CA1101828A

CA1101828A - Composition for chromatography

Info

Publication number: CA1101828A
Application number: CA309,040A
Authority: CA
Inventors: James R. Schaeffer; Roy E. Snoke; Harry W. Harris
Original assignee: Eastman Kodak Co
Current assignee: Eastman Kodak Co
Priority date: 1977-09-09
Filing date: 1978-08-10
Publication date: 1981-05-26
Also published as: JPS5451596A; DE2839170A1; GB2006640B; DE2839170C2; GB2006640A; US4111838A

Abstract

COMPOSITION FOR CHROMATOGRAPHY

Abstract A chromatographic material comprising an inorganic support-polysaccharide particle matrix is described. The matrix comprises an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of a polysaccharide. The free hydroxyl groups of the matrix can be activated, for example, by treatment with cyanogen bromide or sodium metaperiodate, to form active sites where ligands can be attached for affinity chromatography.

Description

Affinity chromatography has found wide application in the purification of various biologically active materials, in-cluding for example, enzymes, proteins, antibodies, nucleotides, small ligands, and the like. It is known that certain poly- -~
saccharide matrices comprise the most useful solid supports for affinity chromatography. Various methods exist to activate a polysaccharide matrix~ e.g., cellulose, starch, and various crosslinked polysaccharide gels such as agarose, Sephade ~ and Sepharose~, for the covalent attachment of, e.g., small ligands and proteins. A widely used technique for the covalent coupling of, for example, protein to insoluble matrices, finding con-siderable application in immunology and enzymology, is the cyanogen bromide method described in Axen et al, Nature, 214, 1302-4 (1967); see also Cùatrecasas et al,''Proc. N'atl'. Aca'd'. Sci.
U.S. r 61, 636-43 ~1968). Another useful activation method, described by Cuatrecasas et al in U.S. Patent No. 3,9~7,352, comprises sodium periodate oxidation followed by reductive amina-tion using sodium borohydride or sodium cyanoborohydride.
~ The use of activated polysaccharides having Iigands ~' attached to purify biologically active material has been found to be a powerful laboratory tool. However, affinity chromatography using these materials is time consuming, especially when it is ~
desired to purify larger volumes, i.e., more than a liter, because '~ -these materials produce columns having slow flow rates.' One way to obtain faster flow rates is to attach a monolayer of a poly-saccharide to an inorganic support, such as a glass bead, as described in U.S. Patent No. 4,006,059, or in U.S. Patent No.
3,983,299. These patents describe the attachment of a monolayer ~'~
of de~tran, starch, giycerol, etc. to an inorganic bead. The '-~
resultant chromatographic material has much improved flow rates o~er the activated polysaccharides described above. However, we

- 2 -.,~

-, .: . ~ .~ .: - : . :
.

have found -that materials having a monolayer of a polysaccharide attached to an inorganic bead do not provide as high a degree of purification as desired (see examples herein). Thus it would be desirable to have a chromatographic material that provides columns with high flow rates and also provicles a high degree of purification.
The present invention provides a chromatographic material characterized by an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of a polysaccharide forming an inorganic support-polysaccharide particle matrix. The matrix can be acti-vated, e.g., by treating with cyanogen bromide or sodium meta~
periodate, etc., and a ligand can be covalently attached to pro-duce a material useful for affinity chromatography. Such material unexpectedly has both high flow rates and high purification ~ ~
capability for biological materials. These high flow rates and ;
the high purification capability of the materials of the present invention are apparently due to the particulate nature of the ~
polysaccharide that is bonded to the support in contradistinction ~ -to prior art materials that have monolayers of polysaccharide on a support.
Examples of some inorganic supports to which polysaccha-rides can be covalently bonded include porous silica, controlled porosity glass, controlled porosity ceramic, alumina, and the like. In principle, polysaccharides can be covalently bonded to any inorganic support containing a high surface density of hydroxyl groups. A particularly useful support material is con-trolled porosity glass beads (CPG) which are available com-merically from Corning Glass Works.
The chromatographic ma-terials of this invention comprise particles of insoluble polysaccharide covalently bonded to an ~ .

