U.S. patent application number 10/602129 was filed with the patent office on 2004-03-11 for methods and compositions for inactivating viruses.
Invention is credited to Shepard, Scot R..
Application Number | 20040047844 10/602129 |
Document ID | / |
Family ID | 25321732 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040047844 |
Kind Code |
A1 |
Shepard, Scot R. |
March 11, 2004 |
Methods and compositions for inactivating viruses
Abstract
The present invention relates to methods and processes of
inactivating viral contaminants in a biological source material
(e.g. a host cell, cell supernatant, cell lysate, blood plasma,
tissue homogenate, or other biological materials) with a solution
containing one or more alkylamine compounds. In a particular
embodiment, the active ingredients are amphipathic, charged amines
or amine oxides coupled to saturated hydrocarbon chains of varying
lengths.
Inventors: |
Shepard, Scot R.; (Clayton,
NC) |
Correspondence
Address: |
INTERVET INC
405 STATE STREET
PO BOX 318
MILLSBORO
DE
19966
US
|
Family ID: |
25321732 |
Appl. No.: |
10/602129 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10602129 |
Jun 24, 2003 |
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09855634 |
May 14, 2001 |
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6635679 |
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Current U.S.
Class: |
424/93.7 ;
514/644; 514/663 |
Current CPC
Class: |
A01N 33/24 20130101;
A61M 1/3687 20130101; A61L 2/0082 20130101; A61K 31/131 20130101;
A01N 33/04 20130101; A61K 35/00 20130101; A61L 2/0005 20130101;
Y10S 514/908 20130101; A61L 2/0088 20130101 |
Class at
Publication: |
424/093.7 ;
514/644; 514/663 |
International
Class: |
A61K 045/00; A61K
031/13 |
Claims
What is claimed is:
1. A method of inactivating a viral or microbial agent present in a
biological source material, comprising the step of contacting the
biological source material with a solution comprising an effective
amount of an active ingredient, wherein the active ingredient is an
amphipathic charged amine or an amphipathic charged amine
oxide.
2. A method of inactivating a viral or microbial agent in a
biological source material comprising the step of contacting the
biological source material with a solution comprising an effective
amount of an active ingredient, wherein the active ingredient is
selected from the group consisting of: dimethyldecylamine,
dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine,
dimethyltetradecylamine, dimethylhexadecylamine,
dimethyldecylamineoxide, dimethylundecylamineoxid- e,
dimethyldidecylamineoxide, dimethytetradecylamineoxide and
dimethyltridecylamineoxide.
3. The method of claim 3, wherein the solution further comprises
glycerol.
4. The method of claim 4, wherein the active ingredient comprises
between 0.001 to 10 percent of the solution.
5. The method of claim 4, wherein the glycerol comprises between
0.6 to 6 percent of the solution.
6. The method of claim 5, wherein the glycerol comprises between
0.6 to 6 percent of the solution.
7. The method of claim 2, wherein the active ingredient is
dimethyltetradecylamine.
8. The method of claim 1, wherein the agent is a member of the
group consisting of: bacteria, yeast, fungi, mycoplasma, mammalian
cells, other animal cells, viruses, or lipid enveloped viruses such
as, Flaviviridae, Retroviridae, Togaviridae, Rhabdoviridae,
Herpesviridae, VSV, SFV, HIV, MuLV, BVDV, and CMV.
9. The method of claim 2, wherein the agent is a member of the
group consisting of: bacteria, yeast, fungi, mycoplasma, mammalian
cells, other animal cells, viruses, or lipid enveloped viruses such
as, Flaviviridae, Retroviridae, Togaviridae, Rhabdoviridae,
Herpesviridae, VSV, SFV, HIV, MuLV, BVDV, and CMV.
Description
1. TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for inactivating viruses present in samples/process streams of
biological origin.
2. BACKGROUND OF THE INVENTION
[0002] It is desirable to use biological materials as sources for
medicinal and industrial intermediates and products. Due to the
very nature of the biological materials or their methods of
production, biological materials may contain unwanted agents of
viral origin that may be pathological or otherwise undesirable. The
intended end-use of materials derived from biological sources may
require a reduction in the biological activity of viral agents
known to be present, or that are potentially present, in the source
material or process additives.
[0003] Reduction of viral activity in materials is commonly
accomplished by a number of techniques including the use of heat,
steam, pressure, chemical treatments and other methods. However,
these techniques may irreversibly alter the properties of the
biological source material or the desired substances to be obtained
from same. In such cases, gentle, non-denaturative and specific
methods are required to reduce the biological activity of viruses
without damaging the desired molecules or substances of
interest.
