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US 12,553,842 B2Granted
Determination of modification degree of therapeutic proteins using 1H-NMR spectroscopy
AssigneeTakeda Pharmaceutical Company Limited
InventorsJean-Baptiste Farcet, Juergen Siekmann
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Abstract
This invention provides, among other things, methods for determining the average number of first polymers covalently attached to protein-first polymer conjugates in a solution.
Claims21
A method for determining the average number of first polymers covalently attached to protein-first polymer conjugates in a solution, comprising:
- a) performing a 1H nuclear magnetic resonance measurement on the solution comprising the protein-first polymer conjugates;
- b) processing said 1H nuclear magnetic resonance measurement;
- c) integrating said 1H nuclear magnetic resonance measurement at a first-type proton peak, wherein said first-type proton peak comprises a signal generated by at least one proton on the side chain of at least one amino acid in the protein-first polymer conjugates;
- d) calibrating said result of c);
- e) integrating said 1H nuclear magnetic resonance measurement at a second-type proton peak, wherein said second-type proton peak comprises a signal generated by at least one proton in the first polymers;
- f) dividing the result of e) by the number of second-type protons per monomer of the first polymer;
- g) dividing the result of f) by the average number of monomers in the first polymer,
- thereby determining the average number of the first polymers covalently attached to the protein-first polymer conjugates in the solution.
5 more elements
A method for determining the average number of polysialic acid polymers covalently attached to Factor VIII-polysialic acid conjugates in a solution, comprising:
- a) performing a 1H nuclear magnetic resonance measurement on the solution comprising the Factor VIII-polysialic acid conjugates:
- with a temperature of between about 15° C. and 35° C.;
- with from about 16 to about 10,000 scans;
- with a spectral width of from about +10 ppm to about −4 ppm;
- with a recycle delay of from about 0.1 seconds to about 120 seconds;
- and
- with an optimized receiver gain;
- b) processing said 1H nuclear magnetic resonance measurement:
- with Fourier transform;
- with automatic phase correction;
- with automatic baseline correction;
- with a line broadening window function of 0.3 Hz;
- with an exponential methods window function;
- and
- with a DSS axis calibration;
- c) center integrating said 1H nuclear magnetic resonance measurement at a first-type proton peak consisting essentially of signals generated by the 6 protons on the methyl groups of valine and the 6 protons on the methyl groups of leucine and the 6 protons on the methyl groups of isoleucine and the proton on the methine group of isoleucine;
- d) calibrating the result of c);
- e) center integrating said 1H nuclear magnetic resonance measurement at a second-type proton peak consisting essentially of signals generated from an H3a proton on a sialic acid monomer in said polysialic acid polymers;
- f) dividing the result of e) by 1;
- g) dividing the result of f) by the average number of monomers in the polysialic acid polymers,
- and dividing the result of step g) by KNMR, wherein said KNMR is obtained from a standard reference or a calibration curve or an alternate assay.
18 more elements
Dependencies are inferred from the claim text.