LT3061B - Process for the preparation of polymeric gels for sorbents manufacturing - Google Patents

Abstract

Invention applies to the chemistry of macromolecular compounds and serves to obtain polymer gels that may be used in biotechnology as a basis for the synthesis of sorbents in chromatography of biomolecules. The method aims to create highly porous polymer gels that would be used as a basis for the synthesis of sorbents in the chromatography of biomolecules eliminating unspecific albumen absorption. The method is achieved by carrying out a radical suspension copolymerisation of allyl alcohol or glycol ether of the same with ethylene glycol-dimethylacrylate at a ratio of (65.34-87.41)/(34.66-12.59) mol percentage of neutral organic solvent - ethyl acetate or mixes of the same with n-alkanes (1-3):1 in the solvent. Copolymerisation is being carried out at a 1/(2-3) ratio of an upper organic phase having co-monomers and a radical indicator and a lower dispersant phase having a suspension stabiliser. There are following n-alkanes: hexane, heptane, octane, decane and dodecane. Dispersing phase uses water solvent of concentrated calcium chloride (30+-2%) with polyvinyl alcohol or solvent of the same and gelatine (0.5+-0.1%).

Description

The invention relates to high molecular weight. of compounds. chemistry, and in particular the method of obtaining polymeric gels. the latter can be used as basic matrices in biotechnology for the synthesis of sorbents for bioblocks / chromogenesis. »/

Methods of preparing high-efficiency currants for purification of biologically active substances using agarose or cellulose matrices are known. Sorbents do not exhibit non-specific sorption effects of proteins on the matrices of these matrices. However, the matrices themselves tend to collapse at high flow rates of eluents due to their low mechanical stability. This limits their use in preparative chromatographic processes for biomolecules / S. Kato, J3-. Hundred, Rates of mass tra. sphere in attinity chromatograpfy- J. Eng. Japan, 13, 151-154 (1980). Synthetic polymer gels such as Asahi gels exhibit sufficient mechanical stability. / EP 0066 * 165, C 08 F 8/12, G 01 N 3.1 / 06, B 01 J 20/26.

1982, SU 1311631, 'Bul. No 18, 1987 "7. Or TSK type gels / Y. Kato, K.. Komiya, H. Sasaki, T. Hashimoto, Separation Range and Separation Efficiency in High-Speec Gel Filtration on TSK-gel SU columns. J.

Chromator., · ISO (1980) 297-303 /. The gels mentioned are

25 prepared by a method of radical suspension copolymerization using vinyl acetate as the monovinyl comonomer and triisyl isocyanurate (Asahigels) or (meta) acrylic acid and pentaerythritol esters (TSK-gels) as crosslinking agents. However, there are a number of drawbacks to the methods of obtaining said gels; - requires /, additional labor costs in transferring them to neutral OH-containing gels, and is characterized by the presence of non-specific (mainly hydrophobic) protein sorption levels on synthetic gels, The presence of a certain amount of residual nonspecific protein sorption in the final gels is negative. responds to the monofunctionality and selectivity of potential sorbents in their substrates,

For suggestion. · Inventing the most technical solution 5. close is the method of obtaining polymeric gels - Spherones.HEMA. Gels of this type are prepared by suspension radial copolymerization of 2-hydroxyethyl methacrylate and ethylenedimethacrylate or ethylenediacrylate in inert organic solvents, cyclohexanol or cyclo10 hexylamine, with octyl or dodecyl alcohols. The dispersing phase is aqueous. polyvinylpyrrolidone or alcohol solution, and starch as a stabilizer. The copolymerization reaction is carried out for 12 15 hours at '70 ° C' / US Pat. 1370477, CO8F 1/11 »..1974 ../.

poivinyl suspensions

The disadvantage of this type of gel preparation is the high level of non-specific protein sorption on the yield gels.

