Note: Descriptions are shown in the official language in which they were submitted.
WO 94/19935 2157118
PCT/US94/01672
-1-
ISOLATION OF COMPONENTS OF INTEREST FROM MILK
Background of the Invention
Due to the long history of their inclusion in the human diet and the
relative ease with which they can be isolated, naturally occurring milk
components have
been studied for many years. (Swaisgood, H.E., Developments in Dairy Chemistry
-I:
Chemistry of Milk Protein, (Applied Science Publishers, NY, 1982)). Recently,
researchers have taken advantage of milk as a convenient source of proteins
not normally
produced in milk by directing the synthesis of particular proteins of interest
to the
mammary tissue of transgenic mammals. For example, researchers have
incorporated a
fusion gene composed of the regulatory sequence from the !3-lactoglobulin gene
and the
coding sequence for the human anti-hemophilic factor IX gene into a sheep
germline.
(Clark, A.J. et al., (May 1989) Biotechnology 7-:487-492). The lactating
transgenic ewes
subsequently secreted factor IX into their milk. Similarly, a goat germline
has been
engineered to include a fusion gene containing the regulatory sequences from
the gene
for murine whey acidic protein linked to the coding sequence for human longer-
acting
tissue-type plasminogen activator. (Ebert, K.M. et al., (September 1991)
Biotechnology
2:835-838). Milk from the transgenic dairy goat contained the modified tissue
plasminogen activator.
The production of proteins of interest, such as therapeutic agents, in the
milk of transgenic livestock offers the possibility of large scale production
of such
proteins with an accompanying reduction of costs which are typically
associated with the
conventional production of complex recombinant proteins in mammalian cell
culture
systems. Although transgenic expression of proteins in milk presents
advantages over
more traditional methods of recombinant protein expression, it also poses
several
challenges. These include the preservation of the activity of the recombinant
protein in
milk and the purification of the protein to the required degree of purity for
feasible
commercial production.
Traditional methods of isolating a protein of interest from milk often
included as one of their initial steps the fractionation of the major milk
components by
either sedimentation (Swaisgood, H.E., Developments in Dairy Chemistry-I:
Chemistry
of Milk Protein, (Applied Science Publishers, NY, 1982), precipitation (Kothe
et al.,
U.S. Patent No. 4644056); Groves, M.L. et al., (1985) Biochem, et. Biophys.
Acta.,
B_4A:105-112; McKenzie, H.A., Milk Proteins: Chemistry and Molecular Biology,
Academic Press, NY, 1971), 88) or enzymatic coagulation using rennin or
chymotrypsin
(Swaisgood, H.E., Developments in Dairy Chemistry-I: Chemistry of Milk
Protein,
WO 94/19935 PCT/US94/01672
2
(Applied Science Publishers, NY, 1982). Problems associated with these methods
include low yields due to the loss of the protein of interest by entrapment in
the
precipitate of the milk component to be removed and loss of biological
activity of the
protein of interest due to the low pH often required for acid precipitation.
See Denman,
J. et al. (Sept. 1991) Biotechnofoav Q: 839-843. Furthermore, because
traditional
methods of isolating proteins of interest from milk involved the
sedimentation,
precipitation, or coagulation of the major milk components, the milk samples
were not
readily processable by filtration.
Summary of the Invention
The present invention pertains to a method of isolating a component of
interest, such as a protein, from milk. The invention is based, at least in
part, on the
recognition that the low yields of biologically active components of interest
obtained
from milk samples using traditional methods were due essentially to entrapment
of the
component of interest in the precipitate of the initial fractionation step.
The method of
the present invention avoids inactivation and entrapment of the component of
interest by
providing a stabilizing agent which stabilizes the solubility of at least a
portion of the
total milk protein. Rather than being sedimented or precipitated out of the
stabilized
milk sample and carrying with it the component of interest, at least a portion
of the total
milk protein remains soluble such that it can be subsequently eliminated
during
purification of the component of interest. In addition, because the method of
the present
invention does not require the low pHs of the prior methods, inactivation of
pH sensitive
components of interest is avoided.
The present invention pertains to methods of isolating a component of
interest in its biologically active form from a milk sample containing the
component of
interest. More particularly, the present invention relates to a method of
isolating a
component of interest from a milk sample by contacting the milk sample
containing the
component of interest with a stabilizing agent under conditions which
stabilize the
solubility of at least a portion of the total milk protein. After
stabilization, the
component of interest can be isolated without significant loss and the
component of
interest can be isolated from the stabilized milk sample in its biologically
active form.
