Note: Descriptions are shown in the official language in which they were submitted.
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Background of the Invention
1. Field of the Invention
This invention relates to the stabilization of
labile enzymes in liquid media.
2. ~escription of the Prior Art
It has recently been estimated that 25% of all
in vitro diagn~stic tests conducted annually in this Country are
not reliable. Unreliable tests can result in unnecessary medical
treàtment, the withho7ding of necessary treatment and lost income.
Because of their high specificity, the use of enzyme determinations
has significantly increased during the last few years and indi-
cations are that this trend will continue. However, rigorous
quality control measures are required to assure the accuracy and
consistency of results. This requirement stems from the fact
that the exact nature of enzymes, as well as the mechanisms of
their action, remain unknown for the most part. At present, the
grcatest limitation on the enzyme reagent manufacturer, by far,
lies in the unstable characteristics of his products. Current
methodologies require the use of numerous labile ingredients, and
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-` . 1091174
these ingredients are more likely to increase, rather than
decrease, in number.
The present commercial state of the art used for
` stabilizing the reactive a~ilitv of enzymes is by locking
S them into a solid matrix eithex by freeze drying, dry
blending such as used for tableting dried powders, primarily
in the pharmaceutical diagnostic and related industries and
immobilization by locking the chemical structure of the
enzyme into a solid matrix. Contrary to the sophistication
these terms imply, these approaches are neither practical nor
desirable and are also expensive. The manufacturer is forced
to remove the water and supply a partial product, thus
relinquishing part of the quality control cycle in the
dilution and use of the final product~ ~aboratories are forced
to pay the high cost of packaging, reagent waste, freeze drying
and dry blending, and usefulness of the product is further
- limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to
achieve. This condition is exemplified by the fact that most
commercial freeze dried controlled sera reference serum list the
acceptable bottle to bottle variation of enzyme constituents at
+ 10% of the mean.
Summary of the Invention
Labile enzymes are chemically modified according to
the invention resulting in long term stability without
affecting enzymatic reactivity in accordance with the invention.
The invention provides reagents where quality control is assured
throughout manufacturing, packaging, storage and use. The
inconvenience of rigid package size is eliminated as is the high
cost of packaging, freeze drying and reagent waste. Liquid
-- 109il~74
enzyme systems provide application flexibility and separation
o~ the ingredients is easily accomplished with negligible
manufacturing cost providing the flexibility of triggering the
desired reaction after all side reactions have been dissipated.
The stabilized enzymes of the invention have been
assessed in studies which compared liquid enzyme reagents with
fresh reagents. The studies show a 1:1 correlation between
liquid and fresh reagents with comparable sensitivity and
precision. Providing enzyme reagents in liquid form enhances
the colorimetric applicability of present day ~AD/NADH coupled
methodologies primarily because the separation of ingredients
is easily accomplished. Liquid reagents are especially
advantageous where NADH consumption is the basis of measure-
ment and the color reagent must be separated from NADH and the
reaction main. In the ultraviolet mode, the liquid enzyme
system offers better reagent homogeneity and packaging, as well
as flexibility in usage, in contrast to the freeze dried or
dry media preparations.
In diagnostic enzymology, the stabilization of
enzyme reagents in a ready-to-use liquid media is a new and
exciting approach to satisfy the needs of the clinical
laboratory and the reliability demands of the regulatory
authorities. The flexibility of liquid enzyme systems insures
their applicability to automated instrumentation, as well as
their convenience in manual testings.
Stabilization of labile enzymes is accomplished in
accordance with the invention by dissolving lyophilized, dry
~nzymes in an aqueous enzyme base including at least 0.05% of
polymer and at least 20~ v/v of organic solvent. The solution
is maintained at a temperature below the denaturing point
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109~174
suitably below 60C and in most eases below 40C for a`t
least 30 minutes, usually 2 to 3 days. The solution is then
diluted with water typically at least a 20 times and usually
a 30 times dilution while adding further polymer to maintain
a level in diluted stage of at least .05 weight ~. The
diluted solution suitably at an enzyme eoneentration from 100
to 10,000 I.U. per liter may then be paekaged in separate
containers and sealed and is stored refrigerated at temper-
atures of 30C or less.
- 10 The diluted solution may also contain substrate
buffer and bacteriastatic agent and other eomponents if
neeessary. If these other ingredients are added the diluted
solution is mixed to obtain a single homogeneous substrate
solution before dispensing into individual containers, sealing
and storage.
Substrates are organie ehemieals of known strueture
whose reaetions or interaetions are catalyzed by enzymes re-
sulting in a ehange in the eompound structure, atomic
eomposition, or stereo chemieal rotation, fo~ example, laetie
acid, L-aspartate or, alphaketoglutarate, L-alanine or the like.
