Method for determination of macromolecular multimers
View Patent ↗A method of determining multimers of a macromolecule monomer in a sample containing the macromolecule comprises the steps of (i) determining the total concentration of macromolecule in the sample, (ii) determining by a biosensor-based detection method, especially mass-sensing, the active concentration of macromolecule in the sample, wherein physical characteristics of the macromolecule monomer are used, (iii) comparing the relationship of determined active macromolecule concentration to total macromolecule concentration for the sample with a corresponding relationship for an at least substantially multimer-free macromolecule-containing sample, and (iv) from a resulting difference determining the presence of multimers in the sample. The method may be used in the purification of macromolecules, such as proteins.
1. A method of determining the presence of multimers of a macromolecule monomer in a sample containing the macromolecule, comprising the steps of:
determining the total concentration of the macromolecule in the sample;
determining the active concentration of the macromolecule in the sample by putting the sample in contact with a sensor surface of a biosensor system arranged to perform a biosensor-based detection method, wherein physical characteristics of the macromolecule monomer are used to determine the active concentration;
providing a reference value for the active concentration of a monomer-standard reference-sample with a total concentration of the macromolecule corresponding to the total concentration of the macromolecule determined for the sample
comparing the determined active concentration of the macromolecule in the sample with the reference value for the active concentration of the monomer reference-sample; and
wherein a lower active concentration of the macromolecule in the sample compared to the reference value for the active concentration of the monomer reference-sample provides a measure of the presence and level of multimers in the sample.
2. The method of claim 1 , wherein the biosensor-based detection method comprises mass-sensing.
3. The method of claim 1 , wherein the physical characteristics comprise molecular weight and diffusion coefficient.
4. The method of claim 1 , wherein the determination of active concentration comprises contacting the sample with a sensor surface at varying flow rates under conditions of at least partial mass transport limitation.
5. The method of claim 4 , wherein the determination is performed without the use of a calibration standard.
6. The method of claim 1 , wherein the method is based on evanescent wave sensing, preferably surface plasmon resonance (SPR).
7. The method of claim 1 , wherein the macromolecule is selected from the group consisting of proteins, polypeptides and nucleic acids.