Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Polymers from only ethylenic monomers or processes of...
Reexamination Certificate
2000-09-18
2002-12-03
Pezzuto, Helen L. (Department: 1713)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Polymers from only ethylenic monomers or processes of...
C526S310000, C526S312000
Reexamination Certificate
active
06489421
ABSTRACT:
The present invention relates to a linear water-soluble quaternary ammonium polymer, a process for the preparation of this polymer, the use of this polymer as a displacer in the displacement mode of chromatography and a method for separating biomolecules through the displacement mode chromatography using this polymer.
The majority of the separation or isolation procedures used in the pharmaceutical field, or other related fields such as the biochemical, the biotechnological and the chemical fields, rely on chromatography, i.e. a differential separation between two phases, a stationary one (usually solid) and a mobile one (usually fluid).
Elution mode chromatography is the most commonly used mode of chromatography. In this method, the sample containing different components to be separated is adsorbed on the stationary phase. The mobile phase, called eluent, is chosen such that the components bind reversibly onto the stationary phase. As the eluent is flowing over the stationary phase, the different components migrate along the column at a speed which reflects their relative affinity for the stationary phase.
Biomolecules such as proteins are generally purified by gradient elution mode chromatography using ion-exchange adsorbents as stationary phase. The elution involves changing the pH of the buffer solution passing through the column or, more common, increasing the salt concentration in the buffer solution passing through the column. When the pH of the solution is changed, the electric charges on the proteins or the ion-exchange adsorbent material are changed and the proteins are released. With an increase of the salt concentration, the salts weaken the bonds between the proteins and the ion-exchange adsorbent material to release the proteins. As the salt level is gradually increased, the proteins having the smaller number of charges, respectively the lower charge density (mass to charge ratio) will generally be released and eluted first and those with the larger number of charges, respectively the higher charge density, will be released later.
Displacement chromatography is a special mode of chromatography. The basic principle of chromatography still applies, but in this case the driving force behind the separation is the push of an advancing front of a so-called displacer, rather than the (increasing) elution strength of a mobile phase. Under the influence of an advancing displacer front, the substance mixture is resolved into consecutive zones of the pure substances.
The displacer is a substance with a higher binding affinity to the stationary phase than the components to be separated. As the displacer front advances, the number of stationary sites available to the binding components are continuously decreased, which engenders competition for the binding sites between the displacer and the components and also between the components themselves. Under ideal conditions, the more strongly bond ones displace the more weakly bond ones until all substances are focused into consecutive individual zones of pure substance that leave the column at the speed of the advancing displacer front in the order of the stationary phase affinities.
One of the important distinctions between displacement and elution mode chromatography is that, in displacement mode chromatography, the displacer front always remains behind the consecutive feed zones, while, in elution mode chromatography, the eluent or desorbent moves through the feed zones with the components to be separated.
The displacer is a unique feature of displacement mode chromatography. At the same time, the choice of the displacer has consequences not only for the success, but also for the economic soundness of the final method. Mathematical simulations, which take into account concentrations in the mobile and stationary phases, allow some predictions concerning the necessary displacer characters for a given substance mixture to be separated.
A displacement mode chromatography method for purifying proteins using, as a stationary phase, a cation-exchange resin and, as a displacer, a cationic species is disclosed in U.S. Pat. No. 5,478,924. These cationic species are poly(quaternary ammonium) salts having a dendritic framework formed by reiterative reaction sequences starting from pentaerythritol. Their structure consists, on the one hand, in a hydrophobic interior zone containing ramifications connected to the initial core pentaerythritol and, on the other hand, in a hydrophilic exterior surface region bearing the quaternary ammonium groups.
The first and second generation pentaerythritol-based dendrimers, all terminated with trimethylammonium groups, having respectively a molar mass of about 2,000 g/mol and 6,000 g/mol, are described as being effective for the purification of a two-protein mixture, i.e. &agr;-chymotrypsinogen A and cytochrome C.
However, it is mentioned that, due to the presence of impurities in the first generation dendrimers contributing to the desorption of the proteins and depression of their isotherms, the cytochrome C zone is considerably less concentrated in relation to the &agr;-chymotrypsinogen A zone.
The preparation of these poly(quaternary ammonium) salts is relatively tedious, time and cost consuming, involving low overall yield multi-step synthesis. The level of purity and homogeneity cannot be high, particularly for the high molar mass second generation pentaerythritol-based dendrimers.
It is also reported that the “zero” generation pentaerythritol-based dendrimers, terminated with trimethylammonium groups and having for instance a molecular weight of 620 g when X=Cl, are equally effective for the separation of the two proteins.
However, S. D. Gadam and S. M. Cramer have reported, in
Chromatographia,
1994, 39, 409-18, that the behaviour of such low molar mass displacers depends to a much higher degree on the chromatographic conditions, for example the salt content of the mobile phase. As a consequence, the switch from displacer to elution promoter is more likely in the case of these substances.
One of the aims of the present invention is to provide a low molar mass polymer capable to be used as a cationic displacer, i.e. having high solubility in an aqueous carrier with a high binding tendency towards a cation-exchange stationary phase. This particular polymer has to be easily available on a large scale with high level of purity and homogeneity.
To this effect, the present invention relates to a linear water-soluble quaternary ammonium polymer having a homopolymeric chain of repeating unit of general formula (I):
where each of R
1
and R
2
independently represents a member selected from the group consisting of linear or branched alkyl, hydroxyalkyl, alkoxyalkyl groups having from 1 to 6 carbon atoms, X
−
represents an anion, n is an integer comprised between 30 and 220. Furthermore, the polymer has a molar mass distribution of less than or equal to 1.5.
The anion represented by X
−
is selected from the group consisting of F
−
, Cl
−
, Br
−
, NO
3
−
, OH
−
, HSO
4
−
, ½ SO
4
2−
, CH
3
COO
−
.
The preferred linear water-soluble quaternary ammonium polymer has the above general formula (I), where both R
1
and R
2
represent a methyl group and X
−
represents Cl
−
, n being an integer comprised between 30 and 220. Such a preferred polymer has a number average molar mass comprised between about 4,800 g/mol and about 35,000 g/mol and a molar mass distribution of less than or equal to 1.5.
Since the 1950s, a number of preparations of linear water-soluble quaternary ammonium polymers having a homopolymeric chain of the same above-mentioned repeating unit have been described. They are based on polymerisation reactions employing various initiation methods, including radically, ionically, or X-ray induced polymerisation, and involving an alternating intra-intermolecular chain propagation.
However, most of these syntheses are oriented towards high molar mass polymers having a number average molar mass of up to 200,000 g/mol,
Christine Wandrey
Ruth Freitag
Browning Clifford W.
Ecole Polytechnique Federale de Lausanne
Pezzuto Helen L.
Woodard Emhardt Naughton Moriarty & McNett
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