Exhibit 2036 Page 1
`
`Enzo Exhibit 2036
`Hologic, Inc. v. Enzo Life Sciences, Inc.
`Case IPR2016-00820
`
`

`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY-[4] 59
`
`
`
`
`
`
`
`[4] Porous Glass for Affinity Chromatography Applications
`
`By H. H. WEETALL and A. M. FILBERT
`
`This report deals with “methods” of attaching ligands to inorganic
`carriers for affinity chromatography.
`An ideal carrier for the aflinity chromatography of enzymes should
`possess a number of desirable characteristics. it should exist as a porous
`network to allow rapid movement of large macromolecules throughout
`its entire structure and be uniform in size and shape to exhibit good
`flow properties. The surface area of the carrier would be necessarily large
`to allow attainment of a high effective concentration of coupled inhibitor,
`and the support surface should exhibit little specific attraction for pro-
`teins. The material should be mechanically and chemically stable to
`various conditions of pH,
`ionic strength,
`temperature, and the presence
`of clenaturants. The availability of controlled-pore glass provides
`a
`material that fulfills many of the above—mentioned “ideal, support cri-
`teria,” and presents a new and unique carrier for use in affinity chro-
`matography applications.
`
`Preparation of Controlled-Pore Diameter Glasses
`
`(see Fig. 1),
`Certain sodium borosilicate glass compositions exist
`which, after heat treatment, can be leached to form a porous glass frame»
`work} During heat treatment the base, glass separates into two inter-
`mingled and continuous glassy phases. One phase, rich in boric oxide,
`is soluble in acids; the other phase is high in silica and is stable toward
`acid solutions. The horic acid~»rich phase may be leached out of the glass,
`leaving a porous structure of Very high silica content. Porous diameters
`for these glasses are in the range of 30 to 60 A, and the pore volume is
`approximately 28%‘of the total sample volume.
`Larger-pore glasses can be prepared from the same sodium borosili-
`cate glass compositions. After the heat treatment and acid leaching steps,
`a mild caustic treatment enlarges pore diameters by removing siliceous
`residue from pore interiors? By carefully controlling the various physical
`and chemical treatment parameters, glasses are produced which exhibit
`extremely narrow pore size distributions, as shown in Fig. 2. The con-
`
`‘M. E. Nordberg, J. Amer. Ceram. Soc. 27, 299 (1944).
`3 H. P. Hood and M. E. Norclberg (1940). U.S. Patent No. 2,221,709, “Borosilicate
`Glass.”
`
`- Exhibit 2036 Page 2
`
`p
`
`Exhibit 2036 Page 2
`
`

`
`60
`
`COUPLING REACTIONS AND GENERAL METHODOLOGY
`
`[4]
`
`No 0
`
`3203
`
`N00
`
`96% SILICA GLASS
`
`
`
`GLASS
`
`302
`
`2 3
`
`40
`30
`
`CONCENTRATION
`
`{MOLES %)
`
`FIG. 1. Phase diagram Of sodium borosilicate system.
`
`
`
`£1.11
`90 be
`
`Q’
`
`6
`LL
`L
`\90'?>o, 0°.‘ 0'} 0\%g%o9e;oQu=°_Oq, 09\0
`.15: Q 4:
`FORE DIAMETER, pm
`1.0 pm 210.0003
`
`0 A
`
`2.0
`
`[.6
`
`l.2
`
`<9
`
`\ 8
`
`2 9 2
`
`0.8
`II.
`9-
`Lu
`
`5;
`n_ L4
`
`0
`
`FIG. 2. Pore size distribution of cOntrO11ed—pore diameter glass supports. All
`values were determined by the mercury intrusion method.
`
`Exhibit 2036 Page 3
`
`Exhibit 2036 Page 3
`
`

`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY'[4] 61
`
`
`
`
`
`
`
`is a 96% silica glass, with 3—5*%
`thus- produced,
`trolled-pore glass,
`B203 content, and trace quantities of several metal oxides.
`
`Surface Area of Controlled-Pore Glass
`
`The surface area of controlled—pore glasses is a function of pore
`diameter and pore Volume. Surface area increases with increased pore
`volume and decreases with increasing pore diameter, as is shown by the
`data in the table.