Nonspecific sorption of proteins is significantly reduced by further modification of the starting gels, e.g., by aminating agents, by introducing high concentrations of ionogenic groups. However, complete elimination of .nonspecific protein s-orbitation is not possible / 0. Mike, Z. Hostomska et al .. _r 'Ion. - exchange derivatives of Spheron, J. Chromatogr. 440: 287-304 (1988). Residual protein non-specific; the presence of sorption in gels is very negatively affected ^- using the latter as a basic matrix for the synthesis of specific sorbents, and for the gel filtration processes of proteins. Synthesis of biospecifinius currants insertable into the ligand matrix as a ^rule: No non-specific protein sorption aukšta.Todėl existence 'can distort biomolecule interactions with the immobilized specific - ligand specificity of the sorbent and reduce efficiency. Non-specific sorption F phenomena in gel filtration of biomolecules. can be eliminated by introducing into the eluent chaotropics

/ ³ agents or additives in organic solvents. However, the use of the latter can lead to conformational changes and denaturation of the biiomolecules, which limits their use of - in particular, the use of starting gels · for the chromatography of biomolecules while retaining their native structure.

It is an object of the present invention to provide high porosity polymeric gels which are suitable for sorbent synthesis and which do not have non-specific sorption and are suitable for chromatography on biomolecules.

The object of the present invention is achieved by slurry radical copolymerization of 'allyl alcohol or allyl glycidyl ether' with ethylene glycol dimethacrylate in the molar ratio {65, 34-87, 41): (34, 66-12, 59) mole percent. The polymerization of the comonomer mixture is carried out in - inert organic solvents in ethyl acetate or mixtures thereof with n-alkanes in a ratio (1-3): 1 and the reaction is carried out for 6 hours at 80 ° C in the upper phase containing the comonomers and Suspension stabilizer, 1: (2-3).

The use of allyl alcohol or allyl glycidyl ether as the major comonomer enables the introduction of a hydrophilic component into the polymeric gel chain having a spatially advantageously neutral OH group on the parent macromolecular chain or an even more remote reactive epoxy group suitable for attachment to various functional groups and specific ligands. In the latter case, an oxypropyl group formed after ligand attachment. plays a role as an interface between the polymer chain and the ligand, promoting a more complete ligand-specific interaction with the + biomolecule.

Inert solvents include ethyl acetate and various solvents

-t use of mixtures with n-alkanes, Λ and coaonomers. changing the molar ratio enables purposeful

= .. visual polymeric polymer; porosity of gels t. y 'pore size and volume, their inner surface / area, / pore size distribution, and remaining non-specific

- protein sorbci.jos on the level of gels.

^; 10; . Upper organic phases containing; :: comgnomers. and.

. polymerization initiators, and / (lower / =: dispersing phase containing suspension stabilizer, <1: 2 to 1: 3 by volume ratio adjustment) allows the geometry of the gel particles to be adjusted.

15 ': {(= // - {/// {=: - - - - {(3

The use of a concentrated calcium chloride aqueous solution with polyvinyl alcohol or a solution of the latter and gelatin is sufficient to stabilize the suspension to obtain spherical particles and to prevent the passage of the hydrophilic comonomer into the lower aqueous phase.

The invention is illustrated by the following examples. =. / -, (::

..25 ((-?: / {: {('(, - :( 3 = / ^ {((((' f-3 .. = / :: ({'((((( :

1. Polymeric gels - copolymers of allyl alcohol with ethylene glycol dimethacrylate = / {conditional, '/ title. FSL-Scales).

