Synthetic oligonucleotides play an important role as novel therapeutic agents.
One of the most important, but also very time-consuming steps in synthetic oligonucleotides production is their purification. Due to their high-resolution power, reversed-phase and ion-exchange chromatography are the most widely used techniques for these purposes. For the reversed-phase separations oligonucleotides need to be kept as 5'-O-dimethoxytrityl derivatives until the purification process is completed and only then the detritylation takes place. Both these steps lower the yield of the production process. In the contrary, ion-exchange chromatography offers applications to deprotected oligonucleotides directly and that is the reason why this chromatography mode is more preferred.
Convective Interaction Media (CIM) are newly developed polymerbased monolithic supports allowing high resolution separations which can be carried out within seconds in the case of analytical units - disks. This is due to predominantly convective mass transport of biomolecules between the mobile and stationary phase and very low dead volumes. Additionally, the dynamic binding capacity is not affected by high flow rates.
In this work weak (DEAE) anion-exchange CIM supports have been successfully applied for the analysis and purification of synthetic oligonucleotides.
Found recently serine protease called, as tissue plasminogen activator (t-PA) is able to dissolve efficiently the blood clots. Thus this protein seems to be extremely useful in clinical practice in the cases of heart attack victims.
Real process of fibrinolysis in human blood system represents very complicated network of simultaneous biological events. It is clear that t-PA has a branched set of functional complements with their own, and probably different, affinity to this enzyme. It seems to be possible and quite interesting to investigate all these pairs separately creating them in vitro. At the same time, it is clear that the affinity chromatography approach could become as the most convenient way to create such biological pairs.
The recently developed High Performance Membrane (Monolith) Chromatography (HPMC) is quite promising in this regard, because of its high capacity and selectivity, combined with low backpressure and short operation times. Due to the inherent speed of the isolation it facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences.
CIM® supports are novel monolithic chromatographic supports. In contrast to conventional particle based chromatographic supports they consist of a single porous polymer. The pores form a highly interconnected network, which enables the flow of the mobile phase through the monolith. Molecules to be separated are transported to the surface by the convection. Since the diffusion is not a bottleneck any more, also the resolution and the dynamic capacity of the monolith are flow independent and an average analysis time is typically below one minute. Furthermore, CIM® columns were successfully applied for the purification of proteins directly from the fermentation broth.
Manganese peroxidases (MnP) and lignin peroxidases (LiP) are a family of glicosilated hemo-proteins, which are excreted into the growth medium during the idiophasic growth of the white rot fungus Phanerochaete chrysosporium. They are both involved in the lignin degradation. For their analysis and separation from the growth medium, HPLC is commonly applied. Besides the separation by Na-acetate concentration gradient (2), also the chromatofocusing can be used (3). A fast method for LiP isoenzyme separation from the growth medium of P. chrysosporium using CIM™ QA disk monolithic columns has been recently developed (1). A modified method was tested on the growth medium containing MnP isoenzymes.
The aim of our work was to study the direct monitoring and purification of proteins from the fermentation broth using ion-exchange CIM® supports. Therefore, we studied the possibility of monitoring and purifying lignin peroxidase extracelular protein isoforms produced by the fungus Phanerochaete chrysosporium. These isoenzymes which also differ in their catalytic properties are able to partially depolymerize lignin and to oxidise several xenobiotics.
The white rot fungus Phanerochaete chrysosporium under nitrogen or carbon limitation produces extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidize several xenobiotics (DDT, PCB, PAH, etc.). By HPLC separation and isoelectric focusing multiple molecular forms of LiP have been isolated from the culture filtrate. For the isolation of LiP from the growth medium, mostly the HPLC technique with ion exchange Mono-Q or DEAE columns is used. The medium should be dialyzed before separation and usually also concentrated. Medium freezing is used to remove mucilaginous polysaccharides which disturb separation. The whole procedure is time consuming and information about isoenzyme content and their relative amounts in the growth medium is delayed for at least 1 day. HPLC separation itself lasts nearly an hour. For the separation of LiP isoenzymes from the culture filtrate, we used the monolithic stationary phase with weak (DEAE-diethylamine) and strong (QA-quaternary amine) ion exchange groups commercially available under trademark CIM (Convective Interaction Media). CIM supports are glycidyl methacrylate based monolithic porous polymer supports. As such they differ from conventional particle shaped chromatographic supports. The liquid is forced to flow through the support channels. Molecules to be separated are transported mainly by convection resulting in travelling times shorter for at least an order of magnitude. As a consequence the resolution as well as the binding capacity remain unaffected with the flow rate and a shorter analysis time can be achieved. This effect is even more pronounced in the case of large molecules such as proteins, which have a low diffusion coefficient. As such, CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.