`
`Surface Properties
`
`The surface properties of controlled-pore glass are greatly affected
`by the thermal history of the glass, heat treatment playing a significant
`role in determining the ratio of adjacent to single hydroxyl groups which
`exist on the glass Surface. Infrared spectra of the glass change as a func-
`tion of temperature. On a wet silica surface three bands can be identified
`as 3747, 3660, 3450 cm”, and 1650 cnrl. The bands at 3450 and
`1650 cm"1 are typical of the vibrations associated with molecular water.
`When temperature is raised to about 150°,
`these two bands disappear,
`but the bands at 3747 and 3660 cm” become more evident. As the
`temperature is raised further, ‘the band at 3660 cm" slowly disappears.
`At 500° it is observed only as a tail on the low frequency side of the
`3747 crrrl band. At 800°, only the band at 3747 curl remains. These
`bands are attributed to the vibrations of, respectively, surface hydroxyl
`groups spaced sufriciently far apart that they do not interact with each
`other, surface hydroxyl groups so close together they are hydrogen-
`bonded to each other, and molecular water physically adsorbed on the
`surface of the silica (Fig. 3).
`The rehydration of a silica surface depends on its previous thermal
`
`CONTROLLED—PORE GrLASS PHYSICAL PROPERTIES
`
`
`Pore Volume
`
`cc/g
`SA (MUg) at
`Pore diameter
`rue
`PV==0J0
`(A)
`____________________________,WMW____________rMa_._______,,s_______..
`
`356
`249
`75
`214
`149
`125
`153
`107
`175
`111
`78
`240
`72
`50
`370
`as
`27
`700
`21
`15
`-1250_
`13
`9
`2000
`
`
`-
`
`Exhibit 2036 Page 4
`
`Exhibit 2036 Page 4
`
`

`
`62
`
`COUPLING REACTIONS AND GENERAL METHODOLOGY
`
`[4]
`
`H
`H
`\ /
`H
`
`/7
`H
`
`\\
`H
`
`H 91,;
`
`
`HEAT
`
`H
`I
`0
`i
`Si + Si
`I
`
`H —
`I
`0’
`o
`I
`/ \
`Si 4- Si
`
`/H
`I
`0
`I
`Si
`
`H
`
`H
`\ /
`,0\
`
`/1
`
`\\
`H
`
`H
`
`0
`|_
`
`O
`|_
`
`O
`|_
`
`0
`I_
`
`0
`!_
`
`REVERSIBLE
`BELOW 400°C
`
`3400 cm"
`
`HEAT
`
`SLOW REVERSE
`HYDRATION
`
`C
`/' \ + / \
`.
`.
`
`/Sui
`
`.
`
`‘°i'\O/ix
`
`S.
`
`/n'\
`
`+
`
`T0
`
`I
`S.
`
`/\
`
`FIG. 3. Dehydration of a silica surface.
`
`history. Up to about 400°, the dehydroxylation of the surface is reversible,
`but above this temperature the removal of the adjacent hydroxyl groups
`from the surface causes the silica to become hydrophobic. Indeed, after
`extensive heating at 850°, a controlled—pore glass surface will not readily
`adsorb water unless the hydroxylated surface is reconstituted by a drastic
`treatment such as boiling in concentrated nitric acid. In this case,
`the
`water adsorption is associated with the hydrogen-bonded hydroxyl groups
`and occurs on these -groups in preference to the freely vibrating group.
`In fact, removal of the freely Vibrating group from the surface by silaniza—
`tion techniques has little effect on the water adsorption that occurs at
`iow partial pressures.
`__
`In addition to the surface silanols, the presence of boron in the glass
`results in the formation of surface Lewis acid sites as shown in Fig. 4.
`Although B303 analysis for the bulk glass is only in the order of 3%
`with a B:Si ratio of 1:20, infrared studies indicate a much higher sur-
`face concentration of boron. Thus, the heat treatment and leaching pro»-
`cedures not only increase surface area of the glass, but also change its
`surface composition. One now finds a surface boron concentration of
`2.4 X 102“ boron atoms per gram of glass and 8 surface silanol groups
`per square Inillimicron. The resulting porous glass, with its
`surface
`hydroxyls and Lewis acid sites, exhibits a slight negative charge in
`aqueous media, and this in large part explains the strong adsorption of
`basic materials.
