Perloza CDI ACA
CHAPTER 8 CONCLUSIONS
Results
The matrix chosen as the focus of this study, Perloza bead cellulose, has been shown to
have good properties for laboratory scale, low pressure chromatography. It has been
found to compare favourably with Sepharose matrices. The results presented here, and
previous reports (Peska et al., 1976; Stamberg et al., 1982; Stamberg, 1 988; Gemeiner et
al., 1 989) have suggested it would also be useful for large scale chromatography.
The chemical reactivity ofPerloza was demonstrated using the anhydrous CDr activation chemistry. The efficiency of this activation was greatly improved by the use of better solvent exchange techniques. Likewise, subsequent ligand attachment was improved by minimising water content of the reaction mixture. However, CDr methods were still not favoured for large scale use because of the solvent requirement, cost and the alkali
sensitivity of the urethane linkage. Aqueous activation and ether linkages were
preferred. Commonly used etherification reactions were not very effective in aqueous media. Carboxymethylation was improved by the inclusion of an organic solvent in the reaction mixture. However, ligand attachment to CM resins was not ideal because it required expensive carbodiimide chemistry. Low activation levels were obtained with standard (and preferred) bifunctional etherification reagents, regardless of the solvation used. The low activation levels were attnbuted to competition from hydrolysis and crosslinking side reactions. Therefore none of these conventional activation methods were considered ideal for large scale use.
The use of bifunctional reagents containing groups of unequal reactivity, would eliminate
or reduce the side reactions. This quality was found with allyl bromide and allylglycidyl
ether. Their reaction with Perloza resulted in covalent attachment of allyl groups by an ether linkage. The allyl groups were relatively stable and no indication of reaction with water or cellulose was found. This simplified the reaction chemistry and high activation levels were obtained (despite aqueous solvation) because side reactions were no longer
limiting. It was demonstrated that the activation level could be easily controlled by the
amount of reagent used.
Two distinct methods were demonstrated for ligand attachment to allyl matrices. The
first of these was nucleophilic substitution with sulphite, amine or thiol ligands, after allyl
groups were modified by bromination (addition of HOBr) of the double bond. Aqueous N-bromosuccinimide (NBS) was preferred because it was anticipated that a side reaction
found with bromine water addition would be eliminated. NBS is also easier and safer to use than bromine water. Nucleophilic substitution of brominated matrices occurred readily at alkaline pH (� 1 0). Reaction ofthiols and sulphite at lower pH values was also effective if the mixture was heated at 60°C. Nucleophilic substitution allowed the preparation of a diverse range of resins for affinity, hydrophobic, ion exchange, IMAC and mixed mode chromatography.
The second method did not require an initial modification of the alkene group. Although the allyl group is comparatively unreactive, it will undergo specific free radical addition reactions other than bromination. Several thiols and the bisulphite ion were found to react readily with allyl Perloza without any (deliberate) catalysis. This was consistent with the facile addition of these molecules reported for simple alkenes, despite the use of a macromolecular alkene substrate and aqueous solvation. Advantages of addition were simplicity and specificity (e.g. glutathione adds readily through its thiol group, without competition from the amino group). Thiol attachment was simplified because there was no requirement to remove oxygen from reaction mixtures, in contrast to conventional methods (Simons and Vander Jagt, 1 977). One less reaction step is required compared to the substitution chemistry. Resins for affinity and ion exchange chromatography were prepared by free radical addition and a wider range of resins might be produced if catalytic methods (e.g. irradiation) were employed. Addition of a mercaptoacid ligand followed by carboxyl group titration was used to analyse allyl activation level. The high activation levels obtained in aqueous media and efficient ligand attachment recommended these allyl chemistries for large scale use especially.
A range of ion exchange resins were produced from allyl Perloza, using simple methods, and high charged group densities were obtained. Wrth one exception the physical properties of these resins were excellent, with only a smaIl loss of performance relative to unmodified Perloza. Flow rates superior to Sepharose Fast Flow resins were obtained. Chromatographic ( bovine serum albumin elution) properties of the strong ion exchange resins were similar to those of Sepharose Fast Flow. Protein capacities were also high, although the BSA capacity of anion exchange resins was lower than that of Q Sepharose Fast Flow. These results indicated that the products of allyl Perloza were useful for protein chromatography.
CDI and allyl Perloza were used for preparation of mixed mode (hydrophobic and ionic) resins (of
high ligand density)
for chymosin purification. Some resins were also produced from CDI and ECH Sepharose. Chymosin was adsorbed to these resins at both high and low ionic strengths. Most forms of chromatography have specific ionicstrength requirements (e.g. low ionic strength for ion exchange chromatography and addition of ammonium sulphate for hydrophobic interaction chromatography). Therefore
an intermediate step to alter ionic strength is typically required before crude fermentation broths can be processed. Therefore, the adsorption found at high and low ionic strength was a preferred characteristic, because an intermediate step was not required.
