Note: Descriptions are shown in the official language in which they were submitted.
-- WO 95/05240 2 1 6 ~ 7 6 7 PCT/US94/09187
COMPOSITE BIOLOGICA~MOLECULE-ACCRETION M~TERIAL
This is a continuation-in-part of priority U.S. Patent Application
Serial No. 08/106,41S filed 13 August 1993.
Background and Summary of the Invention
The present invention relates generally to filters and other devices
for removing undesired chemicals from solution, and more particularly to a
composite biological-molecule-accretion material for extracting desired target
biological molecules from aqueous solution and substantially permanently bindingto those molecules.
There have been several conventional proposals for dealing with
removal of unwanted chemicals from sol~ltion. However, none has proven
adequate for dealing with removal and ultimate disposal of certain chemically
labeled molecules regularly used in connection with basic and applied biologicalresearch. Those molecules will be referred to in the present application as target
molecules, and a list of them is provided below:
Target Biological Molecules
1. peptides;
2. polypeptides;
3. RNA;
4. DNA;
5. enzymes;
6. nucleic acids;
7. proteins;
8. biopolymers; and
9. amino acids.
The essential problem for laboratories performing research with
such molecules is that present environmental regulations no longer allow for
simply pouring them down the drain after they are chemically labeled according
to known procedures. Examples of such labeling involve using radioactivity or
30 fluorescence to label a desired molecule. While radio-labeled molecules,
sometimes referred to as probes, will be referred to throughout, it should be
understood that the to-be-described invention is usable for removing any
Sll~ S~E~ 26)
WO 95/OS240 2 ~ 6 ~ ~ 6 7 PCT/US94/09187
chemically labeled target molecule from aqueous solution and substantially
permanently binding to it.
Basic and applied biological research can also result in waste
products characterizable as ~ lules of toxic organic solutions and radio labeled5 target molecules in aqueous solutions. Such mixtures of solutions will be referred
to herein as "mixed waste". The to-be-described invention is also usable for
removing such radio labeled target molecules from such mixed waste.
Nowhere has there been shown or suggested to provide a composite
biological-molecule-accretion material for (1) extracting desired target b;ological
10 molecules from either aqueous solution or mixed waste, and (2) substantially
permanently binding to those molecules. Accordingly, it is a principal object ofthe present invention to provide such a material that overcomes the drawbacks
of prior-art materials.
Another object is to provide such a material that provides for such
15 removal and binding to a relatively wide variety of molecules such as the target
molecules listed above.
Yet another object is to provide such a material that has a relatively
long working life.
Another important object of the invention is to provide such a
20 material that can be readily and easily disposed of after its working life is over.
It is also an object of the invention to provide such a material that
can be easily and cost-effectively m~m-f~ctured.
In brief summary, one aspect of the invention includes a composite
biological-molecule-accretion material for extracting (or removing) desired target
25 biological molecules from aqueous solution and substantially permanently binding
to those molecules. The material includes a first component designed with a
binding affinity for proteins and peptides in such solution, and a second
component designed with a binding affinity for nucleic acids in such solution. The
material is capable of being formed in a body for passing such solution through
30 it, and such passing has the effect of removing the proteins, peptides and nucleic
acids from such solution and binding them to the body. That binding is
substantially permanent so that subsequent passage of additional aqueous solution
W O 95/05240 ~ 1 ~ 8 7 6 ~ PCT~US94/09187
will not unbind substantially the accreted proteins, peptides and nucleic acids
from the body.
Preferably the first component consists essentially of dextran-coated
activated charcoal particles, and the second component consists essentially of an
S anion-~oYfh~nee resin and a cation-exchange resin. The preferred relative volumes
of both components are 1:1, and the relative volumes of the two ion-exchange
resins is 1:1. As a result, relative volumes for the composition of the preferred
version of the material is 2 parts dextran-coated charcoal particles to 1 part anion-
exchange resin to 1 part cation-çxch~nee resin.
Another aspect of the present invention includes a method of
filtering target biological molecules out of water. The method includes the stepof forming a filter bed by soaking a ~ ule of dextran-coated, activated charcoalin water for a preselected time period, adding to the mixture an ion-exchange
resin with a preselected charge-sensiliviLy, drying the mixture, and rehydrating the
mixture. The method also includes the step of placing the filter bed into a water-
filtration apparatus with an input and an output, and passing water cont~inine the
target biological molecules through the filtration apparatus to cap~-lre
substantially the target biological molecules in the filter bed.