Example 1. A monomeric mixture containing 50 ml. (0.736 mol) of alcohols (AA) AND 20 ML »(0.106 mol) of ethylene glycol dimethacrylate = (EGDMA) =

B7 _Z 41: 12.59 mol%) is dissolved in 75 ml of ethyl acetate. 0.64 g (1% of the total weight of the monomers) of the initiator azo-butyric acid dinitrile (ADN) is dissolved therein and the resulting mixture is blown for 10 minutes. period Gaseous nitrogen for oxygen removal »Mixture obtained

..5 / ./r J.

dispersing with stirring (250 rpm) in a 1% solution of polyvinyl alcohol (molar weight 10,000) in aqueous solution containing. 200 g. of dissolved calcium. . chloride hexahydrate <(volume ratio of upper organic to lower aqueous phases = 1: 2). The reaction mixture is stirred for 6 hours at 90 ° C. · After completion of the reaction, the mixture is cooled to room temperature, ^- discharged onto a filter, -. and the resulting gel is washed on the filter with water, water-isopropanol mixtures with increasing concentration of the latter from 30 to 100%, and mixtures of isopropanol and water isopropanol with decreasing concentration of the latter from 100 to 30%, and water. The resulting copolymer fractionation sits wet sieving for selection of the gel fractions 63-125 and 125 - ¹ - The 500 micron size. ' 140 ml of gel with a particle size of 125-500 mkm and 70 ml of 63 - 125 mkm are obtained.

The main properties of the obtained polymer gel are: water retention, comparative volume of water-swollen gel, non-specific sorption rate of the protein and porosity. parameters.

H ₂ 0 - The delay (W _R , ml / g) is determined as follows.

Equilibrated gel for water removal, weighed, dried and weighed (W ₂ ), calculated according to the formula:

77/7 7/7 { _Λ Wi - W ₂ .

M./-/ -

The comparative 7 cm ³ / g swelling in water is determined by measuring 1 g of wet, (/ dry volume of the gel.) · '''^; · -Y · centrifugation (W.), Followed by gel H ₂ O retention. 'volume (V _R ,

. . · ';</ Γ. ·· 6γ ^; ; γΥ /; γγ, _. · ^ '' γ The non-specific. sorption rate of the protein (N _m , mg / ml) is determined by the amount of sorbent human serum albumin by static sorption. For this purpose, the polymer gel is incubated for 2 hours at room temperature in a protein solution (40-50 mg protein in 1.0 mL of gel) in sodium phosphate buffer containing 3.0 M NaCl.

Porosity of polymer gel, pores. Occupying · volume, comparative surface area, average pore diameter and their polydispersed mouth are determined by reverse gel chromatography / A. A. Gorbunov, L. Ya.

Solovyova, V. A. Pasechnik, Fundamentals of the Theory and Practice of Polymer Gel-Permeation Chromatography.

'' • γ / · '; . · '' · - / ---: · - / · - * '/ · · .y · // - ·' - 'Y; · '^ Yy./y\y _v -y? Γ-γ:.-Υ · · α γ / -' · - / ·. · / · / '· /;''' · Γγ '<- - - //// · · '''.//// e ////'//; / '// · ^! \ · '- · / -, · / γ / 7 .//. as a method of chromatographic porosimetry. -J. Chromatogr., 448, 307-332 (1988), / /

Polymeric gels obtained according to e.g. -1 and the examples below, 'properties' are given in Tables 1-2.

Example 2. The copolymerization of AA and EGDMA is carried out analogously to e.g. 1, except that 75 ml of an equal volume of ethyl acetate / n-hexane are used as the organic solvent. 160 ml of the gel obtained to particle '' Υ / Υ / 'ΥΥ / Υ / γΥΥ // ^/ / ΥϊΥ / ^γ' / Υγ; /:·Γ·///ΥΥγγ;Υ-Υ'ΥΥ.γγ//Υ ^: γγ / Υ · Υ / ΥΥΐΥΥΑΜ / γ /// ν / γ · γ / γ · γγ / · / · / γ-.

size 125-500 mkm if 30 ml - 63-125 mkm.

Example 3. The copolymerization of AA and EGDMA is carried out analogously as e.g. 1, except that an organic solvent is used in a mixture of 75 ml of an equal volume of ethyl acetate and n-heptane. Incoming. I1S ml of gel with a particle size of 125-500 mkm and 65 ml - 63-125 mkm.