`
`Exhibit 2036 Page 5 ‘
`
`Exhibit 2036 Page 5
`
`

`
`
`
`POROUS crass FOR AFFINITY CHROMATOGRAPHY[4]. 63
`
`
`
`V
`
`H
`
`H
`
`H\ ‘ /H
`Ii]
`0/ I
`[I3
`
`/
`
`B
`
`\O
`\
`
`H
`
`t /H
`T
`01/ i \c|
`Cl
`
`B
`
`LEWIS ACID SITE
`SURFACE
`
`LEWIS ACID - BASE
`COMPLEX
`
`FIG. 4. Concept of Lewis acid site on the surface of glass developed byianalogy
`to inorganic Lewis acid halides.
`
`iDurabi1ity of Controlled-Pore Glass
`
`The degree of solubility or the corrosion rate of controlled-pore glass
`(CPG) is a function of temperature, time, solution content, volume, pH,
`glass composition and surface area. Since CPG is essentially pure silica,
`it generally exhibits a high degree of durability, but its high surface area
`contributes to a higher than normal solubility.
`Generally, one can expect solubility to increase by a factor of 1.5
`for each 10° increase in temperature. Solubility of the glass is extremely
`pH dependent and is most stable under strong acid conditions. Controlled-
`pore glass which has been coupled to hydrophilic silanes shows improved
`durability, and its solubility rate in aqueous systems is decreased substan-
`tially. These derivatives (CPG silanes) are excellent starting points for
`use of the glass in affinity chromatography applications.
`
`Preparation of the Silanized Carrier
`
`The inorganic carrier is treated with an organosilane containing an
`organic functional group at one end and a silylalkoxy group at the other.
`Coupling of the silane to the carrier is through the surface silanoi or
`oxide groups to the silylalkoxy groups.3 In the case of the trialkoxy
`silanes, polymerization most likely occurs between adjacent silanes. The
`resulting product is an inorganic carrier having available organic func-
`tional groups. The reaction sequence may be schematically represented as
`in Fig. 5 with a triethoxysilane.
`Typical organosilanes commercially available include epoXy—, vinyl—,
`su1fhydryl—, alkylaminem, al1<y1chlOro—, and phenylsilanes.
`Silanes can be attached to inorganic carriers either in organic sol-
`
`“W. Haller, Nature (London) 206, 693 (1965).
`
`Exhibit 2036 Page 6
`
`Exhibit 2036 Page 6
`
`

`
`64
`
`COUPLING REACTIONS AND GENERAL METHODOLOGY
`
`[4]
`
`O.
`R(CHg),, Si(OCH2CH3)3+ Ho—di—o— m--—>
`5
`HO-S'i—O
`5
`
`Io
`
`OH
`0- |‘—O——S|E(CH2),, R
`«S
`6
`—O—S|i—O- S|i(CH2),, a
`cl)
`:3
`—O—SIi~— O——S|i(CHg),, R
`('3
`OIH
`i
`
`FIG. 5. Silanization of the surface of porous glass. R represents an organic
`functional group.
`
`vents“ or aqueouslyfi In the case of some organosilanes, Organic solvents
`must be used for reasons of solubility.
`
`Organic Silanizarion of an Inorganic Surface
`
`The glass or inorganic carrier is first cleaned by boiling in 5% nitric
`acid solution for 45 minutes and then washed and dried 24 hours at
`
`115°.
`To 1 g of dry, clean carrier is added 75 mi of 10% ~,r—aminOpropyl~
`triethoxysilane (Union Carbide, A—1lO0) dissolved in toluene. The
`preparation is refluxed for 12-24 hours, washed thoroughly with toluene,
`followed by acetone, and air~dried. The resulting product is the alkylamine
`carrier.
`
`Aqueous Silanization of cm Inorganic Carrier
`
`The aqueous procedure, although it does not add as great a quantity
`of amine groups to the carrier, appears to produce a dynamically more
`stable product when exposed to continuous use over long periods of time.