The present invention further pertains to a method of isolating a
component of interest from a milk sample by contacting a milk sample
containing a
component of interest with a stabilizing agent forming a milk sample
processable by
filtration. The component of interest is then isolated from the milk sample
processable
by filtration.
2157118
WO 94/19935 PCT/US94/01672
3
The present invention even further pertains to components of interest
isolated using the above-described methods and methods of stabilizing the
solubility of
at least a portion of the total milk protein in a milk sample coritaining a
component of
interest. The isolated components of interest can be used as additives to cell
culture
media, foodstuff preparations, and medicinal compositions. The invention even
fu.rther
pertains to kits containing reagents used in the above-described methods
together with
instructions for using the kit for stabilizing the solubility of at least a
portion of the total
milk protein of a milk sample containing a component of interest.
Brief Description of the Drawings
Figure 1 is a photograph depicting the extent of solubilization of a portion
of the total milk protein in milk samples over twelve different concentrations
of arginine.
Figure 2 depicts the relative protein concentration versus the longer-
acting tissue-type plasminogen activator (LA-tPA) activity of the supernatant
from the
arginine solubilization step depicted in Figure 1 for the twelve
concentrations of
arginine.
Figure 3 is a photograph depicting the extent of solubilization of a portion
of the total milk protein in milk samples over twelve different concentrations
of
imidazole.
Figure 4 depicts the relative protein concentration versus the LA-tPA
activity of the supernatant from the imidazole solubilization step depicted in
Figure 3 for
the twelve concentrations of imidazole.
Figure 5 is a photograph depicting the extent of solubilization of a portion
of the total milk protein in milk samples over ten different concentrations of
Bis-Tris.
Figure 6 depicts the relative protein concentration versus the LA-tPA
activity of the supernatant from the Bis-Tris solubilization step depicted in
Figure 5 for
the ten concentrations of Bis-Tris.
Figures 7A-7E are five photographs depicting the extent of solubilization
of a portion of the total milk protein in milk samples for various
concentrations of
arginine over a range of pHs.
Figure 8 depicts the relative protein concentration versus the LA-tPA
activity of the supernatant from an arginine-mediated solubilization of a
portion of the
WO 94/19935 PCT/US94/01672
4
total milk protein wherein the arginine was added to the milk sample prior to
adjustment
of the pH of the milk sample.
Detailed Description of the Invention
The present invention pertains to methods of isolating a component of
interest from a milk sample containing the component of interest without
significant loss
of the component of interest. The method includes contacting a milk sample
containing
a component of interest with a stabilizing agent under conditions which
stabilize the
solubility of at least a portion of the total milk protein such that the
component of
interest can then be isolated from the stabilized milk sample. The component
of interest
is then isolated from the stabilized milk sample in a form which is
biologically active.
The language "component of interest" is intended to include materials
present in the milk sample capable of being isolated from the milk using the
above-
described method without significantly affecting the intended function of the
component
of interest in a detrimental manner. The component of interest can be a
material
naturally occurring in a milk sample or can be a material not normally present
in a milk
sample but which is targeted to the mammary tissue of a transgenic mammal and
ultimately introduced into the milk of that mammal. The component of interest
can be in
free form or can be entrapped or encapsulated within another material present
in the milk
sample, e.g. a milk fat globule and/or the membrane encasing the milk fat
globule (see,
e.g. DiTollio et al. (1992) Biotechnologv 1.Q:74-77). Examples of components
of
interest include proteins, such as casein, lactoferrin,l3-lactoglobulin,
immunoglobulins,
lipids, such as glycerides of oleic acid, palmitic acid, and myris+ac acid,
polysaccharides,
such as lactose and more complex oligosaccharides, or any of the above
components in
combination with vitamins, such as vitamins A and those of the B complex, and
minerals, such as phosphorous, potassium, iron, magnesium, copper, and
calcium. For
example, components of interest can be produced in the milk through normal
secretory
pathways. Normal secretory pathways for proteins can include synthesis of the
protein
in the rough endoplasmic reticulum, passage of the protein into the lumen of
the
endoplasmic reticulum followed by transportation to the Golgi apparatus.
Finally, the
proteins (or glycosylated proteins) are packaged into vesicles that are
pinched off into the
cytoplasm and subsequently fuse with the plasma membrane. Another secretion
pathway
involves the blebbing off of milk fat globules from the membrane which contain
the
coriiponent of interest, as in the case of cystic fibrosis transmembrane
conductance
regulator.