In general, substrates are prone to mierobiological
degradation as they serve as food for baeteria, fungi and other
microorganisms. Otherwise, these compounds remain stable in
aqueous media at or near neutral pH typically from 4-10. Thus
if the substrate is added to the enzyme composition the
stabilizing media should also contain a buffer to control
reaction pll such as an al~ali metal acid phosphate and a
bactericidal and/or fungicidal agents which do not chemically
react with the substrate or inhibit the enzymatie reaction of
3~ the substrate. Typieal examples are 0.1~ sodium azide, benzoie
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acid, phenol, thymol, or pentachlorophenol.
It is believed that the selected organic solvent
stabilizes the enzyme in liquid media by protecting the
functional group site, that is the part of the molecule where
S the substrate reaction actua-ly occurs or is catalyzed~and by
protecting the enzyme from microbial contamination and thus
degradation. There is obviously some physical or chemical
reaction occurring in the concentrated solvent media since the
enzyme has no catalytic activity for the substrate at this
solvent concentration. However on dilution the enzyme is
restored to full activity and maintains its full reactivity
at high levels over extended storage periods of from a few
months to several years. The internal chemical structure of
the enzyme molecule need not be preserved. As long as the
reactive site is preserved, the catalytic activity of the
enzyme remains intact.
Microbial degradation can also be controlled by use of
high sale concentrations such as at least 1~ typically 2 to ~ -
welght ~ or higher concentration of salts. The salt molecules
may also protect the active sites by forming electrostatic bonds
protecting the spacial configuration of the enzyme and the
active sites.
These and many other objects and attendant
advantages of the invention will become apparent as the
invention becomes better understood by reference to the follow-
ing detailed description.
Description of the Preferred Embodiments
Enzymes are large molecular weight, complex protein
molecules, usually of unknown chemical structure. They are
presently classified by their catalytic activity and extreme
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~osll74
substrate specificity. Enzymes may be redefined as biological
catalysts, capable of catalyzing a reaction of a single
substrate, or a reaction of a similar group of substrates.
.. . ~
Typical enzymes are LDH, MDH, CPK, and the like. The
enzyme is present in the diluted, stabilized composition in an
amount typically from 100 I.U. to 10,000 I.U.
Substrates are organic chemicals of known structure,
I whose reactions or interactions are catalyzed by enzymes
resulting in a change in the compound's structure, atomic
composition, or stereo-chemical rotation.
In general substrates are prone to microbiological
degradation as they serve as food for bacteria, fungi, and
other microorganisms. Otherwise, these compounds remain stable
in aqueous media at or near neutral p~ (i.e~ pH range of 4-10).
Typical substrates are L-alanine, pyruvate,
L-aspartate, alpha-ketoglutarate~ and the like. The substrates
are usually in salt form and form part of the salt concentration
useful in enhancing stability of the enzyme. The enzymatic
stability increases with substrate concentration. However at
high substrate concentrations over about 8% enzymatic activity
is inhibited. Therefore the substrate concentration should be
optimized, generally at about 2 to 4%.
The buffer salt also provides part of the salt con-
centration discussed above. The buffer salt is added in an
amount necessary to maintain pH between 4-10, typically from
6-8. Generally the buffer is a combination of .1-1% of an
alkali metal hydroxide and 0.5 to 3% of an alkali metal acid
carbonate or phosphate. The total salt concent also effects
thc amount of polymer required. At higher salt content, e.q.
above 4~ by weight, less polymer is required due to the electro-
109~17~
static stabilization provided by the salt. However, at highersalt content, the polymer may cloud the solution or precipitate
requiring warming the solution to redissolve.
The polymer is preferably provided in the diluted
stabilized solution up to an amount that remains in homogenous
suspension under refrigeration without precipitation. The
polymer is present in an amount from 0.01 to 0.5% preferably from
1 0.05 to 0.25%. Water soluble polymers useful as stabilizing
agents in this invention are those that do not inhibit enzymatic
activity, and are capable of entrapping the enzyme in the polymer
matrix. The polymer may be a synthetic organic material such as
polyvinylpyrrolidine or dextran of biologic origin such as
gelatin which is denatured collagen.
The solvent must be miscible with water, of neutral or
alkaline pH, liquid at room and refrigerator temperatures, and
non-degradatively reactive with reactive sites of the enzyme
other than formation of electrostatic bondsO Useful solvents
are generally polar organic solvents such as ethers, ketones,
sulfones, sulfoxides and alcohols such as methanol, ethanol,
propanol, butanol, acetone, dioxane, DMS0, dimethylsulfone
and THF. However, higher activity at lower solvent concen-
tration for the treatment step is found for liquid polyol
solvents containing from 2-40H group and containing from 2-10
carbon atmos such as glyceroI~ propanediol, butane diol,
ethylene glycol and the like.