`The carrier is firs’: cleaned as described for the organic procedure
`- but is not dried. The washed carrier is placed in a 10% solution of
`
`‘M. L. Hair and I. D. Chapman, J. Amer. Ceram. Soc. 49, 651 (1966).
`‘H. H. Weetall, Nature (London) 223, 959 (1969).
`-
`“H. H‘. Weetall, Science 166, 615 (1969).
`‘H. H. Weetall and L. S. Hersh, Biochim. Bfophys. Acta 185, 464 (1969).
`“H. H. Weetall and N. B. I-Iavewala, Biotech. Bioeng. Symp. 3, 241 (1972).
`
`Exhibit 2036 Page 7
`
`Exhibit 2036 Page 7
`
`

`
`[4]-
`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY
`
`65
`
`«panrinopropyltriethoxysilane dissolved in distilled water and the pH is
`immediately adjusted with 6 N HCl to pH -3.45. Th-e suspension is placed
`in a water bath for 2.75 hours at 75°, removed and washed on a Bijchner
`funnel with an equal volume of water, i.e., the same volume as used for
`dilution of the silane. The product is dried overnight at 100°. The resulting
`material has an available alkylamine.
`
`Methods of Activating the Silanizecl Inorganic Carrier
`
`Most of our studies have been carried out using an alkylaminosilane
`derivatized inorganic carrier. This derivative is prepared using commer-
`cially available y-aminopropyltriethoxysilane and aqueous silanizationfi
`
`Preparation of an Arylamine
`
`The arylamine is prepared from the alkylarninosilane carrier by reac-
`tion With p»-nitrobenzoylchloride,
`followed by reduction with sodium
`dithionite. The reaction is represented schematically as shown in Fig. 6.
`To prepare the arylarnine, the alkylamine derivative is refluxed over—
`night
`in chloroform containing 10% triethylamine (v/V) and p-nitro-
`benzoylchloride (5 g/ 100 g of carrier). The product
`is Washed with
`chloroform and allowed to air—dry. Reduction of the nitro groups is
`accomplished by boiling in 10% sodium dithionite solution in water in
`a volume large enough to exceed 1 g of sodium dithionite per gram of
`carrier. The carrier is boiled for 30 minutes and filtered while hot. The
`carrier is washed with slightly acidified water until
`the odor of sulfur
`
`REFLUX
`3-——»
`
`I
`‘-1
`o
`o
`I
`II
`--0-Si (CH2)3 NH2 + N02 C} cc:
`;
`
`0I
`
`SODIUM DITHEONETE
`E""""""""""Ԥ-
`
`Io
`
`oI
`
`0
`I
`H
`-0-Si(CH2)3 NHCQNOZ
`I
`
`CARRIER
`
`CARRIER
`
`CARRIER
`
`o
`ii
`
`I 0
`
`I
`
`Ioa
`
`§—o—sI (CHg)3 NHCQNH3
`
`FIG. 6. Preparation of the arylamine derivative from the alkylamine derivative.
`
`Exhibit 2036 Page 8
`
`Exhibit 2036 Page 8
`
`

`
`2"’?
`
`66
`
`COUPLING REACTIONS AND GENERAL METHODOLOGY
`m.—.-.-__..
`........._...
`
`[4]
`
`CARRIER
`
`I
`O
`O
`0
`o
`o
`O
`E
`ct 1-;
`I
`II
`-0- ‘(cH2)3 NH2 +
`$ ~O-Si(CHg)3NHC(CHgI4 COOH
`1
`I
`(I)
`o1
`
`I
`
`FIG. 7. Preparation of the carboxyl derivative from the alkylamine derivative.
`
`is no longer detectable. The final washes are with distilled water, and
`the product is dried at between 60° and 80°.
`The carrier can be reused by burning off the organic material at
`625° for 12 hours under 02 if possible. The repeat procedure must in-
`clude cleansing and rehydration of the surface with boiling nitric acid.
`
`Preparation of the Carboxyl Derivative
`
`The carboxylated derivative is prepared by reaction of the alkylamine
`carrier with succinic anhydride.
