U.S. patent application number 14/423394 was filed with the patent office on 2015-07-30 for virus purification and formulation process.
The applicant listed for this patent is GE Healthcare Bio-Sciences Corp.. Invention is credited to Edward G. Hayman, Joseph Makowiecki, John J. Vicalvi, JR..
Application Number | 20150210986 14/423394 |
Document ID | / |
Family ID | 48998541 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150210986 |
Kind Code |
A1 |
Vicalvi, JR.; John J. ; et
al. |
July 30, 2015 |
VIRUS PURIFICATION AND FORMULATION PROCESS
Abstract
Disclosed herein is provided a virus purification and
formulation process for purifying a flavivirus represented by one
of a a Yellow Fever Virus, Japanese Encephalitis virus, Dengue
virus, and West Nile virus. The highly purified flavivirus virus
product is characterized as having a low level of sucrose without
significant virus loss such as that which is typically encountered
by prior art virus purification processes. The disclosed process
captures and purifies the virus, separating it from the host cell
proteins and DNA, and leaving the host cell proteins and DNA
behind. The process also can be used to inactivate and/or
concentrate the virus sufficiently for use in formulations.
Inventors: |
Vicalvi, JR.; John J.;
(Shrewsbury, MA) ; Hayman; Edward G.; (Hanover,
NH) ; Makowiecki; Joseph; (Oxford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GE Healthcare Bio-Sciences Corp. |
Piscataway |
NJ |
US |
|
|
Family ID: |
48998541 |
Appl. No.: |
14/423394 |
Filed: |
August 16, 2013 |
PCT Filed: |
August 16, 2013 |
PCT NO: |
PCT/US13/55302 |
371 Date: |
February 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61692956 |
Aug 24, 2012 |
|
|
|
Current U.S.
Class: |
435/238 |
Current CPC
Class: |
C12N 2770/24151
20130101; C12N 7/00 20130101; C12N 2770/24163 20130101 |
International
Class: |
C12N 7/00 20060101
C12N007/00 |
Claims
1. An essentially pure inactivated flavivirus virus preparation
which is purified by the process comprising: a. obtaining a
composition suitable for purification from virus-infected cell
culture; b. clarifying the composition obtained in step "a" by
removing cells and cell debris from said composition by depth
filtration; c. subjecting the filtrate in step "b" to Benzonase
digestion and 0.2 Micron Filtration sufficient to fragment Vera
cell DNA followed by filtration; d. purifying the filtrate obtained
in step "c" via CELLUFINE.RTM. sulfate chromatography to achieve
depyrogenation of said virus in said filtrate; e. stabilizing and
inactivating the viral product obtained in step "d" with an
inactivation amount of .beta.-PL; and f. formulating the product
obtained in step bound to alum.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method of
purifying biologics, such as a virus or modified virus, and a
method of formulating the biologic, for example, a virus and
adjuvant.
BACKGROUND OF THE INVENTION
[0002] In order to purify a virus it is necessary to remove host
cell proteins and DNA from the virus sample. Unfortunately, current
methods of removing host cell proteins and DNA are accompanied by
loss of a significant amount of virus. Typically, for a flavivirus,
recovery of the virus from filtration processes are generally about
ten to twenty percent and typically less than 10 percent. Following
purification, another problem is aggregation or clumping together
of the purified virus particles.
[0003] Prior art methods of purification and concentration of a
virus, e.g., often use ultracentrifugation wherein sucrose is
required in order to run the gradient for the separation. Such
ultracentrifugation generally results in the undesirable presence
of sucrose in the final virus sample. In order to obtain a virus
sample that is free of sucrose, tangential flow filtration (TFF) is
frequently used to separate the virus from proteins and other
compounds. However, use of TFF for such a purification tends to
result in significant loss of virus.