Naturally occurring milk components have been found to be useful as
supplements for the serum-free growth of certain types of cells in culture.
See Steimer,
WO 94/19935 2157118 PCT/US94/01672
K.S. et al. (Nov. 1981) J. Cell Physiol. l.Q2(2):223-234 (epithelial cells and
fibroblasts);
Steimer, K.S. and Klagsbrun, M. (Feb. 1981) J. Cell Biol. $$(2): 294-300
(normal and
transformed fibroblasts). Milk components may also be used in foodstuffs. For
example, the incorporation of milk components into bread can increase the
nutritional
5 value of the final product. e.g. Rudel, H.W. et al. (U.S Patent No.
5178894).
Alternatively, milk components can be added to dairy products, such as frozen
dairy
desserts, to provide body and texture to the compositions and decrease the
sensitivity of
the product to temperature fluctuations. Huang, V.T. et al. (U.S. Patent No.
5175013).
Milk components can also be used to make cheeses. Yee, Jen-Jung et al. (U.S.
Patent
No.5165945).
The language "component of interest" is also intended to include any
compound which is not normally present in a milk sample obtained from a
nontransgenic
mammal but is instead secreted in a milk sample obtained from a transgenic
mammal.
Examples of such components of interest include therapeutic agents. A
therapeutic agent
is an agent having a therapeutic effect on a mammal when administered at an
appropriate
dose under appropriate conditions. The desired therapeutic effect will depend
on the
disease or condition being treated. The afore-mentioned appropriate dose or
conditions
will be determined on an individual basis and will depend on such factors as
the size of
the individual being treated, the severity of the symptoms being treated, and
the selected
route for administration of the therapeutic agent. Examples of therapeutic
agents include
insulin, longer-acting tissue-type plasminogen activator (LA-tPA), anti-
thrombin III, a-1-
anti-trypsin, soluble CD4, interleukin-2, immunoglobulins, cystic fibrosis
transmembrane conductance regulator, and coagulation factors VIII and IX.
The language "transgenic mammal" is intended to include a mammal
whose cells, e.g. germline or somatic, have been genetically manipulated such
that
foreign DNA segments have been introduced therein. Examples of transgenic
mammals
include transgenic cows, sheep, goats, pigs, rabbits, rats, and mice.
Typically, in order to
obtain large scale production of the component of interest at a reduced cost,
a mammal
should be selected according to the amount of milk it produces. Preferably,
large
transgenic farm animals such as cows, sheep, and goats are chosen because of
their
ability to produce large volumes of milk.
The language "biologically active" is intended to include an activity for
the component of interest which allows it to perform its intended function.
The
coniponent of interest does not have to be as active as its naturally
occurring counterpart
but typically has an activity close to or similar to its naturally occurring
counterpart.
WO 94/19935 PCT/US94/01672
6
The language "milk sample" is intended to include samples containing
milk derived from a mammal which contain a component of interest. The milk
sample
can be a sample derived directly from the mammal or a sample which is
processed in a
manner which does not detrimentally affect the activity of the component of
interest or
the stabilizing agent's ability to perform is intended function. The milk
sample further
may be concentrated or diluted using reagents which do not detrimentally
affect the activity of the component of interest or the stabilizing agent's
ability to perform its
intended function. The quantity of the milk present in the milk sample is an
amount
sufficient to ascertain the presence of the component of interest.
The language "stabilizing agent" is intended to include compounds which
render soluble and/or maintain the solubility of at least a portion of the
total milk protein
and/or the component of interest. The language "maintain the solubility of' is
intended
to include the maintenance of the solubility to an extent that prevents the
portion of the
total milk protein from precipitating from the milk sample during later
processing steps,
e.g. pH adjustment, and interfering with the further processing of the milk
sample or the
isolation of the component of interest. This maintenance of solubility does
not have to
be the maintenance of the solubility at a constant value and can be the
maintenance of
the solubility over an appropriate range. Examples of such agents are mono-
and
polyvalent cationic agents such as amino acids and positively-charged buffers.
Useful
positively charged buffers include arginine, imidazole, and Bis-Tris. The pH
of the
stabilizing agent is typically selected such that the agent is positively-
charged.