[0004] Prior methods known in the art for inactivation of viruses
in labile process streams include photochemical treatments in the
presence of Psoralens, solvent-detergent treatments (U.S. Pat. No;
4,481,189), caprylic acid treatments (U.S. Pat. No. 4,939,176), the
use of UVC radiation (Vitex Technologies, formerly NY Blood
Center), ultra short time heating (Charm Technologies,
charmbio.com), photodynamic inactivation in presence of
phenothiazine dyes (U.S. Pat. No. 4,534,972), and the use of
low-molecular-weight electrophilic agents that bind to nucleic
acids (Vitex, Inactine product 4 patents).
3. SUMMARY OF THE INVENTION
[0005] The present invention relates to methods and processes of
inactivating a viral contaminants in a biological source material
or process intermediate. The process of the present invention
involves contacting the biological source material (e.g., a host
cell, cell supernatant, cell lysate, blood plasma, tissue
homogenate, or other biological materials) containing a biomolecule
(e.g., a recombinant or native protein, lipid, nucleic acid, or
carbohydrate) of interest with a solution containing one or more
alkylamine compounds. In a particular embodiment, the active
ingredients are amphipathic, charged amines or amine oxides coupled
to saturated hydrocarbon chains of varying lengths. In a preferred
embodiment, the one or more active ingredients used are selected
from the group consisting of dimethyldecylamine,
dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine,
dimethyltetradecylamine, dimethylhexadecylamine,
dimethyldecylamineoxide, dimethylundecylamineoxide,
dimethyldidecylamineoxide, dimethytetradecylamineoxide and
dimethyltridecylamineoxide. These compounds may be used at
concentrations ranging from 0.001% up to their solubility limit in
the given solution. Preferably, the concentration of the active
ingredients ranges from 0.005% to 5%, 0.1% to 2%, or is
approximately 0.5% of the total solution (weight basis).
[0006] The pH of the solution can range from pH 2 to pH 12.
Preferably, the solution is at a pH ranging from pH 5.0 up to pH
8.0. More preferably, the pH ranges from pH 5.5 to 7.4, from pH 6
to 7.4, from pH 7.0 to 7.4, or is approximately pH 7.2.
[0007] The inactivation of the viral contaminants with the active
ingredient of the invention can be carried out at a temperature of
from about 2.degree. C. to about 50.degree. C. Preferably, the
temperature is from about 2.degree. C. to about 30.degree. C.,
2.degree. C. to about 20.degree. C., 2.degree. C. to about
10.degree. C., about 4.degree. C., about 25.degree. C., or at room
temperature.
[0008] The biological source material may be blood plasma, other
biological tissues or a recombinant source material such as
transformed or transfected "host cells". "Host cells" are cells
containing a biomolecule of interest. A "biomolecule of interest"
is any biomolecule present in the biological source material or the
host cells that one desires to isolate, purify, or formulate for
subsequent processing or application. The biological source
material may be blood, blood plasma, animal tissues, plant tissues
or recombinant host cells or host cell extracts. The host cells may
be of any type, preferably mammalian, bacterial, yeast, fungal,
plant, avian, insect, or reptilian.
4. DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention relates to a process for the facile
and non-denaturative inactivation of viruses present or potentially
present in biological source materials. The process includes
contacting the source material with certain charge-modified
hydrocarbons under appropriate solution conditions.
[0010] In a particular embodiment, solution conditions are adjusted
by sedimentation of an insoluble source material, for example,
recombinant-host cells producing a recombinant protein of interest,
followed by re-suspension, or by solution exchange using filtration
methods, by direct modification of the existing solution
conditions, or by other means of solvent exchange. Source materials
suspended or co-dissolved in the appropriate solution are then
contacted with certain amphipathic molecules that cause the
inactivation of biological agents. Agents that may be inactivated
in this way include bacteria, yeast, fungi, mycoplasma, mammalian
cells, other animal cells and lipid enveloped viruses, for example,
viruses of the families Flaviviridae (BVDV) Retroviridae (HIV,
MuLV), Togaviridae (SFV), Rhabdoviridae (VSV), Herpesviridae
(CMV).
[0011] In a preferred embodiment, a biological source material
containing a retrovirus, or suspected of containing a retrovirus,
for example human immunodeficiency virus (HIV) or murine leukemia
virus (MuLV) is contacted with dimethylamine and/or dimethylamine
oxide compounds with alkyl chains of varying length, depending on
microbe or virus type and solution conditions.
[0012] The alkyldimethylamines or alkyldimethylamine oxides do not
denature individual lipid molecules or other molecules such as
nucleic acids, proteins, carbohydrates, or small molecules such as
organic acids, vitamins, etc. Thus, the alkyldimethylamines or
alkyldimethylamine oxides are particularly suitable for the
reduction of viral or microbial contaminants without the
denaturation or destruction of the biomolecule of interest, such as
a recombinant protein.