[0004] Prior art methods of purification and concentration of a
virus, e.g., often use ultracentrifugation wherein sucrose is
required in order to run the gradient for the separation. Such
ultracentrifugation generally results in the undesirable presence
of sucrose in the final virus sample. In order to obtain a virus
sample that is free of sucrose, tangential flow filtration (TFF) is
frequently used to separate the virus from proteins and other
compounds. However, use of TFF for such a purification tends to
result in significant loss of virus. As well, currently used
methods of virus purification thus have been accompanied by ongoing
problems including low yield or loss of virus, host cell protein
levels higher than desirable, high sucrose levels, and aggregation
of the purified virus particles. Such methods have also been
difficult to use in single use or disposable technologies.
BRIEF SUMMARY OF THE DISCLOSURE
[0005] In accordance with the present invention there is provided a
virus purification process for producing highly purified virus
product having no residual sucrose without significant virus loss
such as that which is typically encountered by prior art virus
purification processes. The disclosed process captures and purifies
the virus, separating it from the host cell proteins and DNA, and
leaving the host cell proteins and DNA behind. The process also can
be used to inactivate and/or concentrate the virus sufficiently for
use in formulations.
[0006] Also disclosed herein is a one-step process for producing a
formulation wherein the concentrated virus particles are not
aggregated or clumped together. The disclosed process utilizes
adjuvants and buffer exchanges to process the virus, and results in
a final, buffered virus formulation. An embodiment of the process
can also be used for formulation of other biologics.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Embodiments of the invention are further described
hereinafter with reference to the accompanying drawings, in
which:
[0008] FIG. 1 is a process chromatogram for Experiment Y1626A, an
embodiment of the new downstream process disclosed herein that is
used to purify and formulate a harvest of modified YF virus.
[0009] FIG. 2 is a process chromatogram for Experiment Y1632A, an
embodiment of the new downstream process disclosed herein that is
used to purify and formulate a harvest of modified YF virus,
comparing elutions using CELLUFINE.RTM. sulphate and CAPTO.TM. Core
700.
[0010] FIG. 3 is a process chromatogram for Experiment Y1637A.
[0011] FIG. 4 is a process chromatogram for Experiment Y1639A using
CELLUFINE.RTM. Sulfate column.
[0012] FIG. 5 is a process chromatogram for Experiment Y1639A using
CAPTO.TM. DeVirS Column.
[0013] FIG. 6 is a flow diagram for an embodiment of the old
downstream purification process and a flow diagram for an
embodiment of the new downstream purification process.
[0014] FIG. 7 is a flow diagram for the embodiment of the old
downstream purification process shown in FIG. 6 and a flow diagram
for an embodiment of the new and improved downstream purification
process.
[0015] FIG. 8 is a flow diagram for another embodiment of an old
downstream purification process and a flow diagram for an
embodiment of the new and improved downstream purification process,
Number 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] The inventors of the present subject matter have now
discovered a process for preparing a highly purified biological
composition, such as, for example, a virus. The process is
particularly useful for purifying a flavivirus, e.g., a Yellow
Fever Virus, Japanese Encephalitis virus, Dengue virus, and West
Nile virus. The examples provided herein are for purifying a Yellow
Fever virus, but with no more than routine experimentation, could
be used to purify other types of viruses.
[0017] The disclosed process significantly reduces the loss of
viral particles as compared to prior art methods for obtaining a
highly purified sample of a biologic. Use of an embodiment of the
disclosed method also significantly lowers the amount of sucrose in
the final purified product as compared to prior art methods of
purification.
[0018] We now describe the development of a chromatographic process
resulting in vaccine material with DNA and host cell contaminant
levels at or below the level of detection while retaining high
levels of virus recovery. Disclosed herein is a Yellow Fever (YF)
vaccine downstream purification process designed to inactivate live
YF virus, fragment Vero DNA, and remove any process contaminants,
such as residual Vero host cell proteins and DNA, residual
BENZONASE.RTM. and beta-Propiolactone (BPL).