These and other objects and advantages of the invention will be
more clearly understood from a consideration of the accompanying drawings and
the following description of the preferred embodiment.
Brief Description of the Drawings
Fig. 1 is an isometric view of the accretion material of the present
invention after being formed in a body (or filter bed) and being placed into
conventional water-filtration apparatus.
Fig. 2 is a side-sectional view through line 2-2 of Fig. 1 with the
accretion material being formed in a body, and with solid lines indicating an
unswelled condition of the material, and dashed lines indicating a swelled
condition of the same.
Fig. 3 is a fragmentary, greatly enlarged, schematic view of the
unswelled body of accretion material shown in Fig. 2.
WO 95/05240 æ 16 8 7 6 ~ PCT/US94/09187
Fig. 4 is a fragmentary, greatly enlarged, schematic view of the
swelled body of accretion material shown in Fig. 2.
Fig. 5 is a greatly enlarged view of unswelled charcoal particles
forming part of the accretion material.
Fig. 6 shows the charcoal particles of Fig. 5 after swelling.
Fig. 7 shows the charcoal particles of Fig. 6 after being coated with
dextran substance.
Fig. 8 shows the charcoal particles of Fig. 7 being mixed with anion-
and cation-exchange resins, with the resins being indicated schematically by
encircled +'s and -'s.
Fig. 9 shows the charcoal particles of Fig. 8 after being mixed with
the resins.
Fig. 10 is like Fig. 2 only that it shows the swelled condition of the
body of accretion material in solid lines, and shows introduction into the
conventional water-filtration apparatus of aqueous solution cont~ining desired
target biological molecules, with the solution passing through the body.
Detailed Description of the Preferred Embodiment
Referring generally to Figs. 1-4 and 10, applicant will describe
generally how he proposes using the invented composite biological-molecule-
accretion material, which material is indicated at 10 being made in accordance
with its to-be-described preferred embodiment. After describing how the inventedmaterial is used, applicant will discuss details of its composition. Material 10 is
formed in a body 12, with body 12 being placed in a conventional water-filtration
apparatus 14. In its place in apparatus 14, body 12 will allow a to-be-describedaqueous solution to pass through it, with such passing having the effect of
removing certain target molecules such as proteins, peptides and nucleic acids
from such solution and binding them to body 12. The binding is substantially
permanent in that subsequent passage of additional aqueous solution will not
unbind substantially the accreted proteins, peptides and nucleic acids from the
body. As will be shown, material 10 does not unbind substantially from the
accreted proteins, peptides and nucleic acids even when subsequent passage of
W O 95/05240 ~ ~ 6 8 7 6~ PCTrUS94/09187
additional aqueous solution includes relatively harsh components such as
detergents.
Still referring to Figs. 1~ and 10, conventional water-filtration
apparatus 14 is constructed with an input 16 and an output 18, and includes a
receptacle 20 for receiving effluent from output 18. Receptacle 20 is formed with
a suitable port 22, which may be an aspiration port, for dispensing effluent from
the receptacle. The port may be fitted with the usual control valve (undepicted).
It should also be understood that a suitable support screen
(undepicted) may be provided within apparatus 14 and around body 12 to hold
10 material 10 in place so that the to-be-described aqueous solution must pass
through body 12 after being poured through input 16.
While undepicted, it should be understood that multiple stages of
filtration are possible, if desired, to perform a backup function as a way of
removing even more chemically-labeled target molecules from waste solutions.
15 In other words, the invention may include an arrangement of two or more
apparatuses, like apparatus 14, with each such apparatus having a body of
material, an input, and output, each like those described above. Preferably, such
arrangement would include a vertical stacking of such apparatuses, with the
bottom apparatus being placed over a receptacle like receptacle 20. As used
20 herein, a one-stage filtration system is one like that depicted in Fig. 1. A two-
stage filtration system is one that has a vertical stack of two apparatuses, like
apparatus 14, with the bottom apparatus being placed over a receptacle like
receptacle 20. To stack such apparatuses, it is presently proposed to use suitable
support structure for stabilizing the stack.
Refocusing on material 10, certain physical characteristics of
accretion material 10 will also be described before describing its composition.