The language "stabilize the solubility" is intended to include
solubilization and/or maintenance of the solubilization of at least a portion
of the total
milk protein. Certain of the major milk proteins, such as casein, are
naturally in a
partially insoluble or insoluble form in milk. Decreasing the pH of the milk
can result in
the precipitation of at least a portion of these milk proteins. However, the
isolation of a
component of interest may require that the pH of the milk sample be adjusted
such that
the component of interest becomes or remains soluble. Adjusting the pH of the
milk
sample may also tend to encourage precipitation of at least a portion of the
total milk
protein. The solubilizing agent solubilizes and/or maintains the solubility of
these
proteins before, during, and after a pH adjustment. Alternatively, isolation
of other
components of interest may not require the adjustment of the pH in order to
become
soluble or to maintain solubility but the solubilizing agent is still required
to solubilize
and/or maintain the solubility of at least a portion of the total milk protein
allowing for
the further processing of the milk sample without interference from
precipitated milk
proteins.
WO 94/19935 , 215711 $ PCT/US94/01672
7
The concentration of the stabilizing agent is selected such that it preserves
the functional properties of the component of interest and stabilizes the
solubility of at
least a portion of the total milk protein. For example, if the component of
interest is an
enzyme, the buffer concentration should be as low as possible since high ionic
strength
may decrease activity of the enzyme. The required concentration of the
stabilizing agent
in the isolation of a particular component of interest from milk will
therefore vary
according to the properties of the stabilizing agent itself and the component
of interest to
be isolated. For example, an arginine concentration of about 0.3M is more
effective at
preserving the functional properties of LA-tPA and stabilizing the solubility
of at least a
1o portion of the total milk protein than does 0.3M of imidazole.
The conditions under which the stabilizing agent is brought in contact
with the milk sa.-nple to form a stabilized milk sample are selected such that
the
functional properties of the component of interest are preserved and the
solubility of at
least a portion of the total milk protein is stabilized. One of ordinary skill
in the art
would be able to manipulate, with only routine experimentation, conditions
such as pH
of the milk sample, the concentrations of the agents used for the isolation,
and other
factors that could affect the functional properties of the component of
interest and the
stability of the solubilization of at least a portion of the total milk
protein.
The pH of the milk sample containing the component of interest should
generally be adjusted to a level selected such that the component of interest
retains its
solubility and functional properties and the solubility of the portion of the
total milk
protein is stabilized. The level to which the pH is adjusted may be dictated
by several
factors. Included among these factors is the pH range within which the
component of
interest is in its natural and/or biologically active form. For example,
certain
components of interest, such as enzymes, may only be active within a narrow
range of
pHs and exposure to pHs outside this range can result in irreversible loss of
activity.
Therefore, in order to obtain the functional form of the component of interest
or at least
the component of interest in its natural form, the pH must be adjusted to
levels only
within the allowable range. A pH range from about 5.0 to about 9.0 is an
example of a
typical range within which many components of interest which have biological
activity
will retain that activity. The term "about 5.0" is defmed below. The term
"about 9.0" is
intended to include pHs close to 9.0 which affect the component of interest in
the same
or similar manner as does pH 9.0, e.g. preferably 8.5 and above, more
preferably 8.8 and
-above.
Another factor to consider in the adjustment of the pH is the level at
which the solubility of at least a portion of the total milk protein is
stabilized. The pH of
WO 94/19935 r} ~~Q PCT/iJS94/01672 ~
(.-~.~~ O 8
the milk sample containing the protein of interest should generally not be
lowered to the
level or below the level at which milk components will precipitate from the
milk. For
example, the caseins precipitate at a pH of about 4.6 at 20 C. The pH should
also be
maintained at a level which prevents the component of interest from becoming
insoluble.
For example, the pH of the milk sample containing human longer-acting tissue-
type
plasminogen activator (LA-tPA) is generally lowered only to about 5.0 so that
LA-tPA
remains soluble. The term "about 5.0" is intended to include pHs close to 5.0
which
affect the component of interest in the same or similar manner as does 5.0
e.g. preferably
4.6 to 5.4., more preferably 4.8 to 5.2.
The language "significant loss" is intended to include losses of the
component of interest which would not allow the above-described method of
isolating
the component of interest to be a commercially feasible approach for producing
the
component of interest. The methods of this invention preferably have a loss of
the
component of interest which is less than 50%, more preferably less than about
20% and
most preferably less than about 10%. The conventional methods for processing
milk
resulted in significant losses in yield of the component of interest because
the component
of interest was trapped in the sediment, precipitate, or coagulum. For
example, Clark,
A.J. et al., (May 1989) Biotechnoloav 7-:487-492 reported a recovery of anti-
hemophilic
factor IX of about 2.0 to 2.5% using acid precipitation to remove milk caseins
from a
milk sample obtained from transgenic ewes. This translates into a loss of
about 98%.