[0019] YF virus may be grown on Vero cells, harvested, inactivated,
and purified.
[0020] Typically, prior art methods for downstream purification of
a virus provide a YF virus recovery of only about 20 percent (%);
and the final virus product for dosing includes a relatively high
amount of Host Cell Protein (HCP). A typical YF vaccine dose for a
phase 1 clinical trial may be about 0.5 milliliters (ml) of YF
virus in a suspension. The remaining HCP in a dose purified by
prior art methods may typically be about forty-five thousand
nanograms (45,000 ng/dose) wherein the dose is about 8.3 log10
Viral Equivalents (VE). VE is the Elisa unit for YF.
[0021] In contrast, the disclosed method provides, for the modified
YF virus purification tests run to date, a downstream virus
recovery of from about 30 percent to about 100 percent; and for a
dose of about 0.5 milliliters (ml) of YF virus, a HCP level that is
below the limit of detection of the commercially used vero cell HCP
assay.
[0022] To purify the final virus product by reducing host cell
proteins while maximizing virus recovery, we designed and executed
a number of experiments.
[0023] Modified Yellow fever virus was prepared from the attenuated
YF 17D virus available commercially as the vaccine, YF-VAX.RTM.
(Sanofi Pasteur, Swiftwater Pa.). The attenuated YF 17D virus was
adapted by serial passage to replicate more efficiently in Vero
cells derived from the WHO Vero 10-87 Cell Bank and passaged in
serum free medium. 10 serial passages were used to modify the
nucleotide sequence of the viral genome virus to develop a seed
virus with enhanced growth in Vero cells for preparation of an
inactivated Yellow Fever virus candidate. See PCT/US2010/043010,
"High Yield Yellow Fever Virus Strain With Increased Propagation In
Cells", filed 23 Jul. 2010 and published 3 Feb. 2011 as WO
2011/014416, and PCT/US2011/022347, filed 25 Jan. 2011. The entire
teachings of the referenced PCT applications are incorporated
herein by reference.
[0024] The master and working virus seeds were manufactured from
the conditioned cell culture medium harvested from stationary
cultures of Vero cells prepared from the Manufacturers' Working
Cell bank (MWCB). For vaccine production, the working virus seed
was used to infect Vero cells prepared from the MWCB grown on
CYTODEX.TM. 1 microcarriers in either a 50 liter, single use
bioreactor (working volume 25 to 40 L) or a 10L glass bioreactor
(working volume 8 L) (Xcellerex, Inc., Marlborough, Mass.). The
virus released into the cell culture medium was harvested from
about 5 to about 7 days after infection. From about 2 days to about
3 days before harvest of the virus, the culture was re-fed with
fresh medium. This re-feeding step has been shown to increase virus
yield. See PCT/US2010/043013, "Drain Down and Re-Feed of
Microcarrier Bioreactor," filed 23 Jul. 2010 and published 27 Jan.
2011 as WO 2011/011660, the entire teachings of which are
incorporated herein by reference.
[0025] Some of our initial experiments in developing a purification
process for the virus are schematically represented in the flow
chart on the left side of each of FIG. 6 and FIG. 7, "Downstream
Old Process." One set of steps was: Harvest Clarification and
BENZONASE.RTM. Addition; Ultra Filtration and DiaFiltration
(UF/DF); BPL Inactivation; CELLUFINE.RTM. Sulfate (Chisso
Corporation, Osaka JAPAN) Column Purification; Eluate Dilution and
Creation of Sub-Lots; Formulation with Alum, and Bulk Vaccine Drug
Product Formulation.
[0026] Additional process development studies were initiated, with
the goals of reducing residual Vero HCP; increasing overall virus
recovery; removing residual sucrose in the virus sample; and
minimizing aggregation of the purified virus particles. The result
of the development work was an improved downstream purification
process, the overall schematic of which is represented in in the
flow chart on the right side of each of FIG. 6 and FIG. 7,
"Downstream New Process."