Example 4. The copolymerization of AA and EGDMA is carried out analogously to Example 1, except that 75 ml of an equal volume of ethyl acetate are used as the organic solvent.

a mixture of n-octane. 140 ml of gel with a particle size of 125-500 mkm and 65 ml of a gel of 63-125 mkm are obtained.

Example 5. The copolymerization of AA and EGDMA is carried out analogously to e.g. 1, except that the organic solvent used is a mixture of 75 ml of an equal volume of ethyl acetate and 'n-decane. 135 ml of gel with a particle size of 125-500 mkm and 35 ml of 63-125 mkm are obtained.

Example 6. The copolymerization of AA and EGDMA is carried out analogously as e.g. 1 except that for the use of organic fertilizers.7 5 ml of a mixture of equal volumes of ethyl acetate and B 'n-dodecane. 85 ml of gel with a particle size of .125-500 mkm and 75 ml of 63-125 mkm are obtained.

Example '7. The copolymerization of AA and EGDMA is carried out analogously to Example 1, except that the monomer mixture in the - phase consists of '50: ml (0.736 mol) of alcohols, 35 ml · (0.185) ethylene glycol dimethacrylate

I \. - /2.()///0¾ahtykšŠ?AA:EGDMAB B = / B // 7 988: 20, 12 mol. %), '' B / - · 75 ml of ethyl acetate and 0.8 g of azoisobutyric acid dinitrile. 220 ml of gel with a particle size of 125-500 microns and is obtained _; 90ml - 63-125mkm. B ^r

Example 8. The copolymerization of AA and EGDMA is carried out analogously as e.g. 1, except that the monomeric mixture used is of the same composition as e.g. 7; o.

. the organic solvent used is a mixture of 75 ml of an equal volume of ethyl acetate and n-hexane. 250 ml of gel with a particle size of 125-500 mkm and 50 ml of 63-125 mkm are obtained.

Example 9. The copolymerization of AA f and EGDMA is carried out analogously, e.g. 1 except that the monomer mixture of B /, / B is of the same composition as e.g. 7 o /.·

Organic solvent 35B uses -75 ml of a mixture of equal volumes of © ethyl acetate and n-octane. 180 ml of gel are obtained; with a particle size of 125-500 mkm and 60ml - 63-125 mkm. 7, and an equal volume of 190 ml gel with g, .g _; . ; .//g/g,,//

Example 10. A copolymer of AA and EGDMA analogously to e.g. except that the monomer mixture is organic in the same composition. Solvent - 75 ml of a mixture of 5 ethyl acetate and n-decane are used. Obtained with a particle size of 125-500 mkm and 40 ml - 63-125 mkm.

Example 11. The copolymerization of AA and 'EGDMA is carried out analogously to e.g. 1, except that the monomeric mixture used is of the same composition as e.g. 7 and 75 ml of an equal volume of a mixture of ethyl acetate and n-dodecane are used as the organic solvent. 180 ml of a gel with a particle size of 125-500 μm and 45 ml of a 63-125 μm are obtained.

Tape 1

Water retention (W _R ), Comparison - Volume of gel swelled in water (V _B ) and Non-specific sorption rate of albumin (N _m )

Example # W _R ml / g V _R cm ³ / g • N _m mg / ml 1. / g / ggg ': / 0 ^F : 18.5 2. 4.07 8.90 5, 9 3. 4.14 - 6.1 4. 3.80 7.25 0.8 5. 2.18 '7.50 2.3 6th 2.95 7.40 2.3 7th //, / -? :: / ge - 20.4 8th / 2.59 5.76 23.4 2, 61 5.65 13.5 10 / -g / g 'Λ- /' 2.49. 5, g 0 '/-../; ^; - / 1 /,: 8 /. ; Uggg ' 2.48 '' ^5/40 // 11.4 ' The prototype Spheron 100,000 27.1