: i . , , , ~

inorganic support such as described above. Examples of useful such polysaccharide particles include crosslinked polysaecharide gels such as agarose gel, polyacr~lamide-agarose gel, and the like. Especially useful polysaccharide particles are agarose gels commercially available from Pharmacia Fine Chemicals Co.
(Piscataway, New Jersey) under the trademarks Sepharos ~ and Sephade ~.
The inorganic substrate having a high surface density of hydroxyl groups and the polysaccharide particles can be covalently bonded together by any known suitable chemical reaction. A eon-venient procedure is outlined below. First the polysaccharide ~
is activated by reaction with cyanogen bromide. ~;

, ~ OH OH O
I *PSP ~ ~ CNBr > ~ O ~ C=NH

*PSP is a polysacchride particle Then a diamine linking eompound is attached to the activated polysaecharide.
,OH O

o / H2~a(CH2)X NE2 , ~OH
> PSP.
\ OCNH(CH2)XNH2 Next the inorganic support material is aetivated with eyanogen bromide.

, ",OH OH O
III *IOS ~ CNBr ~ IOS \ ~ C=NH
~ OH
*IOS in an inorganie support Then the aetivated inorganic support and the polysaccharide par-tiele with the attached linking compound from Equation II are reaeted together.

~ -- .
X

~ ",OH
IV IOS \ C=NH + PSP
O / - OCN~I(CH2) NH2 OH OH HO OH
IOS / ~ PSP
~ OCNH(CH ) NHCO /
" 2 x "

~ 2 ~

In the above equations x is an integer having a value 3 or lQ greater.
The product resulting from the reaction of Equation IV
above is typically granular when the inorganic support is a bead or the like. From photomicrographs of the product of Equation IV
it appears that many polysaccharide particles are typically attached to one bead of inorganic support. However, it is easy to see from the reaction sequence that a polysaccharide particle may be covalently bonded to more than one bead of inorganic support material. Thus, since the polysaccharide particle is multifunctional, more than one point of attachment to the inor~
ganic support may exist and thé polysaccharide may bridge two ox more inorganic support particles.

The quantity of polysaccharide particles present in the -chromatographic matrix material of this invention is dependent, among other thin~s, on the particular inorganic substrate being used, on the structural shape of the substrate, and on the rela-tive size of the polysaccharide partlcles versus the substrate.
Generally, however, the polysaccharide particles are present in an amount of from about 1 percent to about l0 percent by weight of the inorganic support and preferably in an amount of from about 2 tc about 6 percent by weight based on the weight of the inorganic support. Particularly useful results can be obtained when using controlled poroslty glass beads as the support when _ 5 _ , ` ~

.

the polysaccharide particles are present in an amount equal to ~ :
from about 3 to about 5 percent by weight of the glass beads.
The particle size of the polysaccharide and of the in- ~:
organic support can vary depending upon the particular materials, .
the biological material to be purified, the quantities of bio-logical material to be purified, etc. It is generally desired, however, that the mean particle size of the inorgan.ic support material be larger than the mean particle size of the polysaccha-ride. Useful results can be obtained, for example, when the particle size of the inorganic support material is in the range of from about 100 to about 1000 micrometers and the particle size :-`
of the polysaccharide is in the range of from about 25 to about 300 micrometers.
While the chromatographic matrix material described abo~e . .
is useful per se, it can be used to produce even more useful chromatographic materials by activating the hydroxyl groups and attaching a ligand to the activated sites. Such activated matrix materials having a ligand covalently attached to the actlvated :~ ~
site are particularly useful for affinity chromatography. The ~ .
particular ligand selected depends upon the biological material to be purified. As discussed above, the hydroxyl groups can be activated by any known technique, for example, by cyanogen bromide activation or by sodium metaperiodate activation, etc.
Generally considering the use of CNBr-activation, the amount of ligand coupled to the polysaccharide depends on the amount of CNBr added. Typically, this varies between 50 and 300 mg of CNBr per milliliter of inorganic support-polysaccharide particle matrix material. For example, with 200 mglof CNBr per ~ :
milliliter of inorganic support-polysaccharide particle matrix material, if the concentration of low molecular weight ligand, e.g., alanine, is 0.1 M, the amount coupled will be about .
lOf~moles per milliliter of inorganic support-polysaccharide X

~ :. ~ , .