Referring to Fig. 2, body 12 is shown in an unswelled condition with solid linesbetween bracketed area A, and in a swelled condition up to a dashed line
between bracketed area B. The significance of the unswelled/swelled condition
30 will be described. The unswelled condition of body 12 is also shown in a greatly
enlarged, schematic view in Fig. 4. Similarly, Fig. 5 shows the swelled condition
in a greatly enlarged, schematic view.
WO 95/05240 2 ~ 6 ~ 7 61 PCT/US94/09187
Referring to Figs. 5-9, a schematic presentation of a method of
forming material 10 is shown. As will be described, material 10 includes a firstcomponent 10_ (Fig. 7) designed with a binding affinity for proteins and peptides
in aqueous solution such as solution 24 shown in to-be-described Fig. 10.
Material 10 also includes a second component 10_ (Figs. 8-9) designed with a
binding affinity for nucleic acids in such solution.
From an overview, Figs. 5-7 show formation of first component 10~,
and Figs. 8-9 show introduction and then mixing of second component 10_ with
first component 10_. The first component includes, and preferably consists
essentially of, dextran-coated activated charcoal particles, which exhibit the
requisite binding affinity for proteins and peptides. The second component
includes, and preferably consists essentially of, an anion-exchange resin and a
cation-eYçh~nge resin. Those resins exhibit the requisite binding affinity for
nucleic acids. Material 10 is preferably formed of 1 part by volume of first
component 10_ and 1 part by volume of second component 10_. Second
component 10 is preferably formed of 1 part by volume anion-eYrh~nge resin
and 1 part by volume cation-exchange resin. Therefore, the preferred formulationof material 10, by volume, is 2 parts first component 10_ to 1 part anion-exchange
resin and 1 part cation-eyrh~nge resin. The total volume of ion exchange resin
(both anion- and cation-exchange resins) in the preferred embodiment is about
one-third to one-half of the volume of the dextran-coated activated charcoal.
Also, suitable quantities of suitable filler material, such as course sand, may be
added to increase flow rate through material 10.
With material 10 being formed into body 12, it may also be
characterized as a target-biological-substance-retaining filter for use in a filter
system to remove subst~nti~lly and selectively desired target biological substances
from an aqueous solution (e.g. solution 24 of Fig. 10). First component 10_ may
also be thought of as a filter-matrix component formed from first and second
subcomponents. The first subcomponent is preferably activated charcoal particles,
and the second component is a water-soluble, high-molecular-weight substance
such as dextran. Second component 10_ may also be thought of as a charge-
carrier component distributed subst~n~i~lly in the filter-matrix component to
WO 95/05240 21~ 8 ~ 6 7 PCT/US94/09187
provide a plurality of charged sites therein. The filter-matrix component (firstcomponent 10_) and the charge-carrier component (second component l0k) are
usable in a desired thickness (as body 12) in a filter system (apparatus 14) to
provide a filter that defines paths through its thickness for allowing the aqueous
5 solution (e.g. solution 24 of Fig. 10) to pass, and removes substantially the target
biological substances by binding them to the charged sites.
Material 10 being formed as body 12 may also be characterized in
yet another way as a filter bed for use in a filter system to remove target
biological substances contained in an aqueous solution (e.g. solution 24 of Fig.10 10). First component 10_ may be characterized as means for forming a filter
matrix that is porous substantially only to that portion of such solution that does
not include the target biological substances. The filter-matrix-forming means
includes first and second subcomponents, activated charcoal particles and a water-
soluble, high-molecular-weight substance, respectively. The high-molecular-weight
15 substance is preferably dextran, and it is mixed with the charcoal particles to coat
them. Second component lOk may be characterized as a charge-carrier
component distributed subst~nti~lly in the filter-matrix-forming means to provide
a plurality of charged sites therein. The filter-matrix-forming means and the
charge-carrier component are usable in a desired thickness (i.e. body 12) in a
20 filter system (i.e. apparatus 14) to provide a filter bed that defines paths through
its thickness for allowing the aqueous solution (solution 24 of Fig. 10) to pass, and
removes substantially the target biological substances by binding them to the
charged sites.