Convective Interaction Technology (CIM®) offers a number of benefits for the purification of large molecules in comparison with conventional chromatography. The innovative matrix, cast as a single homogeneous piece, means that monolithic columns have a high pressure tolerance and allow fast operating flow rates.
Because the matrix structure is composed of large pores, mass transfer is essentially convective in contrast to conventional chromatography beads, where mass transfer is essentially diffusive. Therefore, CIM can be used at high flow rates without compromising binding capacity.
For these reasons, a monolithic column with anion exchange properties (CIM® QA) was selected to purify a very large protein (8 Mega Dalton) extracted from a marine mollusc.
Because 150 g of protein was required to perform preclinical trials, a scale-up of the process had to be designed and implemented. Early stage process development was carried out on an 8 mL column to determine the column loading capacity as well as the yield and the process reproducibility.
To improve binding on the column, stabilising agents had to be removed prior to this purification step. The protein had been observed to precipitate within hours of the removal of these reagents. Therefore, a suitable time frame for protein processing had to accommodate this instability.
Isolation and purification of proteins, peptides and polynucleotides as well as fractionation of biological mixtures are of great importance both for the solution of theoretical problems in chemistry and biology and the
realization of practical plans connected, in particular, with the production of medicines on the basis of large biomolecules. An important problem in the production of biological substances for medicine is to work out the step of their isolation and fine purification, e.g. creation of high performance separation methods, particularly, the chromatographic techniques. Here, fast and efficient affinity separations based on dynamical interaction of biocomplements play very important role.
High Performance Membrane (Monolith) Chromatography (HPMC) allows to solve all problems of High Liquid Chromatography (HPLC) demonstrating a number of number of distinct advantages. A small thickness of separation layer and opened structure of throughput channels where the separation takes place cause minimum difussion resistance for normal mass transport of the substances as well as low working back pressure and thus, the possibility of use of high elution flow rates.
In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immonoglobulin G represent more than 75 % of all abundance proteins. There are many strategies developed for an efficient removal of these two main proteins, the majority of them rely on highly selective, yet expensive affinity techniques. In this work an affinity monolithic column was used for the depletion of IgG. For the removal of HSA we tested an alternative - complementary approach, where an ion-exchange mode was used as one of the depletion steps. the results were compared to the ones obtained by by using the prseudoaffinity columns.
White rot fungus Phanerochaete chrysosporium produces under nitrogen limitation extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidise several xenobiotics (DDT, PCB, PAH, ) and synthetic dyes. Trough HPLC separation and isoelectric focusing multiple molecular forms of LiP have been determined and isolated from the culture filtrate. Depending on growth conditions, separation technique, strain employed and culture age 2-15 different LiP izoenzymes were observed in culture media of Phanerochaete chrysosporium. They are structurally similar but differ in stability, quantity and in catalytic properties. For the isolation of LiP from growth medium, mostly the procedure employing HPLC ionexchange columns as shown on Scheme 1 is used. For the separation of LiP isoenzymes from the culture filtrate, we used CIM (Convective Interaction Media) units. Their advantage is very fast separation of macromolecules due to their particular threedimensional structure. In contrast to particle supports containing closed pores, CIM units consist of monolith porous material containing flow through pores. Therefore, macromolecules to be separated are transported to the active site by convection rather than by diffusion. As a consequence, the separation resolution and dynamic binding capacity are flow independent. As such CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.