Z~ :

particle matrix material. ~he actual coupling efficiency will depend on the specific ligand used.
The quantity of CNsr and the exact composition of any buffer used in the coupling reaction should be adapted to the specific system under study. These conditions have been de scribed in detail, Cuatrecasas, J. Biol. Chem., 245, 3059 ~1970).
A standard condition is the use of 200 mg of CNBr per milliliter of matrix material and of 0.2 M sodium bicarbonate at pH 9.5 as a buffer for the coupling reaction. Smaller quantities of CN~r, lower pH values, and high concentrations of ligand will decrease the probability of multipoin-t attachments of proteins (especially those of high molecular weight) to the matrix, a condition that may lead to decreased or altered biological activity.
In many cases, the interposition of spacers between the matrix and the ligand greatly increases the effectiveness of the adsorbent. A variety of spacer molecules can be attached to polysaccharides, and many chemical reactions exist that can be used to couple ligands and proteins to these derivatized poly-saccharides, Cuatrecases, J. Bi_l. Chem., supra. Diaminodi-propylamine has been one of the most useful spacer moleculesbecause it is relatively long and because it exhibits very mini-mal hydrophobic properties as compared to strictly methylenic diamine compounds such as hexamethylenediamine. Whenever possible, it is advantageous to attach such spacers first to the ligand rather than to the polysaccharide since the adsorbents prepared in this way are less likely to exhibit nonspecific or ionlc properties that can interfere in subsequent affinity chromatography procedures.
A typical reaction for adding a ligand to the matrix material of *his invention is illustrated by the following equations that depict the coupling of an amino-ligand to the composite material. First, the matrix material is actlvated by reactin~ the cyanogen bromide~
~ -7-.. . . .

HQ / OH
V ~ S-CNH(CH2)x NHCO-PSP \ ~ CNBr HO~ / O
~ ~:)S-OCNEI ~CH2 ) XNHC"O \o,~

Next/ the amino-ligand is covalently bonded to the activated site.

VI HO~S OCN ( 2)x ,, < o / ~2N( 2)y ~`

HO OH
~ I~S~OCNH(CH2) NHCO-PSP ~
~ NH2 ~ NH2 OCNH(CH2)yR
In the above equations x is an integer having the value 3 or more, y is an integer haviny a value from 2 to about 30, and R is CH3, NH2 or NR2 where each R' is independently selected from the group consisting of lower alkyl groups having from 1 to about 4 carbon atoms. Of course, other known chemical reactions can be used to couple these and other ligands to the inorganic support-polysaccharide particle matrix material. Although the equations above illustrate only the bonding of the ligand to the poly-saccharide portion of the matrix, i-t is realized that the in-organic support also has free hydroxyl groups that may be activated and available for bonding with the ligand.
The inorganic support-polysaccharide particle matrix material may be used as a chromatographic materiàl, as is, or it may be activated so that ligands may be attached to the matrix as described above. An activated inorganic support-polysaccharide particle matrix can be prepared and stored for use, at which time a suitable ligand can be attached and the resulting material used for purifying biologically active materials. The inorganic support-polysaccharide particle matrix can be activated for storage and future use by a variety of known reactions, for '.

example, by the cyanogen bromide method described in U.S. Patent

3,914,1~3, or by the sodium metaperiodate method in U.S. Patent 3,947,352.
The following examples are provided to further illustrate the present invention.
Example 1 Preparation of CPG550-A~ Sepharose~ 4B
In 50 ml of distilled water, 4.0 g of CPG550 glass (a borosilicate base controlled porosity glass having a particle size diameter in the range of from 170 to 840 micrometers manu-factured by Corning Glass Works and distributed by Pierce ChemicalCo., Rockford, Illinois) was suspended. The pH of the magneti-cally stirred suspension was adjusted to 11 with 6 M potassium hydroxide and the temperature was adjusted to 18C by addition of pieces of ice. Cyanogen bromide (0.5 g) was added and the pH
was maintained at 10.5-11 for 35 min. by addition of 6 M potas-sium hydroxide as needed; the -temperature was maintained at 18C.
by addition of ice. The reaction mixture was filtered (vacuum), and the solids were washed with 400 ml of ice water. The solids were suspended in 50 ml of distilled water and 2.0 g of com-merical AH Sepharose~ 4B (including associated dextran) wasadded. The pH of the suspension was adjusted to 8.5, and the reaction mixture was stirred at 11C for 72 hr. The product was washed with cold distilled water. AH Sepharose~ 4B is a bead-form agarose gel having a wet bead diameter in the range of 40 to 190 micrometers available from Pharmacia Fine Chemical Co., Piscataway, New Jersey. The commercial AH Sepharose~ 4B as supplied by Pharmacia contains a large quantity of dextran and lactose. The weights used include the weight of the dextran mixture associated with the A~ Sepharose~ 4B. The dextran mix-ture does not take part in the reaction and is removed when theproduct is first washed.