Referring to Figs. 5-7, preparation of material 10 is shown
25 schematically with first component 10_ being formed by soaking a mixture of
activated charcoal 10_, and dextran 10_2 in water (undepicted) for a preselectedtime period. Figs. 5-6 show the swelling of charcoal 10_1 in water prior to
addition of dextran 10_2. The presently preferred time period is about one hour. Next, referring to Figs. 8-9, second component 10k, taking the form of preselected
30 anion- and cation-exchange resins (shown schematically as encircled +'s and -'s),
is mixed with first component 10_. The resins forming second component 10k
have a preselected charge-sensitivity, and preferred ones will be described below
WO 9S/05240 2 ~ ~ 8 7 6 7 PCT/US94/09187
in connection with example formulations. After mixing the first and second
components, the mixture is aspirated to remove residual water and allowed to dry,
and then rehydrated with a suitable amount of water so that it swells. Applicanthas found that such rehydration promotes formation of the above-described filter5 matrix within body 12, and lessens the possibility that some target molecules will
be missed when the first amount of the aqueous solution (e.g. solution 24 of Fig.
10) is passed through body 12.
EXAMPLE I
Material 10 was prepared using 45% activated charcoal such as that
marketed under the trademark "NORIT A", 255'o Wh~tm~n DE52 pre-swollen
microgranular anion exchanger (diethylaminoethyl cellulose, catalog number 4057
050), 25% Rolm & Haas Co. Amberlite CG-50 (weakly acidic cation çx~h~nger,
carboxylic type, hydrogen form, wet mesh 100-200), and 5~o course sand. An
amount of that material having a wet volume of 100 ml was formed in a body like
body 12 and placed in a filter structure like apparatus 14. 20,uCi of 3sS-ATP in800ml of Tris/Borate EDTA buffer was passed through the body of material 10.
After subsequently passing 800ml of aqueous solution cont~ining no radio-labeledbiomolecules through the material, the total effluent contained only .37,uCi of
radioactive isotope, indicating a permanent binding efficiency of appr--xil"~tely
98~o for ATP.
EXAMPLE II
Material 10 was prepared as in example I and placed in a filter
structure like apparatus 14. Solutions cont~ining 24,uCi of 32P-CTP DNA probe
in 0.1~o sodium dodecyl sulfate solution were passed through material 10.
Despite the harsh character of a detergent like the sodium dodecyl sulfate
solution, only .2~Ci of the 32P-CTP DNA probe passed through the material,
indicating a permanent binding efficiency of greater than 99~o.
EXAMPLE III
Material 10 was prepared as in example I and placed in a filter
structure like apparatus 14. 49,uCi of 32P-labeled, 1450 base-pair DNA fragment
in a 50~o formamide solution was passed through the material. Analysis of the
WO 95/05240 2 1 6 8 ~ 6 7 PCT/US94/09187
effluent from apparatus 14 revealed .3~Ci of radio-isotope passed through the
material, indicating a permanent binding efficiency of greater than 99~o.
EXAMPLE IV
Material 10 was prepared as in example I and placed in a filter
5 structure like apparatus 14. A mixed waste solution of HPLC waste (40%
acetonitrile/60% water - by volume) Cont~ining radiolabeled peptide was passed
through the material. Specifically, 300-mL of the solution containing
approxi~ tely 4.5,uCi 12sI-,~EP (~-endorphine) was passed through the material.
Analysis of the effluent from apparatus 14 revealed less than 0.0004 ,uCi of radio-
10 isotope passed through the material, indicating a permanent binding efficiency ofgreater than 99.99%. Over a 48-hour time period, water was intermittently passed
through the material, and analysis of the effluent after such time period indicated
no change in the 99.99+ % permanent binding efficiency.
A prolonged use of material 10 in a one-stage filtration system was
15 run over an 8-month period to remove 35S radioactive nucleotides. Specifically,
an amount of material 10 having a wet volume of 100 ml was formed in a body
like body 12 and placed in a filter structure like apparatus 14. 58 units of
aqueous solutions cont~ininE 35S radioactive nucleotides were passed through theamount of material 10, and the average % of radioactive material absorbed by the20 amount of that material was 91.3%.
Additional, similar experiments involving radiolabeled proteins,
peptides and nucleic acids revealed similar results, with apploxi~ tely 98-99% of
such molecules becoming subst~nti~lly permanently bound to the accretion
material as the aqueous solution cont~ininE such molecules was passed through
25 an amount of the material formed into a body such as a filter bed. The
additional experiments have involved solutions with l25I-labeled peptides, 35S-
labeled amino-acid monomers. When using a body of material 10 having a wet
volume of 100 ml, the time for passage through the body of aqueous solution
containing target molecules ranged from about 10 minutes with aspiration, to
30 overnight under gravity feed.