Despite the high ligand density (and hydrophobicity) which imparted strong adsorption properties, near homogeneous (by SDS PAGE) chymosin was recovered, in one step, by a small pH change. High capacities were also found. Fouling of resins by crude broths was significant for amine but not carboxylate containing resins and the latter resins also had higher capacity. Mercaptoalkyl carboxylate derivatives of allyl Perloza were preferred for large scale use.
The features of high ligand density, adsorption at high and low ionic strengths and elution by a small pH change were retained for a new form of hydrophobic chromatography. This relied on weakly ionisable ligand groups which titrated in a pH range near neutrality. At adsorption pH these resins were neutral and hydrophobic, while at elution pH they had mixed mode characteristics. The use of a variety of ligands (and functional groups) allowed production of resins with differing hydrophobicity and titration range. The latter property meant a range of titration "windows" were available and a rational selection of ligand/resin could be made to suit the preferred pH requirements of a target protein.
Salt promoted adsorption of various proteins to ionisable (pyridyl) resins of low ligand density, in comparison with Phenyl Sepharose, suggested the uncharged forms
functioned as HIC adsorbents. At moderate ligand density, lysozyme and
chymotrypsinogen were not completely eluted and at high ligand density they were adsorbed at high and low ionic strength. The ionisable character was then exploited, by a pH change, to obtain rapid elution. High ligand density (pyridyl) resins were also used for chymosin adsorption independent of salt concentration, at pH 5.5-6.5. Elution was again obtained by a pH change (to 2).
Chromatography of two other crude enzymes was investigated. The more hydrophobic of these (a-amylase) was also adsorbed at low and high ionic strength to the mixed mode (carboxylate) and hydrophobic ionisable resins used for chymosin. Rapid elution was again obtained by a small pH change. Eluted a-amylase was near colourless and homogeneous (by SDS PAGE). Subtilisin is less hydrophobic and was only retarded at high ionic strength on the carboxylate resins. It was however adsorbed by the more
hydrophobic of these resins at an intermediate ionic strength, similar to that of the crude broth. Elution from these resins was also more difficult than was found for a-amylase and chymosin. Stronger adsorption was found with the uncharged form of hydrophobic ionisable (especially pyridyl) resins. Nevertheless, rapid elution was found following a pH change to the partially ionised form. Although purification was less evident (by SDS PAGE), eluted subtilisin was largely colourless and separated from the bulk of material absorbing at 280 nm.
Catalase, an enzyme which required 50% ethylene glycol for elution from a conventional (phenyl Sepharose) resin, was chromatographed on low ligand density pyridyl resins. Similar adsorption properties were found using a salt gradient at pH 7.5 « 1 0% activity eluted). However, catalase was recovered (rapidly) by a pH change to 4. No elution was found at this pH using Phenyl Sepharose. Regeneration of the pyridyl resins was apparently superior to that of Phenyl Sepharose.
Future
Further applications of allyl chemistry could exploit the high ligand density potential of
this method. High ligand density has been considered unfavourable in many applications because non-specific interactions become more significant and therefore resolution is
reduced. However for some large scale applications, it may be preferred to increase capacity at the expense of resolution. High ligand densities have already been obtained for IMAC and glutathione resins and these might be useful for large scale, high capacity chromatography, especially for purification of fusion proteins. Allyl chemistry appears to be particularly suited to use with Perloza, because low activation levels are obtained with the conventional etherification reagents.
Lower ligand densities can still be obtained with efficiency and accuracy using allyl chemistry. Mercaptoacetic and mercaptopropionic acid derivatives of allyl matrices should provide cheap, chemically stable "spacer arm" resins, for attachment of amine ligands for affinity chromatography. These would be alternatives to aminocaproic acid resins, and results here (subtilisin chromatography at high ionic strength) indicate that the mercaptoacid spacer arm would be less hydrophobic. If a hydrophobic spacer arm is
required, this can still be obtained by using a longer mercaptoacid (e.g. mercaptohexanoic acid). Allyl matrices are relatively stable in aqueous media, facilitating storage and shipment. Another application of allyl chemistry would be to test a wider range of chromatographic matrices.
Specific ligand addition reactions may be useful for directed attachment of thiol containing ligands such as peptides (Englebretse� 1 992). This attachment would be simplified by lack of competition from amine groups and water for reaction with the allyl
matrix. The scope of addition possibilities will be determined by the effectiveness of irradiation catalysis of general thiol ligand additions.