The solvent must be present in an amount of at least
20% during the treatment step typically from 25 to 50%. Some
solvents require concentrations as high as 70% in order to
maintain stabilized activity above 60% enzymatic reactivity.
Specific examples of practice follow:
109117~
EX~5PLE 1
Enzyme Base
Material Amount
Gelatin ~ 0.1% W/W
1,2 propane diol 30% V/V
Water 70~ V~V
Ammonium sulfate suspension (2.2M) or dry
lyophilized LDH enzyme in an amount equivalent to 22,500 IU/l
was dissolved in the enzyme base and held at 4-30C for 2-3
10 days.
Substrate Reagent
Material Amount
L-Alanine 22 g/l
Alpha-ketoglutaric acid 1.6
H2PO4 14
NaO~ 5
Na~2
Gelatin
The enzyme base was diluted thirty-fold by addition
to the substrate reagent suspension and mixed to obtain a
homogenous suspension. The suspension is stored refrigerated.
Projected shelf life under refrigeration is three years with
50-90% activity remaining.
In the clinical diagnostic field the commercial
application of these stabilizing methods is represented by,
but not limited to, the diagnostic reagents used to determine
and quantitate the following constituents in biological fluids:
1. Glutamic-oxalacetic transaminase (SGOT):
2. Glutamic-pyruvic transaminase (SGPT)
3. Lactic dehydrogenase (LD~
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4. Creatine phosphokinase (CPK):
5. ~-Hydroxybuterid dehydrogenase (~-HBD)
6. Glucose (via Hexokinase-G-6-PDH).
These reagents react similarily, contain some common labile
.;, .
ingredients, and some of the chemical reactions involved are
common. The following chemical reaction scheme is presented
as a model to illustrate the general nature of the reactions
involved:
; REACTION SCH~ME 1. --GENERAL MODEL
. 10 Enzyme 1
(1.) SUBSTRATE(S) PRODUCT(S)
pH
-~ ~ - Enzyme 2
¦ (2.) PRODUCT/SUBSTRATE + NAD-NADH -- NADH -NAD+PRODUCT
_ 2 - 2
pH
Catalyst
15 (3.) NADH2 + CHROMOGEN - ~ CHROMOGEN + NAD
(oxidized) - ~ (reduced)
All enzymatic reactions listed above will follow this
general scheme, where reaction (2.) is usually referred to as the
coupling reaction, reactions (2.) or (3.) are the measuring
reactions, and reaction (1.) may be characterized as the primary
reaction. It is understood however, that not all three reactions
are required for measurement in fact, they may ~e limited to two,
or one. In the case of the ultraviolet measurement of lactic
dehydrogenase (LDH) activity, only reaction (2 ) is involved,
25 as follows:
RE~CTIO~ SCHEME 2. -- LDH
_
LDH
Pyruvate + NADH2 -- ` NAD + Lactate
Conversely, more than the three reactions listed may
be involved as in the case of Creatine phosphokinase (CPK):
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REACTION SC~E~E 3. -- CPX
CPK ATP + Creatine
HK
S (2,) ATP + Glucose - Glucose-6-Phos. + ADP
; G-6-PDH
(3.) Glucose-6-Phos. + NAD ~ -~ NADH
PMS
~4.) NADH2 + INT ~ INT + NAD
(ox) ~ ~red)
SYMBOLS: .
CP 3 Creatine phosphate
ADP = Adenosine-5'-diphosphate
ATP = Adenosine triphosphate
lS HK = ~exokinase
NAD = nicotinamide-adenine dinucleotide
NAD~2 = nicotinamide-adenine dinucleotide, reduced
G-6-PDH = Glucose-6-phosphate dehydrogenase
INT = tetrazolium salt
PMS = phenazine methosulfate.
In this case, reactions (2.) and (3,) may be considered the
coupling reactions, reactions (3.) or (4.) the measuring
reactions, and reaction (1.) the primary reaction,
Referring to REACTION SCHE~IE 1, --GE~ERAL MODEL, it
becomes obvious and is general ~nowledge that the use of the
reaction sequence permits the analytical quantitation of either
the reacting substrates/products or the catalyzing enzymes.
The quantitation oif these constituents in
biological fluids is a well accepted and widely ~sed diagnostic
tool in diagnosis and treatment of human and animal disease states.
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It is to be realized that only preferred
embodiments of the invention have ~een described and that
numerous substitutions, modifications and alterations are
permissible without departing from the spirit and scope of
S the invention as defined in the following claims.
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