Material 10 worked as described above with acidic or basic aqueous
solutions. It has also indicated a relatively long working life, i.e. an amount of
W095/05240 216 8 7 6 ~ PCT/US94/09187
material 10 with a wet volume of 100 ml continues to exhibit the above binding
efficiency of 98-99% after week~y use in a biological laboratory for approxim~tely
six months.
For situations where it is important to control the pH of solutions
5 that are passed through material 10, buffer-like components may be added to
material 10. It is presently proposed to use solid forms of acid- or base-le~ç~ing
substances. For example, solid masses or nuggets of NaOH may be uniformly
distributed in the material, using suitable mixing apparatus, to act as a bufferwhen acidic solutions are passed through material 10. Likewise, suitable weak
10 acids may be used, in solid masses/nuggets, to act as a buffer when basic solutions
are passed through material 10.
Operation and Preferred Method of Practicing
Using the above-described biological-molecule-accretion material
10, which is a solid, a method may be practiced for extracting desired target
15 biological molecules from aqueous solution and substantially permanently binding
them to that solid. The method includes the step of forming a solid with a
binding affinity for such molecules. The forming step is preferably practiced bym~king the above-described biological-molecule-accretion material 10. The
method also includes the steps of shaping the solid into a body and placing the
20 body into a water-filtration apparatus with an input and an output, and passing
aqueous solution conl~ini~g such molecules through the filtration apparatus to
remove them subst~nti~lly from such solution, and substantially permanently bindthem to the solid. Next, there is a step of repeating the passing step with
additional aqueous solution and rn~inl~ ing binding of the accreted proteins,
25 peptides and nucleic acids to the solid.
Use of material 10 also involves a method of filtering target
biological molecules out of water. That method includes a step of forming a filter
bed by the substeps of soaking a mixture of dextran and activated charcoal in
water for a preselected time period, adding to the mixture an ion-exch~nge resin30 with a preselected charge-sensitivity, drying the ~ lul e, and then rehydrating the
lure. The second step involves placing the filter bed into a water-filtration
apparatus with an input and an output, and the third step involves passing water
W095/05240 2 1~ 8 7 6 7 PCT/US94/09187
containing the target biological molecules through the filtration apparatus to
ca~Lure subst~nti~lly the target biological molecules in the filter bed.
It is presently proposed to m~int~in material 10 in a wet condition
throughout its working life, although if the material were to dry out, a simple
5 rehydration step could be performed as described above. Such rehydration was
performed in connection with Example IV above and that amount of material 10
exhibited 99.99+ ~ permanent binding efficiency.
When material 10 has exceeded its working life, it is dried by
aspiration, removed from water-filtration apparatus 14, and disposed of as solid10 radioactive waste. Any effluent from aspirating may be poured down a drain ifsufficiently low radioactivity is detected. It is presently contemplated that state
and federal regulations do, or may soon, require extremely low or no radioactivity
for effluent poured down a drain to a public water supply. With those regulations
in mind, multiple-stage filtration systems utilizing material 10 may be used until
15 a sufficiently low radioactivity is detected.
It should also be understood that material 10 could be tailored to
be selective to certain types of molecules as opposed to being designed for
removal of the entire class of target molecules listed above. For example, if one
wanted to remove only those molecules with negative charges in aqueous solution
20 (such as DNA compounds), then only a cation-exchange resin would be used as
second component 10_.
The present invention achieves the above objects by providing a
composite biological-molecule-accretion material for extracting desired target
biological molecules from aqueous solution and substantially permanently binding25 to those molecules. That material overcomes the drawbacks of prior-art
materials, and provides for such extraction/removal and binding to a relatively
wide variety of molecules such as the target molecules listed above. The invented
material has presently shown to have a relatively long working life, and when its
working life is over the material can be disposed of readily and easily. Based on
30 its preferred embodiment, the material can also be easily and cost-effectively
m~nllf~ctured.
WO 95/05240 PCT/US94/09187
~16876'~
12
Accordingly, while a preferred embodiment of the invention has
been described herein, it i5 appreciated that modifications are possible that are
within the scope of the invention.
