Loading patent…
Assigneei3 Membrane GmbH
InventorsKenneth RENFREW, Stephan BRINKE-SEIFERTH
Estimating the term…
The Invention relates to a method for purifying, separating, selecting and/or isolating biomolecules by means of affinity chromatography, wherein a stationary phase containing, in particular selective, binding sites formed by ligands and/or receptors is provided. There is a need to provide a suitable method for purifying, separating, selecting and/or isolating biomolecules in which desorption via change of the pH value, in particular using acidic or alkalic buffer solutions, and or via application of salt solutions is avoided, thus eliminating potential harm of degradation of the separated biomolecules, which especially, if degraded by any means, could provide health risks in medical applications or alike. This might be solved by applying a voltage between two electrodes, in particular with the stationary phase placed between those electrodes.
Method for purifying, separating, selecting and/or isolating biomolecules by means of affinity chromatography comprising the following steps:
The method according to any of the preceding claims, wherein at least one of the electrodes is formed by at least one electrically conductive, in particular metal, coating and/or metallic net, wherein in particular the at least one coating and or net is formed and/or disposed on at least one non-conductive carrier, in particular formed by porous substrate(s), in particular polymeric membrane(s), and/or on the at least one affinity substrate, in particular membrane.
The method according to any of the preceding claims, wherein at least one of the electrodes, in particular the conductive coating and/or metallic net forming the electrode(s), consists of metal, in particular metal coating(s), preferably gold or platin, in particular consisting of a single metal, preferably gold or platin.
The method according to any of the preceding claims, wherein at least one of the electrodes and/or the at least one conductive coating and/or metallic net forming the electrode(s) and/or the at least one non-conductive carrier is/are permeable.
The method according to any of the preceding claims, wherein the stationary phase contains at least one of the, preferably at least two or all of the, electrodes, in particular formed by metal coating(s) and/or the metallic net(s) disposed on the at least one affinity substrate, in particular membrane, and/or on the at least one non-conductive carrier, wherein in particular ligands and/or receptors forming binding sites are immobilized on the at least one electrode contained in the stationary phase.
The method according to any of the preceding claims, wherein the at least one affinity substrate, in particular membrane and/or the at least one non-conductive carrier acts as an insulator between electrodes and/or an insulator is placed between the electrodes, in particular formed by the at least one affinity substrate, in particular membrane and/or the at least one non-conductive carrier.
The method according to any of the preceding claims, wherein the ligands and/or receptors are antigen ligands and/or receptors.
The method according to any of the preceding claims, wherein the ligands and/or receptors, in particular antigen ligands and/or receptors, are proteins or aptamers, in particular oligopeptides or oligonucleotides.
The method according to any of the preceding claims, wherein the ligands and/or receptors are immobilized on, in particular on surfaces, in particular outer and/or inner surfaces of, the stationary phase, in particular the at least one affinity membrane, in particular by EDC or DCC coupling and/or radical coupling, in particular through electronic beam treatment.
The method according to any of the preceding claims, wherein the biomolecules to be purified, separated, selected and/or isolated are proteins, in particular immunoglobulins, or antibodies, in particular, natural or monoclonal antibodies, or enzymes or nucleic acids.
The method according to any of the preceding claims, wherein the first fluid contains at least a first biomolecule and a second biomolecule, wherein the ligands and/or receptors forming binding sites are chosen, so that the first biomolecule has a higher binding affinity to the ligands and/or receptors compared to the second biomolecule, in particular compared to all other biomolecules contained in the first fluid, and/or so that the binding affinity of the second biomolecule, in particular all other biomolecules contained in the first fluid being different from the first biomolecule, is/are low and/or zero.
The method according to any of the preceding claims, wherein, especially during elution by applying a voltage and/or during a least some time during elution by applying a voltage, no elution by means of pH-value amendment is performed and/or wherein any fluid added within the elution step, especially during elution by applying a voltage and/or during a least some time during elution by applying a voltage, has a pH value equal to the first fluid and/or differ in pH value by less than 1.0, in particular less than 0.5, in particular less than 0.2, in particular less than 0.1 compared to pH value of the first fluid.
The method according to any of the preceding claims, wherein no fluids with salt concentrations exceeding 150 mM are added for elution of the bound biomolecules.
The method according to any of the preceding claims, wherein the voltage applied for elution is in the range from 0.1 to 50 volts, in particular from 0.1 to 3 volts, in particular 1.5 to 3.0 volts, in particular to 2.5 volts.
The method according to any of the preceding claims, wherein the stationary phase, consists of at least two specific stationary phases, each specific stationary phase in particular being formed by at least one, in particular a multitude of, specific affinity substrate(s), in particular membrane(s), wherein each specific stationary phase, in particular each specific affinity substrate, in particular membrane, contains specific binding sites formed by ligands and/or receptors, wherein the specific binding sites of a first specific stationary phase, in particular first specific affinity substrate(s), in particular membrane(s), are formed by first ligands and/or receptors and the specific binding sites of a second specific stationary phase, in particular second specific affinity substrate(s), in particular membrane(s), are formed by second ligands and/or receptors, wherein the first and second ligands and/or receptors are different.
The method according to any of the preceding claims, wherein the stationary phase and/or the specific stationary phase(s) each comprise(s), in particular is/are formed by, at least two, preferably a multitude of, affinity substrates, in particular membranes and/or specific affinity substrates, in particular membranes.
Method according to any of the claims wherein at least two stationary phases, in particular specific stationary phases are placed one behind the other in terms of flow direction and/or the first fluid is passed sequentially over the at least two stationary phases, in particular specific stationary phases.
Method according to any of the preceding claims, wherein the affinity substrate(s), in particular membrane(s), and the electrodes and/or the stack is/are pleated and/or wound, in particular together, in particular as a stack.
Affinity chromatography device for purifying, separating, selecting and/or isolating biomolecules comprising a stationary phase characterized in that the stationary phase comprises, in particular is formed by, at least one porous affinity substrate arranged between at least two electrodes, particularly in a stacked arrangement, wherein the at least one affinity substrate comprises at least one substrate as stationary phase with binding sites formed by ligands and/or receptors immobilized on the porous affinity substrate.
Dependencies are inferred from the claim text.