_ g _ Example 2 Preparation of CPG550-AH Sepharose~ 4B
1,6-Diaminohexane The CPG550-AH Sepharose~3 4B product from Example 1 was resuspended in 50 ml. of distilled water and 1.0 g of cyanogen bromide was added. The pH of the reaction mixture was maintained between 10.5 and 11 by addition of 6 M potassium hydroxide, and the temperature was maintained at 18C by acldition of ice ~reaction time 30 min). The solids were co]lected, washed with 400 ml of ice water and resuspended in a mixture of 40 ml of 10 dioxane and 8 ml of water containing 8.0 g of 1,6~diaminohexane.
The suspension was stirred at room temperature for 18 hr. 4.0 g of ethanolamine was added, and stirring was continued for an additional 18 hr. The product was collected, washed with 800 ml of 70% aqueous dioxane, 50 ml of dioxane, 300 ml of distilled water and 50 ml of 2 M potassium chloride (negati~e 2,4-dinitro-benzenesul~onate test). The product was stored under 50 ml of 2 M potassium chloride.
Example- 3 Puri'fic`ation of Uricase on CPG550-AH Sepharo'se~
4B-1,6-Diaminohexa'ne An aliquot of the material prepared in Example 2 was placed in a Pasteur pipette containing a glass wool plug. The column volume was 0.7 ml. The column was washed with water then with 50 mM potassium phosphate buffer plus ethylenediamine tetracetic acid (pH 8.6). The uricase sample, extracted from Micrococcu's luteus, was prepared by ammonium sulfate precipita-tion followed by solution and dialysis in buffer. A sample con-taining 6.2 units uricase and 1101 units catalase was applied to the column. Fractions were eluted with 50 mM potassium phos-phate buffer containing 0.2 M, 0.5 M, 1 0 M and 2 0 M sodium chloride as fractions 1-4,respectively. Each fraction was assayed for uricase and catala.se activity and the results are reported in Table 1 below.

J~ . .

Tabl'e 1 Chromatographic Purification of Ba'cterial Uricase with CPG550-AH Sepharos'e~3 4B-1,6-Diaminoh'exane Activi-ty~_~o Fraction Uricase Catalase Wash 18 7 * Activity is expressed as percent of units applied.

Example 4 Purification of Cholesterol Oxidase on CPG550-AH
Sepharose~ 4B-1,6-Diaminohexane An aliquot of CPG550-AH Sepharose~ 4B-1,6-diaminohexane ~ -prepared as in Example 2 was placed in a Pasteur pipette con-taining a glass wool plug. The column volume was 0.7 ml. The column was washed with water then with 0.1 M phosphate buffer, pH 7Ø A cholesterol oxidase sample, extraeted from Nocardia cholesterolicum, was prepared by ammonium sulfate precipitation .:
followed by solution and diaIysls in buffer. A sample containing 18.0 units cholesterol oxidase was applied to the column.
Enzyme was eluted with 10 mM Tris-Cl, pH 8.0, containing 1.0 M
sodium chloride (Fraction 1) and 1.0 M sodium chloride - 1%
desoxycholate (Fraction 2). Each fraction was assayed for ~; ~
cholesterol oxidase activity and the results are reported in ~ ' Table 2 below.
Tab'l'e 2 Chromatogra'phic Purific'at'ion o'f Chol'e's'te'ro'l'Oxidase on CPG550-~A~H Seph'ar'os'e~g'4B'~ D'i'aminohexane Fraction Activity,'%

Wash 1 1 ' * Activity is expressed as percent of UIlitS applied. '~

- . . . - .. : . . . .