Denman, J. et al., (Sept. 1991) Biotechnology 2: 839-843 reported a recovery
of LA-tPA
of about 25%, and thus a loss of about 75%, using acid precipitation to remove
milk
caseins from a milk sample obtained from a transgenic dairy goat. Denman et
al. also
reported that other initial fractionation steps such as rennin treatment and
acid
precipitation provided no significant increases in LA-tPA yields. Denman, J.
et al.
(Sept. 1991) Biotechnologv 2: 839-843.
The language "at least a portion of the total milk protein" is intended to
include at least a portion of at least one protein which is normally present
in milk in a
partially insoluble or insoluble form. The portion of the total milk protein
of which the
solubility is stabilized must be in aniount such that its removal constitutes
more than an
insignificant step in the isolation of a component of interest. The
solubilities of portions
of different milk proteins can be stabilized or the solubility of entire
portions of one or
more milk proteins can be stabilized. In the examples below, arginine and
imidazole
stabilized the solubility of at least a portion of the total milk protein in
the examples
described below, stabilized the solubility of at least the casein family of
proteins, i.e. the
entire portion or substantially the entire portion of at least one type of
milk protein.
WO 94/19935 2157118 PCT/US94/01672
9
It should be understood that the component of interest can be isolated
from the stabilized milk sample using conventional techniques. The selection
of a
separation or isolation technique is dependent on such factors as the type of
component
of interest being isolated and the amount of the component of interest present
in the milk
sample. Further, the isolation or separation of the component of interest from
the
stabilized milk sample can be performed using a single technique or a
plurality of
techniques in succession. These techniques include filtration and
chromatographic
techniques. Examples of filtration include dead-end filtration filter unit and
cross-flow
filtration. Examples of chromatographic techniques include ion exchange,
hydrophobic,
1o affinity, and gel-filtration chromatographic techniques. Similarly, further
processing by
fractionation of the milk sample prior to or after the isolation of the
component of
interest can be conducted using the above-described separation techniques. For
example,
fractionation of proteins and other components can be carried out by any of a
variety of
chromatographic methods depending on the properties of the component to be
isolated.
Proteins are most often fractionated by column chromatography, in which a
mixture of
proteins in solution is passed through a column containing a porous solid
matrix, and
they can be separately collected, in their native functional state, as they
flow out of the
bottom of the column. Depending on the type of matrix used, proteins are
separated, for
example, according to their charge (ion-exchange chromatography), their
hydrophobicity
(hydrophobic chromatography), their ability to bind to particular chemical
groups
(affinity chromatography) and their size (gel-filtration chromatography). For
higher
degrees of resolution, high performance liquid chromatography may be employed.
Those skilled in the art will be able to vary the order and number of the
above-described
separation techniques with routine experimentation to isolate the component of
the milk
sample in which they are interested to the purity that they require.
The present invention further pertains to a method of isolating a
component of interest from a milk sample by contacting a milk sample
containing a
component of interest with a stabilizing agent forming a milk sample
processable by
filtration. The component of interest may then be isolated from the milk
sample
processable by filtration. The terms component of interest and stabilizing
agent are as
defined above.
The language "processable by filtration" is intended to include a milk
sample in which the solubility of at least a portion of the total milk protein
is stabilized
to an extent such that the stabilized milk sample is capable of passing
through a filter and
becoming part of the filtrate. The filters are of a type typically used to
process milk or
substances similar to milk. The filter generally contains pores of a size that
typically
would not be used for milk processing because the pores would become blocked
by
I . Y
CA 02157118 2004-09-17
50409-17
components in the milk. Examples of types of filters which can be used to
process the
milk samples of this invention include filters having pore sizes ranging from
about 0.2
m to about 5.O m. The component of interest can then be isolated from the
filtrate by
any of the methods for isolating proteins and in any order known to those of
ordinary
5 skill in the art. Some of these methods are described above.
The present invention further pertains to a method of stabilizing the
solubility of at least a portion of the total milk protein in a milk sample
containing the
component of interest. The method involves contacting a milk sample with a
stabilizing
10 agent under conditions which stabilize the solubility of at least a portion
of the total milk
protein. The component of interest can be isolated from the stabilized milk
sample
without significant loss of the component of interest. The language
"stabilizing agent",
"under conditions which stabilize the solubility," and "at least a portion of
the total milk
protein" are as defined above.