[0027] In one embodiment of the invention, the New Downstream
Process outlined in the right side of FIG. 6 can have the following
steps, each represented by a rectangle in the Figure: Following
Virus Harvest, (1) Clarification using a depth filter, and buffer
adjustment. (2) BENZONASE.RTM. digestion and 0.2 Micron Filtration,
producing a live virus bulk. (3) Purification using CELLUFINE.RTM.
Sulfate Chromatography and Dilution for inactivation step. (4)
Virus Inactivation and 0.45 Micron Filtration. (5) Sucrose Gradient
UltraCentrifugation. (6) Identify Fractions; Pool Fractions; Warm
to from about 25.degree. C. to about 30.degree. C.; 0.2 Micron
Filtration, forming Bulk Drug Substance, a purified, inactivated
virus. (7) Alum Binding and Formulation.
[0028] In another embodiment of the invention, the New and Improved
Downstream Process outlined in the right side of FIG. 7 can have
the steps as in the New Downstream Process shown in FIG. 6, with
the following changes. Just prior to the virus inactivation step,
there is a Purification Step using GE CAPTO.TM.Core 7FT with one
percent (Human Serum Albumin) HSA or rHSA and adjustment to 20
percent sucrose/0.0005 percent TWEEN.TM.-20. Then following the
virus inactivation, there is an adjustment with one percent human
albumin or rhuman albumin and 0.2 micron filtration to yield the
bulk drug substance which can then be formulated with Alum, buffer
exchange, and stabilizers.
[0029] It should be noted that the virus data determined by the
2E10 monoclonal antibody is elevated when bound to alum. While not
being bound by theory, we postulated that the virus particle is
arranged on the surface of the alum hydrogel so that the epitope
may be presented in a more open form. Another hypothesis is that
the particles are arranged in a more symmetrical manner, thus
precluding the formation of aggregates which could mask epitope
exposure.
[0030] These experiments indicate that the recovery of intact virus
may be in some way dependent upon the presence of a small amount of
"chaperone" protein. We have discovered, unexpectedly, that
increasing the post sucrose gradient purified pool temperature
prior to filtration results in significantly greater recovery of
virus prior to alum binding, thereby resulting in an increase in
virus recovery. This effect of temperature may be dependent upon
the presence of the "chaperone" protein at a sufficient
concentration.
Virus Harvest and BENZONASE.RTM. Treatment
[0031] The conditioned cell culture medium containing virus was
removed from the bioreactor and clarified by depth filtration. A
Millipore DE50 depth filter was used to clarify the Virus Harvest.
The depth filter was flushed twice, once with USP purified H.sub.2O
followed by buffer with the target formulation 20 mM Tris, 145 mM
NaCl, pH 8. The harvest material was passed through the depth
filter at a flow rate of approximately 500 mL/min and a pressure
not to exceed 25 psi.
[0032] Filtered material was collected into a bioprocess single-use
bag. The depth filter was chased with the same buffer and the chase
volume was combined with the original filtrate. Following depth
filtration, the material was adjusted to a target formulation of 50
mM Tris, pH 8 and 2 mM MgCl2 in preparation for the subsequent
BENZONASE.RTM. treatment step. The adjusted clarified harvest was
mixed for approximately 10 min at room temperature.
[0033] The adjusted clarified virus intermediate was treated with
BENZONASE.RTM. in order to fragment Vero cell DNA. BENZONASE.RTM.
was added to the adjusted clarified virus to a final target
concentration of 3 units/mL, and the suspension was mixed for 16 to
18 hours at room temperature. After BENZONASE.RTM. treatment the
product pool was 0.5 .mu.m filtered.