Example 5 Preparation of CPG550-AH Sepharose~ 4s-Oleylamine In 500 ml of distilled water, 40 g of CPG550 glass was suspended. This suspension was mechanically stirred (150 rpm~, and the pH was adjusted to 11 with 6 M potassium hydroxide. ~'he temperature was adjusted to 18C. with ice. Cyanogen bromide (5 g) was added and the pH was maintained between 10.5 and 11 for 35 min. by addition of 6 M potassium hyclroxide as needed.
The temperature was maintained at 18C. by addition of ice. The ~' reaction mixture was filtered (vacuum) and washed with 4 liters of ice water. The solids were suspended in 500 ml of distilled water and 20 y of commercial AH Sepharose~ 4B (including asso-ciated dextran) was added. The pH was adjusted to 8.5 and the reaction mixture was stirred at 11C for 72 hr.
To this suspension, 10 g of cyanogen bromide was added.
The pH of the reaction mixture was maintained between 10.7 and 11.3 by addition of 6 M potassium hydroxide. The temperature of the reaction mixture was maintained at 18C by addition of ice to the reaction mlxture (reaction time 30 min). The solids were collected and washed with 4 liters of ice water. The solids were suspended in a mixture of 80 g of oleylamine dissolved in a composition of 400 ml dioxane and 80 ml water. The reaction mixture was stirred (150 rpm) for 18 hr at room temperature, then 40 g of ethanolamine was added and the reaction mixture was stirred an additional 18 hr. The product was collected, washed with 8 liters of 70% aqueous dioxane, 500 ml dioxane, 3 liters distilled water, then 500 ml 2 M potassium chloride. The pro-duct was stored under 500 ml of 2M potassium chloride.

Example 6 Par'tial'purific'ation of L'1pas'e'M by Affinity Chromatography Crude lipase M powder (obtained from Enzyme Development Corp.) was suspended in distilled water (10~ w/v) and centri-fu~ed. The supernatant fraction was dialy~ed by diafiltration .~
.

, until the permeate was clear, then this material was lyophilized.
A 668 ml bed volume column (10 x 8.5 cm) of CPG550-AH Sepharose~
4B-oleylamine was prepared and e~uilibrated with 0.1 M Tris-Cl, pH 8Ø A 15.0 g sample of lipase M dissolved in 1600 ml of Tris ~.
buffer was applied to the column. This column was eluted sequentially by five column volumes of Tris buffer (Fraction 2), four volumes of 1.0 M sodium chloride in Tris buffer (Fraction 3), three volumes of Tris buffer (Fraction 4), and three volumes of Tris buffer containing 0.1 M sodium chloride and one percent :
desoxycholate (Fraction 5). Fraction 5 was dialyzed against water, then lyophilized prior to storage. Cholesterol esterase (CE) and triglyceride esterase (TE) activities of Fraction 5 were assayed. Protein was assayed as described by Layne (Methods of Enzymology, 3, 451, 1957). ~ summary of the affinity coIumn purification is presented in Table 3. Elution patterns of pro-tein, CE, and TE activites are illustrated in Table 4.
Tab'l~e 3 ~' Affini*y Co'lumn Pu'r'i'ficat~io'n`of Lipa'se M
Activity~
Total Units per ~Purifica- :
~Sample Enzyme Units mg Protein tion Partially CE 3154 0.86 . Purified Enzyme ~:
: Applied to Column TE 1501 0.33 1 ;;~
Column CE 4000 6.0 7 .~' Purified Enzyme TE 2151 3.2 9.7 T _ e 4 Affinity Column Pur _ cation of Lipase M
Activity, units x 103 cholesterol Triglyceri.de Fraction Prot'ein,g esterase esteràse ~ - .
1 2.1 0.1 0.5 '`
2 2.3 0.7 0.8 :-' 3 0.4 0.15 0.5

4 0 0 0.4 .~:' ' 0.72 4.0 2.1 '.

- 13 - : ~ ' X

82~ ~

Example 7 Preparation of Glycophase G/CPG150'0-Oleylamine .~
In 30 ml of distilled water, 25 ml of glass (Glycophase G/CPG1500) was suspended. To the magnetically stirred mixture, 6.3 g of cyanoyen bromide was added in small portions. The pH
was maintained at 9-10 with 0.1 N sodium hydroxide and the temperature at 27C by addition of ice treaction time 30 min).
The solids were collected and washed wi-th 2 liters of ice water then placed in 30 ml of distilled water. To this suspension, ~
15 ml of oleylamine dissolved in 20 ml dioxane was added, and the ~ ' 10 reaction mixture was shaken for one hour at 25C. Ethanolamine ~ ;~
(5 ml) dissolved in 25 ml of potassium phosphate buffer tpH 7) was added, and the mixture was shaken an additional hour. The product was collected and washed with 2 liters of dioxane followed by a liter of buffer. The wash was repeated, and the solids were ~'~
stored under 25 ml of buffer.