Table 2

Porosity parameters of allyl and ethylene glycol dimethacrylate-based gels

For example No. Pore Volume · fraction Vp / Vp + V _Q Equal to m ² / ml pore volume  t Moderate pairs diameter (nm) /.<////./ /// 5 / '-' · Polidis- Frakciona Optimal ' persis- *. gossip vimo inter. under- molecular am. (nm) dextral no Mol. mass 1. 0.43 82 . 24th 2.0 3.1-59 .103000 2. - 0.43 120 16.5 19th 2.3-300 .330000 0.45 15th 140 6.1 17-1000 8123000 4. 0.40 23rd '87 165 17-9000 '27024000 5. 0.29 62 32 48 5-1200 ' 3152000 € .. 0.23 100 i 20 2.1 - 2.5-50 76000 7, 0.39 93 ' 21.5 '1.8 2.8-45 81000 8th 0.44 · 130 15th 30th 2 / 2-390 431000 9th 0.44 88 - 23rd 55 4-1000 187300 10th 0.44 36 56 90 10-3500 11415000 11th 0.44 92 21.5 81 4-1250 1674000 Spheron 100000 prototype Exclusion limit for dextran 10 ⁸ Data , submit in the tables 1-2, shows that hpgliiieerlhiA to receive ally alcohol and

ethylene glycol dimethacrylate has very large pores that can be penetrated not only by large protein macromolecules but also by viruses. The present method of synthesis of this type of gel enables a targeted alteration of their porosity and consequently of the non-specific sorption rate of proteins. These types of gels can be

- be used in the synthesis of sorbents based thereon on all types of biopolymer chromatography other than gel chromatography. Non-specific sorption of human serum albumin on Spheron 100000 (27.1 mg / ml) and an analogue of dextran in its exclusive range of gel obtained according to e.g. Comparison of Table 4 (0.8 mg / ml), (Table 1), shows that the overall level of non-specific protein sorption on polymeric gels obtained according to the proposed method is significantly lower. and may be directionally controlled by varying the composition of the monomer mixture, the solvents and the composition of the mixtures thereof.

2. Polymeric gels 10 ethylene glycol dimethacrylate name FSL-SE-gels) allyl glycidyl copolymers in ether and (conditional acid-dinitrile) gas

Example 12. Into a 2 liter triple-bottomed flask, add a monomeric mixture consisting of 100 ml (Ό, 694 mol) of allyl glycidyl ether (AGE) and 40 ml (0.212 mol) of ethylene glycol dimethacrylate in a mechanical stirrer, thermometer and reflux condenser. AGErEGDMA = 76.61: 23.39 mol%) Then 150 ml of ethyl acetate and 1.40 g of initiator - azo-butter are added to the mixture. nitrogen and dispersed by stirring (250-300 rpm) in 870 ml of a 2% aqueous solution of polyvinyl alcohol (molar weight 100000) containing 4, 35 g of gelatin (1: 3 organic / aerosol phase). The reaction mixture was stirred for 6 hours at 80 ° C. The reaction mixture was then cooled to room temperature and filtered. Polymeric gel. washed on the filter with water, water isopropanol mixtures with increasing concentration of the latter from 25 to 100%, isopropanol, yandensisopropanol mixtures with decreasing concentration of the latter from 100 to 25% and water. The resulting gel is fractionated by wet sieving, selecting fractions with particle size 63-125. and 125-500 mkm.

3.5

The resultant is the concentration (C _E , mkmol / ml) and porosity groups after imaging of the gel.

The epoxy groups are determined by incubating a gel sample in sodium thiosulfate solution, titrating / liberating the hydroxyl ions during the reaction with standard -HCl solution / J. Turkova, Affinity Chromatography, p. 2415. 242, '1980. /.

C _E = 24-25 mkmol / ml.