Other applications of the mixed mode resins described are also likely to be for large scale protein purification. Hydrophobic ionisable resins may be useful for analytical (FPLC and HPLC) as well as preparative chromatography. This would require adjustments of matrix (to higher performance) and ligand density. At lower ligand density a more
traditional chromatographic separation on the basis of retention time rather than the strong adsorption methods used here. Therefore decreasing salt gradients could be used for elution. A pH step or gradient could be used concurrently or after a salt gradient to provide further resolution. Catalase results suggested that low ligand density resins would be useful for HIC of very hydrophobic proteins (simple recovery) and/or HIC applications where severe hydrophobic fouling occurs.
High ligand density mixed mode and hydrophobic ionisable resins may be applied to adsorption of a wider range of proteins, particularly for industrial separations. Another possible application is non-covalent enzyme immobilisation (to the neutral resin form). If the pH is maintained in an appropriate range, the enzyme should be strongly retained. Hydrophobic resins have been used before (Butler, 1 975; Nemat-Gorgani and Karimian, 1 983; Hutchinson and Collier, 1 986) for non-covalent immobilisation. Ionisable resins have the advantages that the enzyme may be recovered and the resin regenerated (foulants removed) by pH adjustment.
The activation chemistries and hydrophobic ligands described may also be used for modification of polymers other than chromatographic resins. Possibilities include membranes for protein adsorption (Heath and Belfort, 1 992) and soluble polymers for two phase liquid extraction (Jensen et al., 1 993).
APPENDIX 1 : Molar prices for activation reagents and ligands Activation allyl bromide allylglycidylether bromine N -bromosuccinimide butadiene diepoxide butanediol diglycidylether carbonyldiimidazole chloroacetic acid cyanogen bromide cyclohexylmorpholinoethylcarbodiimide divinylsulphone epichlorohydrin ethoxycarbonylethoxydihydroquinoline ethyldimethylaminopropylcarbodiimide ethyleneglycoldiglycidyl ether
fluoromethylpyridinium paratoluene sulphonate tosyl chloride
tresyl chloride
Ion and arm
amino caproic acid cysteamine Hel diethylamine mercaptoacetic acid mercaptopropionic acid mercaptosuccinic acid sodium (meta)bisulphite sodium sulphite trimethylamine $3.50 $6.40 $3.50 $8.50 $3 1 0 $ 1 57 (95% grade) $200 $ 1 . 1 0 $47 $ 1 200 $ 1 54 $ 1 .00 $ 1 90 $ 1 000 $238 $3290 $6.00 $4250 $8.00 $70 $0.90 $2.20 $5.40 $30 $0.67* $ 1 . 1 0 $ 1 . 1 0
Mixed mode and ionisable 2-( aminomethyl)benzimidazole 2-( aminomethyl)pyridine 3 -( aminomethyl)pyridine 4-( aminomethyl)pyridine 1 -(3-aminopropyl)imidazole 4-(3-aminopropyl)morpholine 3-chloro-4-hydroxypheny1acetic acid 3,5-dichlorosalicylic acid histamine histidine 4-hydroxybenzoic acid 4-hydroxy-3-nitrobenzoic acid 4-hydroxyphenylacetic acid hYdroxythiophenol 2-mercaptobenzimidazo1e 2-mercapto- 1 -methylimidazole 2-mercaptopyridine 4-mercaptopyridine 3-nitrotyrosine thiolacetic acid# tyramine Hel 4-vinylpyridine#
# raw materials for mercaptoethylpyridine synthesis
$588 $75 $24 $57 $40 $ 1 6 $ 1 1 5 $86 $530 $47 $5.70 $7 1 $90 $355 $6. 1 0 $74 $ 1 1 0 $ 1 020 $620 $9.90 $270 $ 1 0
APPENDIX 2 : Structures of hydrophobic ionisable ligands
2-( arninomethyl)pyridine 3-( aminomethyl)pyridine
4-( aminomethyl)pyridine 2-( arninomethyl)benzimidazole
4-(3-arninopropyl)morpholine 1 -(3-aminopropyl)imidazole
2-mercaptobenzimidazole 2-mercapto- l -methylimidazole
©lSH
N�CH2CH2)SH
U
H2NH2 . N I H histamine HO OOH 3,5-dichlorosalicylic acid 4-hydroxy-3-nitrobenzoic acid HO-Q-c
OOH 4-hydroxybenzoic acid 3-nitrotyrosine HO-Q-
CH2CH2NH2 tyramine HO 3,5-dibrODlotyrarnll1e Cl HO�
H2COOH 3-chloro-4-hydroxyphenylacetic acid HO-Q-c
H2COOH 4-hydroxyphenylacetic acid histidineREFERENCES
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