CELLUFINE.RTM. Sulfate
[0034] The virus was further purified and concentrated by
CELLUFINE.RTM. sulfate chromatography. CELLUFINE.RTM. sulfate
(Chisso, Tokyo, Japan) is a virus affinity resin designed to
concentrate, purify and depyrogenate virus. This process step
significantly reduces Vero cell proteins, endotoxin and Vero cell
DNA. The 2E10 ELISA that detects a YF viral envelope epitope is
used to measure the virus concentration on a sample of the
clarified and BENZONASE.RTM.-treated live virus to ensure the
column is appropriately sized to process a virus mass challenge of
no more than 5-6E+09 VE per /ml of CELLUFINE.RTM. sulfate
resin.
[0035] Prior to loading the live virus material, the column was
charged with 0.1 M NaoH/0.5 M NaCl buffer at an approximate linear
flow rate of 200 cm/hr. The column was then equilibrated with
equilibration buffer, 10 mM Tris, 145 mM NaCl, pH 7.5 at an
approximate linear flow rate of 200 cm/hr. Post equilibration, the
appropriate volume of the virus was loaded onto the column at an
approximate linear flow rate of 200 cm/hr.
[0036] After loading, the column was washed with 10 mM Tris, 145 mM
NaCl, pH 7.5 buffer at an approximate linear flow rate of 200
cm/hr. The bound virus was eluted from the column using 10 mM Tris,
1.5 M NaCl, pH 7.5 buffer at a reduced linear flow rate of
approximately 100 cm/hr. Decreasing the elution flow rate increases
buffer residence time and, therefore, decreases elution volume.
[0037] The elution pool was immediately diluted 2.times.(1 part
eluate to 1 part dilution buffer) with 62.5 mM HEPES, pH 8 (target
formulation) buffer to reduce precipitation of virus. Post elution,
the resin was cleaned with 0.1 N NaOH/0.5 M NaCl at an approximate
linear flow rate of 200 cm/hr. The column was stored in 0.1 N
NaOH/0.5 M NaCl at room temperature.
GE CAPTO.TM. Core 700
[0038] The 2.times. diluted CELLUFINE.RTM. Sulfate elution pool was
then loaded onto a CAPTO.TM. Core 700 column. Prior to loading, the
column was regenerated with 0.1 N NaOH/0.5M NaCl at a linear flow
rate of 300 cm/hr. One column volume of a re-equilibration buffer
of 500 mM Tris/145 mM NaCl, pH 7.5 was applied prior to the
equilibration buffer consisting of 20 mM MES/100 mM NaCl, pH 7 at a
linear flow rate of 300 cm/hr.
[0039] After loading, the column was washed with 20 mM MES/100 mM
NaCl, pH 7 at a linear flow rate of 300 cm/hr. The flow through and
wash was collected as the product. This fraction was then diluted
2.times. with 50 mM HEPES/20% Sucrose/0.001% TWEEN.TM.-20, pH 8.
The resin was cleaned with 1 N NaOH/1M NaCl at a linear flow rate
of 300 cm/hr. The column was stored in 0.1 N NaOH/0.5M NaCl at room
temperature.
.beta.-PL Inactivation
[0040] A 10% solution of BPL was made by diluting BPL with water
for injection (WFI). The 10% BPL was stored in single use aliquots
at <-60.degree. C. The concentration of BPL in this 10% solution
was confirmed by gas chromatography (GC) analysis.
[0041] Human serum albumin (HSA) was added to the sample to adjust
the concentration to 1 mg/mL HSA. A sufficient amount of 10% BPL
was thawed and added while mixing to the live virus pool to bring
the BPL concentration to approximately 0.1% (v/v) BPL.
[0042] The inactivation mixture was mixed for approximately 3 hours
at room temperature on a low heat-generating stir plate. This
material was then incubated at 30.degree. C. for 60 minutes, and
filtered using a 0.2 .mu.m PES filter.