Polymeric gels obtained according to e.g. for descriptions of the examples below, the properties are given in Table3. *

Example 13. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that 150 ml of a mixture of equal volumes of ethyl acetate and n-octane is used as an organic solvent. 200 ml of gel with a particle size of 125-500 mkm and 50 ml of - 63-125 mkm are obtained. C _E = 6-15 mkmol / ml.

Example 14. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that 150 ml of a mixture of organic solvents such as e.g. 13 at a volume ratio of 3: 1. 400 ml · '»gel with a particle size of 125-500 mkm and 150 ml - 63-125 mkm are obtained:

C _E = 20 mkmol / ml.

Example 15. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that 150 ml of an organic solvent mixture consisting of

112.5 mL of ethyl acetate and ’37.5 mL of n-hexane. 400 ml of gel with a particle size of 125-500 mkm are obtained. and 150 ml - 63125 mkm. _{C E} = 30 mkmol / ml.

Example 16, copolymerization of AGE and EGDMA is carried out analogously as e.g. 12 except that the organic solvent used is 150 ml of a mixture of equal volumes of ethyl acetate and n-decane. 260 ml of particle size gel are obtained

1/2

125-500mkm. and 120 .ml. - 63-125km. C _E = 8 - 12.5 mkmol / ml.

Example ^ '17> The copolymerization of AGE and EGDMA is carried out' analogously as e.g. 12 except that 150 ml of a mixture of 112.5 ml of ethyl acetate and 37.5 ml of organic solvent are used. of n-decane. 500 ml of gel with a particle size of 125-500 mkm are obtained. and 15 ml - 63-125 mkm. C _E = nicol / ml.

Example ^ 18. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that the organic solvent used is 150 ml of a mixture of equal volumes of ethyl acetate and n-dodecane. 200 ml are obtained. gel with a particle size of 125-500- mkm. and 30 ml - 63-125- mkm. C _E = 5.5-6.0 mkmol / ml.

Example 19, - AGE. and EGDMA copolymerization is carried out in an analogous manner to. e.g. 12 except that the 'monomer mixture' phase consists of 100 ml (0.694 mol) of allyl glycidyl ether, 70 ml (0.370 mol) of ethylene glycol dimethacrylate (ratio AGErEGDMA = 65.34: 34.66 mol%) in 150 ml of ethyl acetate. and 1.86 g of azinobutyric acid dinifcryl, and the volume of the aqueous phase containing polyvinyl alcohol and gelatin is equal to .y r. ·? ..: 1/7/77: //. .; / 7: / -: · / ·. 7777-7 / ^ 7: / :: 7 :: -, 77: 7 777 /: 7 7: · 7 // 7- 77'777 / 777 // 7 /: 7/77 ^ 7 - / - 7 " 77 / 7-77 / 777-,: 7 / '/ 7-7-: 7'

960 ml. 280 ml of gel with a particle size of 125-500 mkm and 60 ml of 63-125 mkm are obtained. C _E = 30-38 mkmol / ml.

Example 20. · The copolymerization of AEG and EGDMA is carried out analogously as e.g. 12 except that the monomeric mixture used is of the same composition as e.g. 19, and an organic solvent is used ^_ 150 ml of an equal volume of -etilacetato and n-hexane. 200 ml of gel with a particle size of 125-500 mkm and 50 ml of 63 - 125 mkm are obtained.

-35, C _E = 20 - 24 mkmol / ml.

- X'- - '\ I? X- '.

- Example 21. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, · except that a monomeric mixture of the same composition as e.g. 19 and 150 ml of a mixture of equal volumes of ethyl acetate and n-octane are used in the organic solvent. 220 ml of gel with a particle size of 125-500 microns and 50 ml is obtained. - 63-125 mkm.

C _E = 14-20 mkmol / ml.