Example 8 E'f'f'e'c't of'Var'i'at'ion of A'mount of AH Sepharose~ 4B
to Glass on the Rec'overy 'of Enzym'at'i'c'Act.ivity and'Colu'mn Flow Ra'tes -Material was prepared as in~Example 4, except that the ;
columns contained~varying amounts of CPG550-AH Sepharose~ jB
modlfied with oley~lamine. The results for two runs are tabulated below in Table 5.

:~ , . . . . . .

*
O 1- ~
~ o ~ o o ~ ~ : :~

~ ~ ~ 30 ~
~ o 'O ~ C~ ~ U~
h Y a) n~
~ U ~ ~ ~
u~ ,~ u~ ~H
~ ~ ~ : ~ ~1 o , ~ .
. ~1 ,~ ~ ~ ~ ~
U~ ~ O C~ O'¢ P~ O
O 'O .~ ~ O
. ~ ~ $ ~'o ~.~ ~ O t~
: E~. ~H .0 fL'; U ~ ~1 *
O 3 p:; N ~ o O GO O N
.~ 'O ~:: :~ Il~
.rol ~ ~ \ F~ ~ a) .
~J ~ . ~oH
h . ~
~ 0 ~
~ u?
O ~ ~ rl ~ :
-1 tQ :(d U ~ O ~1 ~ ~ :
a~ s~ w ~ ~ ~ ;-.
4~ $ ~ ~ ~
Q. O
U~ ~ r~l (d ` ' a) o s~ :
::~ u~ ~H
~ ~ l O
D~O 1¢ 1i4 3 * .` -: ~, ~` ' .
, ' ' ~; ` '.
' ~' .
- 1 5 & 1 6 .~
.
,~:. -. " : - : :

'2!3 Also, thls comblnation of agarose beads c~alently bonded to glass beads exhibits a 6ubstantl~1 difference ln separation capability (3.6 to 7-fold purification of enzyme) compared wlth commercially available glass coated w~th a monolayer of carbohydrate (2-fold purification o~ enzyme) as shown in Table 6.

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C) rl r~ ~1 h ~ ~4 ~ r~l td n~ tlS
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c~ C ~ ~ QC~
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4~ ~ .~ ~ a~ ~ ~
q-l ~ h C~ t~ ~ ~t r~
~ ~ ~ ~) ~ e) O : ~ O a~ C

5~ ~; ~3 ¢
a~ ~ ~; ~
~ ~ ~ ~ ~ ~ ~ p C to ~ ~1 ~ ~` E
O ~ ~B ~) C * O s~
co G) C~ ~ ~ ~ ~
,i :~ v ~ C~ 3 0 ~ ~d ~d a~
0 o r : oq h~i h ~) c: h :I r-l ~ ,¢ o ~, ~1 $
E3 ~ ~ :~: ~ 0 ~
O ~ ::4 ~ ~ ~ ~ X ~ X O S~
Ei O ~ X r l ~ ~1 ~I C) Lr~ O cs ~rl ~rl a) ~i :
:~ c) O ~ O ~ ~-- O
~1 h O-- C ~ a) ~ C ~ c~ l h ~ ~ t O ~I Ll~ O ~ ~ ~ ,0 ~11 p, p ~

, . -:

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Example 9 _Preparation of CPG500-~H S'eph'arose~ 4B-3-Diethyl-minopropylamine To a stirred suspension of CPG550-AH Sepharose~ 4B
prepared as described in Example 1, 2.0 g of cyanogen bromide was added. The pH of the reaction mixture was maintained between 10.5 and 11 by addition of 6 M potassium hydroxide. The tempera-ture of the reac-tion mixture was maintained at 18C by addition of ice (reaction time 30 min). The solids were collected and washed with 200 ml of ice water and suspended in a composition of 40 ml of dioxane and 8 ml of water containing 8.0 g of 3-diethylaminopropylamine. The reaction mixture was shaken at 11C
for 24 hr; the product was collected and washed with 4 liters of distilled water (negative 2,4-dinitrobenzenesulfonate test). The product was stored under 50 ml of 2 M potassium chloride.