`To each gram of alkylamine derivative is added 0.3 g of succinic
`anhydride dissolved in 50 mM sodium phosphate at pH 6.0. The reac«
`tion is continued for 12 hours and is followed by exhaustive washingwith
`distilled water. The final product,
`the carboxyl derivative, may be dried
`and stored. The reaction is schematically represented in Fig. 7.
`
`Preparation of the Aldehyde Derivative
`
`The active aldehyde of the alkylamine carrier is prepared by reac—
`tion with glutaraldehyde. The product readily reacts with amines.
`The reaction is carried out initially at room temperature. To 1 g of
`alkylamine glass, add enough 2.5% glutaraldehyde solution (25% glu—
`taraldehyde diluted 1
`to 10 in 0.1M sodium phosphate at pH 7)
`to
`cover the glass. The entire reaction mixture is placed in a desiccator and
`attached to an aspirator to remove air and gas bubbles from within the
`particles. Reaction is continued in the aspirator for 60 minutes.
`The product is removed and filtered on a Biichner funnel and Washed
`with distilled water. The resulting derivative is the activated aldehyde.
`Schematically the reaction is represented in its simple form in Fig. 8.
`
`Ii
`
`,
`
`,
`
`I
`
`CARRIER —o—sI(cH2I._.. NH2 + <eH213 $ $-0—~S|{ (CH2); N=CH(CH2l3CH0
`
`CH0
`I
`
`CHO
`
`I
`0
`I
`
`I
`<|3
`
`I
`0
`I
`
`cl)
`
`FIG. 8. Preparation of the aldehyde derivative from the alkylamine derivative.
`
`Exhibit 2036 Page 9
`
`Exhibit 2036 Page 9
`
`

`
`[4]
`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY
`
`67
`
`Methods of Covalently Attaching Proteins and Other Organic
`Molecules to the Silanized Derivative
`'
`
`The three derivatives previously described may be utilized for the
`covalent attachment of any organic moiecule of choice. In additionhthese
`three derivatives can easily be converted to other derivatives having func-
`tional groups of different characteristics. Several methods for coupling an
`organic moiety are presented here although others may be found through—
`out this volume, particularly in Articles [1] and [2].
`
`A lkylamine Carrier
`
`Amide Linkage. The alkylamine may be covalently coupled. to any
`free carboxyl group using a carbodiirnide.-"-11 In the case of proteins or
`other Water—soluble materials, one may utilize a soluble carbodiimide. In
`the case of organic soluble materials, an organic solvent may be suitable.
`In this case N,N’—dicyclohexyl carbodiimide would be useful.
`WATER—SOLUBLE CARBODIIMIDES. The coupling is carried out at room
`temperature or at 0°. With an enzyme,
`the 0° approach may be pre-
`ferred. To 1 g of alkylamine-glass is added 50 mg of protein and 10 ml
`of distilled water to which 40 mg of 1—cyc1oheXy1—3(2—morpholinoethyl)
`carbodiirnide metho—p—toIuenesulfonate had been added and adjusted to
`pH 4.0. After addition of the diimide solution,
`the mixture is adjusted
`to pH 4.0 and monitored for 2030 minutes. Continuous agitation is
`recommended for maximum coupling. After the first 30 minutes,
`the
`suspension is placed in an aspirator for 20 minutes to ensure complete
`wetting and is refrigerated overnight. The following morning, the solution
`is decanted and the derivative is washed several times with distilled water.
`
`in some instances,
`The product may be stored at 6° as awet cake or,
`dried. With some derivatives activation is best achieved at pH 10-11.
`If the carrier is a carboxy derivative, one can form a pseudourea of
`the carboxyl group, remove excess carbodiimide, and in this way prevent
`possible cross—linking wherever both carboxyl and amine groups are
`available.
`
`To form the pseudourea of a carboxyl derivative, one simply adds a
`large excess of carbodiimide to the derivative, adjusts to pH 4.0, and
`allows the reaction to take place for several hours. The excess is removed
`by filtering with distilled water slightly acidified. The product should be
`
`9N. Weliky, H. H. Weetall, R. V. Gilden, and D. H. Campbell, Inmizmc-chen7z'st.-‘y
`1, 219 (1964).