Example 22. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that a monomeric mixture of the same composition as e.g. 19 and 150 ml of organic solvent are used. of a mixture of ethyl acetate and n-dodecane. 150 ml of gel with a particle size of 125-500 mkm and 30 ml of gel are obtained. 63-125 mkm.

'C _E = 11-13 mkmol / ml.

Example 23. The copolymerization of AGE and EGDMA is carried out analogously as e.g. 12, except that a monomeric mixture of the same composition as e.g. 19, while yy \ .y'.i ··. ·· · / x χ'x;';;xy?' 7xij in · '··''-.X; ^: xx. '. 'i x.';. 'x. ·' x. '^; ·· '·'<..χ'·. χ χχχ'i -. 'y

- Organic solvent used in a mixture of 150 ml of iodine acetate and n-decane. 600 ml of gel with a particle size of 125-500 mkm are obtained. C _E = 18-19 mkmol / ml.

ΜΜχ-'ΐχ '' ·

Table 3

Porosity parameters of allyl glycidyl ether and ethylene glycol dimethacrylate-based gels

For example: Couples Like · '- Middle • Poly Factional Optimal jio no occupied surface. tinis' dispe- vima dextran aunts sius pairs rsiš- inter. Mol. part m ² / ml slide gossip according to mass:. · '.. / V _p / V _p + pairs ///// meter 7 Mol. + Υθ ......... volume (nm) diam. (nm) 1 2 3 7 '//' ^ ++ '+ / +++++ 5 + /// + // · / ^: + ^ ^Ί 7 // + / - / - + // :: - /: 7- / 7 '/' 12, 0, 39 67 30th * 1.44 4.2-50 138000 13th 0.47 30th /////.O/ 4.30 8-330 1439000 14. ' 0.46 33 + - / ++ / -:.: / +: /: 61 + / 1.18 10-81 499000 15th 0.41 50 40 1.50 5.5-71 245000 / 16th 0.38 57 35 '7.80 4.3-300 662000 17th .0.32 34 60 3.30 7.2-230 906000 18th 0.40 360 5.5 1.20 0.9-7 '5000 19th 0.39 /::./+/1+15:/ 17.4 1.03 3.3-19 47,000 20th 0.37 180 11.1 1.12 '1.9-14 19000 21st 0.43 43 47 27th .6, 8-1150 4103000 22nd 0.45 148 13, 5 1.00 2.7-14 28000 23rd 0.37 58 ^ - // + :: / - /// 35- + 15.5 4.6-530 797000

The data in Table 3 show that polymeric gels based on allyl glycidide ether and ethylene glycol dimethacrylate also have high porosity. sufficient for the penetration of large biomolecules. Gels of this type are more monofunctional with respect to pore size and are suitable for gel-filtration processes of biomolecules.

The suitability of the resulting polymeric gels for sorbent synthesis and the suitability of the latter for biomolecule chromatography are illustrated by the examples below.

Example 24. Synthesis of carboxymethyl derivative of FSL-S-42 gel.

<k. . . ·

In 100 ml · wet precipitated gel obtained according to e.g. 8th

Add 5 ml of the description, stirring in small portions, 55 ml of a 46% sodium hydroxide solution and leave the resulting suspension to incubate for 1 hour. The suspension is then transferred to a triglyceride flask containing a reflux condenser, a thermometer and a mechanical stirrer, and a solution of 27 g (0.145 mol) of monoiodoic acid in 50 ml of water is added under stirring. The reaction mixture was stirred for 30 min. min at room temperature for 6 hours at 80 ° C. The resulting sorbent is filtered off and washed with water, 2N HCl solution and * water to neutral pH of the washings.

The ionic capacity of the cation exchanger is 50 mcg / ml based on Na ⁺ ions. *

The suitability of the 20 '' Serbent for protein chromatography is assessed by reference to Cytochrome c (0.02 M Na phosphate buffer pH 8.0), human serum albumin (0.02 M Na o acetate acetate buffer pH 4.0) and trypsin. (0.02 M Na 'phosphate buffer pH 7.1). static sorption,

25 · · incubation of sorbent samples in protein solution for 2 hours.

at room temperature, and desorbing the protein with 1 4 M NaCl. The sorbent sorption capacity and desorption yield of the protein are shown in Table 4.

