clarification Followed by Concentration,
Conditioning and Capturing
[0174] The concentration of lysate leads to a reduction of the
volume of 4 M ammonium sulfate solution (if HIC is the following
chromatography step) needed for conditioning and the duration of
column loading. The clarified lysate was concentrated with a hollow
fiber membrane (UFP-100-E4X2MA, Quix Stand) of Amersham Biosciences
(100 kDa cut off).
[0175] For this Example, 70 g biomass as obtained by the method
described in Example 1 was processed according to the description
in Example 2 in the lab scale system (pumps: 15 ml/min; contact
time: .about.1 min). The resulting lysate was collected in a glass
vessel. The glass vessel was connected with a peristaltic pump to
feed the ultrafiltration membrane. After 100 ml of lysate were
collected in the glass vessel ultrafiltration was started and
continuously continued. Therefore the pump speed and the trans
membrane pressure (.about.0.3-0.4 bar) were adjusted in a way that
permeate and retentate flow were similar (20 ml/min respectively)
resulting in a pDNA-concentration factor of 2-fold in the
retentate. The obtained retentate is shown in FIG. 15 as analytical
HPLC-chromatogram. While the lysis took 50 minutes (without washing
of the flocks) the parallel concentration took about 60 minutes (to
be sure not to run out of lysate). The retentate was collected in
an intermediate vessel, which was used as feed tank for the
following continuous steps conditioning, filtration and capturing,
which are described in Example 5. The flow-rate of the pumped
concentrated lysate (retentate) was therefore adjusted to 15 ml/min
and for the ammonium sulfate solution to 30 ml/min). The column
used for capturing had an inner diameter of 5 cm and a bed height
of 25 cm).
* * * * *