The present invention also pertains to kits for stabilizing the solubility of
at least a portion of the total milk protein of a milk sample containing a
protein of
interest. The kits include a container holding a stabilizing agent and
instructions for
using the stabilizing agent for the purpose of stabilizing the solubility of
at least a portion
of the total milk protein of a milk sample containing a component of interes.t
to an extent
which allows isolation of the component of interest from the stabilized milk
sample
without significant loss. The kits also may include a container for holding an
agent for
adjusting the pH of the nzilk sample to a level selected such that the
biological activity of
the component of interest is preserved and the solubility of the portion of
the total milk
protein is stabilized.
The language "agent for adjusting the pH" is intended to include
compounds that are used by those of ordinary skill in the art to adjust the pH
of a
solution, such as a miLk sample. Such agents include acids, such as HCI and
acetic acid,
and bases, such as NaOH. In addition, pH can also be regulated by gases, such
as CO2.
In the case where the agent for adjusting the pH of the milk sample is in the
form of a
liquid, examples of containers which provide convenient means of delivering
the pH
adjusting agent include vials, bottles etc. that have caps which incorporate
pipettes or
medicine droppers.
The present invention is further illustrated by the follo:ving examples
which should in no way be construed as being further limiting.
+. lx.b.l,
CA 02157118 2004-09-17
50409-17
11
The present invention is further illustrated by
the following examples.
Example 1: Stabilization of the Solubilitv of Milk Protein from Milk of
Transgenic
I?airv Goats Containing Longer-Acting Tissue-Type Plasminogen
Activator (LA-tPA) Using Arginine as the Stabilizing Agent
Milk samples obtained from lactating transgenic dairy goats generated
according to the method of Ebert, K.M. et al. (Sept. 1991) Biotechnologv Q:
835-838
were stored frozen at -20 C and thawed in a 37 C water bath prior to analysis.
The
component of interest produced in the milk of the dairy goats was LA-tPA, a
glycosylation variant of human tissue-type plasminogen activator which
exhibits a
longer circulating half-life than wild-type tPA. (See Denman, J. et al., cite
for a
characterization of LA-tPA produced in the milk of transgenic dairy goats).
The pH of the milk sample was then adjusted from 6.7 to 5.0 using 1.OM
acetic acid. The milk sample was subsequently divided into twelve 500m1
aliquots
which were placed in 1.5m1 polypropyIene tubes. A different amount of 2.OM
arginine-
HCi (pH 5.5) was added to each tube to obtain arginine concentrations ranging
from_
O.OM to 1.OM (Table 1). Phosphate buffered saline (M) was added to bring the
final
volume up to 1.0ml.
ARGININE pH 5.5
Final Arginine
Coneentration j11i1j{{l 2.OM Arrinine (ul) Pas (m Final Yolume
An ll 11 1mi1
1.00 500 500 - 1.0
0.90 S00 450 50 1.0
0.80 500 400 100 1.0
0.70 S00 350 1S0 1.0
0.60 500 300 200 1.0
0.50 500 250 250 1.0
0.40 500 200 300 1.0
0.30 500 150 350 1.0
0.20 500 100 400 1.0
0.10 S00 50 450 1.0
0.05 500 25 47s 1.0
0.00 500 - S00 1.0
TABLE I
CA 02157118 2004-09-17
50409-17
12
The tubes were centrnged for ten minutes at 1000 rpm in a Brinkman
5315 centrifuge. The tubes were removed and photographed to illustrate the
extent of
solubilization by examining the size of the casein pellet for each arginine
concentration
(Figure 1). The supernatant,%vas removed for quantitative analysis. Figure 1
depicts the
extent of solubilization of a portion of the total milk protein in milk
samples over twelve
different concentrations of arginine. For concentrations of arginine lower
than 0.4M,
protein (probably consisting mostly of casein) pellets were clearly visible.
For
cuncentrations of arginine greater than about 0.4M, there was little or no
protein pellet.
The protein concentration of the supernatant was calculated by an
absorbance measurement at 280nm. The absorbance at 280nm was taken by diluting
each of the twelve supernatants 1:50 with PBS and reading on a Beckman DU-6*
spectrophotometer. Each of the 12 samples had its own control blank diluted
1:50 with
PBS wherein PBS was substituted for milk. LAt-PA activity was measured in an
indirect amidolytic activity assay using the plasmin substrate Val-Leu-Lys-
para-
nitroanilide (5=2251, Helena Labs, Inc.) as described by Lau, D. et al.