• 30 /., ........ 1β

Table 4,. //

Sorbent sorption susceptibility to proteins

Protein and its concentration mg / ml Volume of protein solution Suction sorbent The sorbent susceptibility, mg of protein per ml sorbent Protein of desorption yield,% Cytochrome c 4.7 1.0 ml 4.7 98 Album - 50.0 1.0 ml 37.2 95 Trypsin- 22.5 1.0 ml 13.0 · '95

Example 25. Synthesis of a sulfopropylated derivative of FSL-SE-81 gel.

.

. 20th

Transfer 100 ml of the gel obtained according to e.g. 22, 11.1 g (0.09 mol) of distilled + +1+; + h // anhydrous Na ₂ SO ₄ and 27.25 ml of the reaction mixture is left at a temperature and the reaction is continued by stirring a suspension of 4 .vai 95 ° C. The reaction mixture is cooled to room temperature, the sorbent is filtered off and washed on the filter with water. The sorbent is then transferred to a beaker and washed with water in a decanter. The sorbent sorption by Na ⁺ 'is determined by titration of the H ⁺ form with a standard solution of 5 mH NaOH and is equal to 12 mkmol / ml.

water. room

The suitability of the sorbent for biomolecule chromatography is evaluated by the bovine Tripsin static sorption in 0.02 M Na in pH 5.0 buffer. For this purpose sorbent

- the sample was washed with the indicated buffer, incubated for 2 hours at room temperature in protein solution (initial concentration 20 mg / ml, 1.5 ml solution in 1 ml sorbent) and; sorbent protein eluted with 1M (NaCl · / buffer buffered at pH 5.0 /) The sorbent has a trypsin uptake of -16 mg / ml, with a protein yield of 86-90%.

- -? IT f /

The present method of polymeric gels, using instant mixtures of allyl alcohol 'or allyl-glycidyl ether and ethylene glycol dimethacrylate and organic solvent mixtures of ethyl acetate and n-alkanes, enables the maturation of the porosity of the gels and the non-specific sorption rate of the proteins. in turn, use the gels to synthesize currants that are monofunctional to the attached ligand. .The details are given in Table 4 and eg. 25 shows that the gel-based sorbentase obtained is suitable for chromatography on biomolecules, providing practically quantitative desorption yields for the latter.

Claims (4)

Method 1 - Obtained polymeric gels for the synthesis of sorbents by suspension. radical Polymerization of a mixture of 5 'kSmoriomers containing a single monomer and a double bond crosslinker in an inert organic solvent in the presence of an initiator and a process in an aqueous dispersing phase containing 10 suspension stabilizers, characterized in that the main monomer having a single double bond uses allyl alcohol or allyl glycidyl ether as the crosslinking agent. ethylene glycol dimethacrylate in the molar ratio (65.34-87.41) r (34, 66 15 / 12.59) mol%; using ethyl acetate or mixtures thereof with n-alkanes in an organic ratio (13): 1, and copolymerization. is carried out at a volume ratio of the upper organic phase containing comonomers and initiator to the lower dispersing phase containing sus20 pension stabilizer! : (2-3), 2. A process according to claim 1, wherein hexane,, heptane, octane, decane and dodecane are used as n-alkanes. 3. A process according to claim 1, wherein the dispersing phase comprises an aqueous concentrated solution of calcium chloride and polyvinyl alcohol, or an aqueous polyvinyl alcohol and 30 gelatin solution. 4. The method of claim 3, characterized in that the calcium chloride concentration _t is - 30 ± 2% of polyvinyl alcohol - 1-2% gelatin - Q 5 + 0.1% ..