[0013] Once a biological source material has been obtained, it is
contacted with the inactivation reagents of the present invention.
The inactivation reagents comprise one or more active ingredients.
In a particular embodiment, the one or more active ingredients are
amphipathic, charged-amines or amine oxides coupled to saturated
hydrocarbon chains of varying lengths. In a preferred embodiment,
the one or more active ingredients used are selected from the group
consisting of dimethyldecylamine, dimethyltridecylamine,
dimethylundecylamine, dimethyldidecylamine,
dimethyltetradecylamine, dimethylhexadecylamine,
dimethyldecylamineoxide, dimethylundecylamineoxide,
dimethyldidecylamineoxide, dimethytetradecylamineoxide and
dimethyltridecylamineoxide. Active ingredients may be used at
concentrations ranging from 0.001% up to their solubility limit.
Preferably, the concentration of the detergents ranges from 0.05%
to 5%, 0.1% to 2%, or is approximately 1% of the total
solution.
[0014] In addition to the one or more active ingredients, in a
preferred embodiment, the biological source material may also be
contacted with polyols, such as glycerol, to enhance the activity
of the active ingredient or to protect the molecules of interest.
Preferably, the glycerol concentration is at least 0.6%, or ranges
from 0.6% to 20%, 0.6% to 12%, 0.6% to 6%, 0.6% to 3%, or 0.6% to
1%.
[0015] Preferably, the solution is at a pH ranging from pH 5.0 up
to pH 8.0. More preferably, the pH ranges from pH 5.5 to 7.4, from
pH 6 to 7.4, from pH 7.0 to 7.4, or is approximately pH 7.2.
[0016] The microbial and/or viral inactivation can be carried out
at a temperature of from about 2.degree. C. to about 50.degree. C.
Preferably, the temperature is from about 2.degree. C. to about
3020 C., 2.degree. C. to about 20.degree. C., 2.degree. C. to about
10.degree. C., about 4.degree. C., about 25.degree. C., or at room
temperature.
[0017] The amount of time allowed for inactivation after contacting
the biological source material with the inactivation reagent may be
determined by one of skill in the art. For example, the biological
source material may be incubated in the presence of the
inactivation reagent for 40 minutes, 90 minutes, or 150 minutes.
Shorter and longer times may also be appropriate. In general, the
amount of time can be increased when the concentration of detergent
is low and decreased when the amount of detergent is high. For
example, an inactivation reagent with a 1% detergent concentration
is effective after 40 minutes, while an inactivation reagent with a
0.1% detergent concentration should be incubated for 150 minutes or
longer. For optimal inactivation, the exact amount of time
necessary can be determined by a simple time-course experiment at a
given concentration of active ingredient, where viability of the
microbial or viral contaminant is determined over time. After a
certain time point, no further decrease in viability will be
observed. This time point is the optimal time necessary for
inactivation.
[0018] Typically, if the biological source material is comprised of
cells, the cells will lyse after contact with the inactivation
reagent of the invention. After lysis of the cells, the solution
can be centrifuged to collect cellular debris in the pellet,
leaving the released protein or biomolecule of interest in the
supernatant. The supernatant may be processed according to methods
known to those of skill in the art to further isolate and purify
the protein of interest. The methods utilized to further isolate
and/or purify the protein of interest are highly dependent upon the
characteristics and properties of the particular protein of
interest, and must be determined for each protein.
[0019] This invention is further illustrated by the following
example which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
5 EXAMPLES
5.1 Inactivation of a Viral Contaminant in a Biological Source
Material
[0020] Chinese hamster ovary (CHO) host cells are grown to
3.times.10-5 cell density and are then harvested from the reactor
vessel. Cells are removed from the suspension to produce a
clarified culture broth. Tetradecyldimethylamine is then added to
the clarified culture broth to a concentration of 0.5% (w/w). The
clarified culture broth is then incubated for a period of time
during which membrane bound organisms and viruses are
inactivated.
[0021] The same approach is taken for blood plasma, tissue
extracts, and cell extracts. The amount of detergent, time of
incubation, temperature of incubation, and other parameters are
readily ascertained by those skilled in the art. Appropriate
solution conditions and incubation times are identified and
validated by the intentional addition, or "spike", of microbes or
viruses of known titer into the biological source material prior to
the addition of the charge-modified hydrocarbon. The
charge-modified hydrocarbon is then added to the biological source
material and samples are withdrawn at specific time points. The
samples are then analyzed by appropriate methods, known to those
skilled in the art, that measure the biological activity, growth
characteristics, or infectivity of the microbe or virus initially
"spiked" into the biological source material.
* * * * *