((Sept. 1987)
BiotechnoloQV 5: 953-958). The results are set forth in Figure 2. The dilution
necessary
was 1:500000. Figure 2 depicts the relative protein concentration versus LA-
tPA
activity of supernatants over twelve concentrations of arginine (pH 5.5). The
results in
Figure 2 demonstrate that as the amount of arginine increased, there was a.
concomitant
rise in both the total protein concentration of the milk sample and the LA-tPA
activity.
These results demonstrate that arginine is able to stabilize the solubility of
at least a
portion of the total milk protein, thereby allowing isolation of increased
amounts of LA-
tPA.
Ex=le 2: Stabilization of the Solubilitv of Milk Protein from Milk of
Trantgenic
Dairv Goats Containing LA-tPA Using Imida?ole as the Stabilizing Agent
The procedure described in Example 1 was followed except that 2.OM
imidazole (pH 7.0) was substituted for arginine. Table II sets forth the range
of
concentrations of imidazole tested. Figure 3 is a photograph illustrating the
extent of
solubilization of the milk sample over the range of imidazole concentrations.
For
concentrations of imidazole lower than about 0.7M, protein (probably
consisting mostly
of casein) pellets were clearly visible. For concentrations of arginine
greater than about
0.7M, there was little or no protein pellet. Figure 4 depicts the relative
protein
concezttration of the supernatant versus the LA-tPA activity over the twelve
concentrations of imidazole. As in the case of arginine, imidazole was able to
stabilize
the solubility of at least a portion of the total milk protein, thereby
allowing an increased
yields of LA-tPA. Figure 4 shows that there was a concomitant increase in the
total
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13
protein concentration of the milk sample and the LA-tPA activity as the
concentration of
imidazole in the milk sample increased.
IMIDAZOLE pH 7.0
Final Imidazole
Concentration Milk (Li 2.OM Imidazole (u PBS (u. Final Volume
D11 !1 1~ Il ~
1.00 500 500 - 1.0
0.90 500 450 50 1.0
0.80 500 400 100 1.0
0.70 500 350 150 1.0
0.60 500 300 200 1.0
0.50 500 250 250 1.0
0.40 500 200 300 1.0
0.30 500 150 350 1.0
0.20 500 100 400 1.0
0.10 500 50 450 1.0
0.05 500 25 475 1.0
0.00 500 - 500 1.0
TABLE II
E~=le 3: Stabilization of the Solubility of Milk Protein from Milk of
Transgenic
Dairy Goats Containing LA-tPA Using Bis-Tris as the Stabilizing Agent
The procedure described in Example 1 was followed except that 1.7M
Bis-Tris (pH 6.0) was substituted for arginine and the range of concentrations
of Bis-Tris
tested was narrower than the range of concentrations of arginine tested. Table
III sets
forth the range of concentrations of Bis-Tris used in the experiment.
BIS-TRIS pH 6.0
Final Bis-Tris
Concentration ASi1k(it 1,7M Bis-Tris (ul) PBS (u Final Volume
f1~2 !1 11 tmR
0.85 500 500 - 1.0
0.80 500 471 29 1.0
0.70 500 412 88 1.0
0.60 500 353 147 1.0
0.50 500 294 206 1.0
0.40 500 235 265 1.0
0.30 500 176 324 1.0
0.20 500 118 382 1.0
0.10 500 59 441 1.0
0.00 500 - 500 1.0
TABLE III
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PCT/US94/01672
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Figure 5 is a photograph illustrating the extent of solubilization of the milk
sample over
the range of Bis-Tris concentrations tested by depicting the size of the
protein pellet. For
concentrations of Bis-Tris as low as 0.2M, there was little to no protein
pellet visible.
Figure 6 depicts the relative protein concentration of the supematant versus
the LA-tPA
activity over the ten concentrations of Bis-Tris. Figure 6 shows that there
was a
concomitant increase in the total protein concentration of the milk sample and
the LA-
tPA activity as the concentration of Bis-Tris in the milk sample increased
with a decrease
in LA-tPA activity at Bis-Tris concentrations of about 0.65M and greater.
1o Example 4: Stabilization of the Solubilitv of Milk Protein from Milk of
Transgenic
Dairy Goats Containing LA-tPA Using Arginine at DifferentpHs as the
SolubilizingAgent
The initial pH of the milk sample was 6.7. From this point the pH of the
sample was adjusted to the desired pH, e.g. 4.3, 6.0, 7.0, and 8Ø The pH of
the milk
sample was not adjusted in the case where the pH of the stabilizing agent to
be tested
was 6.7. The 2.OM arginine was similarly adjusted to the pH of the sample to
which it
was to be added to prevent fluctuations in the pH of the final sample. The pH-
adjusted
milk samples were then divided into 500ml aliquots which were placed in 1.5m1
polypropylene tubes. Varying amounts of 2.OM arginine were added to the milk
samples
to obtain milk samples containing arginine at various concentrations and at
various pHs.