Example 10 Purification of ~-GLyc'e'ropho phate Oxidas'e on CPG550-AH Sepharos'e~4B'-3'-D'i'ethy'laminopro'pylami'ne An aliquot of CPG550-AH Sepharose~ 4B-3-diethylaminopro-pylamine was placed in a Pasteur pipette containing a glass wool plug. The column volume was 0.7 ml. The column was washed with water then with 0.1 M potassium phosphate, pH 7Ø The ~-glycerophosphate oxidase sample extracted from Streptococcus faecalis was prepared by ammonium sulfate precipitation followed _ by solution and dialysis in buffer. A sample containing 0.728 unit ~-glycerophosphate oxidase (GPO) and 0.146 unit lactate oxidase ~LO) was added to the column. Activities were eluted with 0.1 M potassium phosphate buffer, pH 7.0, containing 0.05 M, '-0.075 M, and 0.3 M sodium chloride as fractions 1 through 3, respectively. The fractions were assayed for GPO activity and , LO activity and the results are reported in Table 7.
' `

_ ~ g _ : ' :
~( :

~able 7 Chromato~raPhic Purificatlon of ~Gl~ceroPhosphate Oxidase with CPG550~AH Sepharos~hB-3-Dlet~lamino-propylamine Fraction * Actlvity is expressed as percent Or units applied.

~ ';
. .

~ .
' :~

Claims

WE CLAIM:

1. A chromatographic material characterized by an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of polysaccharide forming an inorganic support-polysaccharide particle matrix.

2. A chromatographic material as in claim 1 wherein said inorganic support is selected from the group consisting of porous silica, controlled porosity glass, controlled porosity ceramic, and alumina.

3. A chromatographic material as in claim 1 wherein said particles of polysaccharide are comprised of agarose gel or polyacrylamide-agarose gel.

4. A chromatographic material as in claim 1 wherein said inorganic support is a bead having a particle size in the range of from about 100 to about 1000 micrometers.

5. A chromatographic material as in claim 4 wherein said particles of polysaccharide have a particle size in the range of from about 25 to about 300 micrometers.

6. A chromatographic material characterized by an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of polysaccharide forming an inorganic support-polysaccharide particle matrix, the hydroxyl groups of said inorganic support-polysaccharide particle matrix being activated for reaction with and covalent bonding with, a ligand.

7. A chromatographic material as in claim 6 wherein said hydroxyl groups have been activated by treatment with cyanogen bromide.

8. A chromatographic material as in claim 6 wherein said hydroxyl groups have been activated by treatment with sodium metaperiodate.

9. A chromatographic material characterized by an inorganic support-polysaccharide particle matrix having active sites comprising activated hydroxyl groups and ligands attached to said active sites; said inorganic support-polysaccharide particle matrix comprising an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of polysaccharide.

10. A chromatographic material as in claim 9 wherein said ligands are attached to said active sites of said inorganic support-polysaccharide particle matrix by means of a spacer molecule.

11. A chromatographic material as in claim 10 wherein said spacer molecule is diaminodipropylamine.

12. A chromatographic material as in claim 9 wherein said activated hydroxyl groups are activated by treatment with cyanogen bromide.

13. A chromatographic material as in claim 9 wherein said activated hydroxyl groups are activated by treatment with sodium metaperiodate.

14. A chromatographic material characterized by an inorganic support-agarose gel matrix having active sites comprising activated hydroxyl groups and ligands attached to said active sites, said inorganic support-agarose gel matrix comprising an inorganic support having a high surface density of hydroxyl groups and, covalently attached thereto, insoluble particles of agarose.

15. A chromatographic material as in claim 14 wherein said ligands are comprised of diaminoalkane.

16. A chromatographic material as in claim 15 wherein said diaminoalkane is 1,6-diaminohexane.

17. A chromatographic material as in claim 14 wherein said ligands are comprised of oleylamine.

18. A chromatographic material as in claim 14 wherein said ligands are comprised of 3-diethylaminopropylamine.

19. A chromatographic material as in claim 14 wherein said inorganic support is selected from the group consisting of porous silica, controlled porosity glass, controlled porosity ceramic, and alumina.

20. A chromatographic material as in claim 14 wherein said agarose is present in said inorganic support-agarose gel matrix in an amount in the range of from about 1 to about 10 percent by weight based on the weight of the inorganic support.