`“D. G. Hooveand D. E. Koshland, Ir., J. Biol. Chem. 242, 2447 (1967).
`“N. Weliky, F. S. Brown, and E. L. Dale, Arch. Biochem. Biophys. 131,
`1 (1969).
`
`Exhibit 2036 Page 10
`
`
`
`
`
`Exhibit 2036 Page 10
`
`

`
`
`
` 68 COUPLING REACTIONS AND GENERAL METHODOLOGY [4]
`
`
`
`CARRIER §~o— Sit (CH2)3 NH2+lI\|l + H* + Hooc— R” m-—>
`
`I
`‘?
`
`O
`I
`
`ii
`
`c
`
`nNI R
`
`I!
`
`3 ‘
`
`“.”“‘
`.9
`?
`-«om '(CH2)3NHCR'" + o=c + H *
`I
`1
`9
`NHR"
`
`FIG. 9. Preparation of an amide-linked ligand with the ailcylamine derivative
`and a carbodiimide. R’ and R" represent groups on the carbodiimide, e.g., cyclo-
`hexyl groups; 11”’ represents the ligand having a free carboxyl group.
`
`capable of reacting with amine group or a ligand. With some derivatives,
`best results are achieved at pH 10-11.
`WATER-INs0LUBLE CARBODIIMIDES. Coupling between a derivative
`having free carboxyl or amine groups is carried out in a manner similar
`to that described above. However, the reaction is carried out in ethanol.
`As an example, N,N’—dicyclohexyl carbodiimide is soluble in ethanol and
`may be used to link amines to carboxyls as described for the water-
`soluble derivatives. The carbodiimide reaction is
`schematically repre-
`sented in Fig. 9.
`'
`Thiourea Linkage. An alkylamine carrier may be converted to an
`isothiocyanate and covalently attached to a free amine group?“ The
`isothiocyanate will react with free amine groups. The reaction is sche-
`matically represented in Fig. 10.
`To 1 g of allcylamineglass is added enough 10% thiophosgene in
`CHCl3 to reflux for at least 4 hours; usually 75-125 ml is adequate. The
`carrier is Washed with CHCI3, quickly air—dried, and used immediately.
`Fifty milligrams of protein or ligand dissolved in 50 mM NaHCO,.;,
`
`CARRIER X-O-Si(CH;;_}3 NH2 + Cl WC -Cl Q -O-Si(CHgi;-,NCS
`
`S
`
`O
`1
`
`I
`O
`I
`
`O
`I
`
`I
`O
`l
`
`FIG. 10. Preparation of the isothiocyanate from the alkylamine derivative.
`
`‘2 L. Wide, R. Axén, and I. Porath, Iimmnzodiem. 4, 381 (1967).
`‘“ L. Wide and I. Porath; Biochim. Biophys. /{cm 130, 257 (1967).
`
`Exhibit 2036 Page 11
`
`
`
`
`
`Exhibit 2036 Page 11
`
`

`
`[4]
`
`.
`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY
`
`69
`
`1.
`
`C.’
`
`IOI
`
`j—o— 'lCH2l3,NHa + NHZ-NHQ E———-e
`
`CAF-.'RiER
`
`Cu’
`—O—Si (CHz)3NH -NH2
`I
`0
`I
`
`,
`
`Nc|N02+H
`»———>
`
`0
`e
`-G—~SE(CH2)3 N;
`I
`o
`J
`
`FIG. 11. Preparation of the hydrazide from the alkylamine derivative.
`
`is con»
`is added to the isothiocyanate derivative. It
`buffer pH 9-10,
`tinuously agitated with the use of an aspirator for 30 minutes and for
`an additional 2 hours at room temperature. The product is washed with
`distilled Water and stored as a wet cake at 6°.
`
`Hydrazide Linlcage. This reaction can be used for the covalent attach-
`ment of an aikylamine carrier to a compound containing an available
`primary aliphatic amine.” The reaction is schematically represented in
`Fig. 11.