PBS was added to bring the final volume up to 1.0m1.
The tubes were centrifuged for 10 minutes at 1000 rpm in a Brinkman
5315 centrifuge. The tubes were then removed and photographed to illustrate
the extent
of solubilization by examining the size of the protein pellet for each
arginine
concentration at pHs 4.3, 6.0, 6.7, 7.0, and 8Ø These photographs are
depicted in
Figure 7. As shown in Figure 7, the arginine at pH 6.0 acted as the best
stabilizer of the
solubility of the milk protein over a range arginine concentrations from about
0.5M to
about 1.OM. Arginine at pH 7.0 at concentrations ranging from about 0.6M to
1.OM was
also a good stabilizer of the solubility of the milk protein. Arginine at pH
4.3
demonstrated poor stabilization of the milk protein over a range of
concentrations from
about 0.1 M to about 1.OM.
Example 5: Isolation of LA-tPA from the Milk of Transgenic Dairy 7oat By First
Adding Arginine and Then Adi u ing the nH of the Milk Sam,ple
The procedure described in Example 1 was followed except that 2.OM
arginine (pH 5.5) was added prior to the pH adjustment. The range of
concentrations of
arginine tested in the experiment was the same as that tested in Example 1 and
is set
CA 02157118 2004-09-17
50409-17
forth in Table I. Figure 8 depicts the relative protein concentration of the
supetnatant
versus the LA-tPA activity over the twelve concentrations of arginine. A rise
in both the
total milk protein concentration and the LA-tPA activity accompanied the
increase in
arginine concentration. LA-tPA activity, however, peaked for an arginine
concentration
5 of about I.OM at a level (about 1.20e + 6 IU/ml) as compared to the peak LA-
tPA
activity (about 1.50e + 6 IU/ml) in Example 1 for the same arginine
concentration.
Exa=le 6: Isolation of Anti-Thrombin HI from the Milk of a Transgenic Mouse
lting AXginine as the Stabilizing Agent
Milk samples obtained from a lactating transgenic mouse were stored
frozen at -80 C and thawed at room temperature prior to analysis. The
component of
interest produced in the milk of the mouse was human anti-thrombin III.
The pH of the milk sample was not adjusted because the volume was too
small. The milk sample was subsequently divided into two 500 1 aliquots which
were
placed in 1.5m1 polypropylene tubes. A 500m1 aliquot of 2.OM arginine, pH 5.0,
was
added to one tube and 100 1 of I.OM epsilon-amino caproic acid (EACA) was
added to
the other tube with 400 1 of PBS to equalize the volumes at 1.Oml in each
tube. The
'tube contents were filtered using a 0.45 m filter unit (Millipore, Bedford,
MA). Results
showed that the arginine containing sample was easy to filter and the recovery
was 83%
of the starting anti-thrombin M. The material with the EACA was extremely
difficult to
filter and the recovery of anti-thrombin III was 50%.
Eya=le 7: Isolation of LA-tPA from the Milk of Transgenic Dai Coats Using
Arginine as the Solubilizing Agent
The milk was thawed at room temperature, adjusted to pH 5.0 using 1.OM
acetic acid, diluted with an equal volume of 1.OM arginine and dead-end
filtered through
a 0.22 m nominal filter (Sartorius, Bohemia, N.Y.) followed by a 0.201im
absolute filter
(Millipore, Bedford, MA). LA-tPA was purified from the filtrate by initially
binding the
enzyme to a TSK-butyl column. The column was equilibrated and washed in 20mM
sodium phosphate (pH 6.0), 100mM arginine-HCI, 0.01% Tween 80 and step eluted
using 20mM sodium phosphate, 100mM arginine-HCI and 70% ethylene glycol.
Elution
pool collection was accomplished by monitoring by absorbance at 280nm at 1.0
AUFS.
Collection started at the first deflection above baseline. This initial step
provides a
means of concentrating and substantially purifying the enzyme. Recovery of LA-
tPA
from this procedure was 95% of the starting activity compared with 25 to 50%
recoveries
observed without the addition of arginine.
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WO 94/19935 PCT/US94/01672 ~
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Fquivalents
Those skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
embodiments of the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