Alum Binding and Formulation
[0043] The 0.2 .mu.m filtered purified inactivated virus was bound
to "alum" [Aluminum aluminum Hydroxide hydroxide (Alum
ALHYDROGEL.RTM.)] and buffer exchanged into the final formulation
buffer. All process steps were aseptically performed.
[0044] One part of 2% Alum alum was added to 9 parts of 0.2 .mu.m
filtered sucrose gradient-purified inactivated virus to achieve a
final alum target concentration of 0.2%. We refer to the resulting
product as the original alum-bound virus pool. The pool was mixed
for a target of 1-4 hours at room temperature.
[0045] The alum-bound virus was aseptically buffer exchanged into
10 mM Tris/1.2 mM MgCl2/10 mM L-glutamic acid/0.11 mM D-mannitol/2
mM Trimethylamine-N-oxide dihydrate, pH 7.5. The alum-bound virus
was settled by centrifugation or membrane filtration. After
settling, the supernatant was decanted. The alum-bound virus pellet
was re-suspended with formulation buffer to a volume equal to the
original alum-bound virus pool volume and mixed for a target of 10
minutes at room temperature. This process was repeated 3-4.times.
until the alum-bound virus pool was exchanged into the final
formulation buffer.
[0046] The alum-bound and buffer exchanged virus pool was stored at
2-8.degree. C. This is referred to herein as the "Bulk Drug
Product." The potency of the Bulk Drug Product was measured using
ELISA that detects alum-bound YF.
[0047] Process recovery chromatograms for Experiments Y1626A,
Y1632A, and Y1637A are shown in FIG. 1 through FIG. 4,
respectively.
[0048] A process recovery chromatogram for Experiment 1639A using a
CELLUFINE.RTM. Sulfate Column is shown in FIG. 4 and for Experiment
1639A using a CAPTO.TM. DeVirS column in FIG. 5.
[0049] Two other embodiments of the disclosed process are shown in
FIG. 8, "Downstream new and Improved process #3."
[0050] It should be noted that the experiments were run using
frozen/thawed materisl and that there was no non-GMP material
remaining. We recommend using stored GMP material for further
development of the disclosed process. However, the experimental
results obtained with the non-GMP material demonstrate that the
disclosed process significantly reduces HCP and DNA levels.
[0051] The disclosed chromatographic purification process yielded
sufficient virus recovery and reduced the host cell protein levels
to below the limit of detection of the commercial Vero cell HCP
assay. These virus recoveries and HCP results were comparable to
results achieved by a similar purification process utilizing
sucrose gradient ultracentrifugation. A benefit of the disclosed
process is that, unlike centrifugation methods, it is suitable for
use in single-use systems.
[0052] The next phase of the Yellow Fever purification development
is to replace the Chisso CELLUFINE.RTM. Sulfate resin with an
equivalent GE Healthcare resin. The CELLUFINE.RTM. sulfate resin
can potentially be replaced with GE Healthcare's CAPTO.TM. DeVirS
resin. The CAPTO.TM. DeVirS is part of GE Healthcare's Custom
Designed Media program, and is an affinity chromatography resin
with the ligand dextran sulfate, which is known to have an
affinity-like behavior to different types of virus. CAPTO.TM.
DeVirS offers the a number of benefits for purification of virus,
including, for example, excellent productivity, good chemical
stability, and affinity-like behavior to various viruses.
[0053] The next planned experiments include following the use of
CAPTO.TM. DeVirS with GE CAPTO.TM. Core 700. We will also
investigate the use of an anion exchange membrane such as a Q
Monolith (BIA) or a Q membrane (Natrix, Pall, Sartorius) between
the step using the CAPTO.TM. DeVirS and the step using GE CAPTO.TM.
Core 700.
Equivalents
[0054] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith. All of the features
disclosed in this specification (including any accompanying claims,
abstract and drawings), and/or all of the steps of any method or
process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. The invention is not restricted to the details
of any foregoing embodiments. The invention extends to any novel
one, or any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
* * * * *