`’
`This activated carrier should react with free primary aliphatic amines
`under slightly alkaline conditions. However, in our laboratories we have
`not attempted this method of activating and coupling compounds to
`inorganic carriers.
`
`The Aldehyde Carrier
`
`The aldehyde carrier will react directly at neutral pH with primary
`aminesfi The reaction is gentle, but should be carried out in ice. How-
`ever,
`it
`is imperative that excess glutaraldehyde be washed out before
`the coupling step. ‘The preparation of the aldehyde derivative has been
`described above.
`
`Place the derivative in the solution containing the protein or ligand
`to be coupled. As little liquid as possible should be used, and the reac-
`tants should be maintained at pH 7-9. Continue the reaction for at least
`2 hours. Wash and store. This compound may be reduced with agents
`such as 1.0% sodium borohydride at acid pH. The reaction sequence
`in its simplest form is represented in Fig. 12.
`
`“J. K. Inman and H. M. Dentzis, Biochemistry s, 4074 (1959).
`
`Exhibit 2036 Page 12
`
`!
`
`5 I I
`
`.
`
`it
`.
`
`l
`
`F. g:
`
`
`
`Exhibit 2036 Page 12
`
`

`
`70
`
`COUPLING REACTIONS AND GENERAL METHODOLOGY
`
`[4]
`
`I
`‘I’
`~«O—S[i(CH2)3 N =CH(CH2)3 CHO + NH2 -5‘ pm»
`
`0a
`
`CARRIER
`
`I
`REDUCTION
`(.3
`-0- '(CHgL~,N=CH(CHg)3CH=N-R m——-»
`
`I0I
`
`I
`‘I’
`-0- six — NHCHg(CHg)3 CHNH-R
`
`OI
`
`
`
`
`
`ligand with the alde-
`FIG. 12. The covalent coupling of an amine functional
`hyde derivative followed by reduction with sodium borohydride.
`
`Carboxylated Carriers
`
`The carboxylated carriers may be used to couple both aryl and
`alkyl amines.
`Amide Linkage. The mechanism is similar to that shown for the
`alkylamine-carriers using the carbodiitnide. A major advantage of using
`the carboxyl derivative is that the carboxyl group can be activated and
`the excess carbodiirnide removed before coupling to the amine (Fig. 13).
`
`R
`I
`
`H
`c
`II
`
`NI R
`
`"
`
`RI
`
`O
`II
`
`O
`II
`
`_
`
`IO
`
`I
`
`I
`0
`I
`
`CARRIER §~O-Si {CHZB3 NHC (Ci-ia)2C-OH + N D-—-—>
`
`Io
`
`NH
`O
`O
`I
`II
`II
`2
`—o— Si (CHg_)3 NHC (CH2); c —O—c
`I
`a
`Q
`NH
`illR
`
`FIG. 13. The preparation of the pseudourea of a carboxyl derivative by reac-
`tion with a carbodiimide.
`
`Exhibit 2036 Page 13
`
`Exhibit 2036 Page 13
`
`

`
`[4]
`
`POROUS GLASS FOR AFFINITY CHROMATOGRAPHY
`
`71
`
`'
`
`o
`0
`I
`II
`CARRIER -O~»SEi(CH2l3 NHC
`c
`I
`
`0
`o
`NONO2
`I
`aI
`:sH2m—---> -0-Si(CHg}3NHC
`H*
`I
`o
`i
`
`Na+CE'
`
`FIG. 14. The preparation of the diazonium chloride from the arylarnine derivative.
`
`The pseudourea formed in this reaction will react with amines form-
`ing an amide linkage. The reaction can be carried out under slightly acid
`conditions by addition of the ligand dissolved in either Water or organic
`solvent to the activated carrier at pH 4.0.
`
`Arylamine Derivative
`
`react with phenolic compounds and other
`Diazonium salts will
`heterocyclics.5J1’*'1S These salts willalso react with alkylamines to form
`triazines. However,
`the triazine is not a preferred linkage because of
`rather poor stability. The reaction is generally conducted under slightly
`alkaline conditions.
`
`The diazotization procedure is carried out in an ice bath. To 1 g of
`arylamine glass add 10 ml of'2N HCl and 250 mg of solid NaNO2.
`Place the entire reaction mixture in a desiccator and attach to an aspirator
`so as to remove air and gas bubbles in the particles. Continue diazotiza-
`tion in aspirator for 20 minutes, maintaining the suspension packed in
`ice. The reaction is represented schematically in Fig. 14.
`Remove and filter on a Biichner funnel and wash with ice——cold water
`containing 1% sulfarnic acid if desired. Place the diazotized glass in the
`ligand solution previously adjusted to pH 8-9,; ‘using as little liquid as
`possible; a slurry is the preferred state. Maintain pH by the addition of
`2N NaOH. The use of a bufier such as 50 mM NaHCOa or 50 mM
`Tris will aid in pH control. If possible, use 50-100 mg of protein or
`other ligand per gram of glass. Allow the reaction to- continue for at
`least 60 minutes. Samples of the solution may be withdrawn at intervals
`for quantitative determinations in order to establish when the maximum
`coupling rate decreases; usually this occurs within 60 minutes. Continuing
`the reaction may add an additional
`l5—20% of bound ligand. Wash in
`
`in “Biochemical Aspects of Reac-
`‘5 R. Goldman, L. Goldstein, and E. Katchalski,
`tions on Solid Supports” (G. R. Stark, ed.), pp. 1-72. Academic Press, NEW York
`1971.
`
`“‘ H. H. Weetall, Res./Develop. 22, I8 (1971).
`" N. Weliky and H. H. Weetali, Irmnzmochenzistry 2, 293 (1965).
`‘‘‘D. H. Campbell and N. Weliky,
`in “Methods in Immunology and Immunochern-
`istry" (C. A. Williams and W. Chase, eds.), Vol. I. Academic Press, New York,
`1967.
`
`Exhibit 2036 Page 14
`
`Exhibit 2036 Page 14
`
`

`
`[5]
`COUPLING REACTIONS ANDGENERAL METHODOLOGY
`72
`
`
`distilled water and store in closed container immediately after filtration;
`the product usually contains 50-»70% water by weight.
`There are a large number of additional reactions that can be utilized
`for coupling of enzymes, antigens, antibodies, and ligands to inorganic
`supports. Such methods are reviewed in more detail eisewhere.15=13’“’
`
`‘” A. Bar-Eli and E. J. Katchalski, Biol. Chem. 238, 1690 (1963).
`
`[5] Polymers Coupled to Agarose as Stable and
`High Capacity Spacers
`By MEIR W1LcnEp§iiiand TALIA MIRON
`
`m.
`
`the polyrner—bound
`For successful use of affinity chromatography,
`ligand must be sufficiently distant from the polymer surface to minimize
`steric interference. This is achieved by introducing a spacer between the
`
`solid matrix and the ligand}
`Since the only efiective method available for binding various mole-«
`cules to Sepharose is by its activation with cyanogen bromidef the spacer
`must contain a free amino group through which the binding can talce place.
`Two different approaches for introducing the spacer may be used.
`First,
`the ligand can be prepared with a long hydrocarbon chain con-
`taining an amino group, and this derivative is coupled to the solid
`matrix.‘ The long chain serves as the spacer. The second approach is to
`bind a spacer, such as eaminocaproic acid, hexamethylenediamine, cysta—
`mine, p—arninObenzOic acid,
`tyramine, or p—hydroxymercuribenzoate, di-
`rectly to Sepharose so that a ligand can be attached to these derivatives
`by various methods.“
`In both methods the ligand is coupled monovalently to Sepharose
`through N-substituted isourea bonds, which are not completely stable.
`
`S9Pha1‘059
`
`+
`‘F-H2
`«OH + CNE-r + NI-{ER ————>— —o—~c::NHR
`
`Sepharose
`
`-OI-I
`
`--OH
`
`‘P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61,
`. 636 (1968).
`2]. Porath, R. Axén, and S. Ernback, Nature (London) 215, 1491 (1967).
`3 P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
`‘M. Wilchek and M. Rotman, Isr. J. Chem. 8 (E970).
`
`Exhibit 2036 Page 15
`
`Exhibit 2036 Page 15