Abstract: The present invention relates to a method for analysing protein variants of a recombinant protein of interest, such as antibodies or Fc-fusion proteins, in a liquid sample of a mammal. Specifically the method comprises a step of affinity purifying the recombinant protein of interest from the sample together with an internal standard, and analyzing the protein variants using an analytic separating method such as HPLC, capillary electrophoresis or MS. This method is particularly suited to measure pharmacokinetic parameters of a recombinant protein of interest, such as a biopharmaceutical, in a mammal in clinical or pre-clinical studies. It allows for the use of a small sample volume and the possibility to operate with high throughput, such as in a 96-well plate sample preparation. It also provides high sensitivity and allows analysis of protein variants individually.

Abstract: The present invention uses anthranilic acid (2-AA) to label N-glycans prior to separation using a reversed-phase liquid chromatography (RP-LC) column under acidic conditions using formic acid. Negatively charged 2-AA offers stronger retention on the reversed phase column than 2-aminobenzamide (2-AB) in RP-LC and allows efficient ionization and detection of 2-AA labeled N-glycans. The acidic conditions used for the RP-LC leads to an efficient separation of acidic 2-AA N-glycans carrying terminal sialylation without the need for an ion-pairing reagent. The present invention may be used with RP-nano-LC-MS and a 96-well plate sample preparation, which allows attomolar sensitivity and high throughput.

Abstract: The present invention relates to a method for analyzing glycans of a recombinant glycoprotein in a liquid sample of a mammal. Specifically the method comprises a step of affinity purifying the recombinant glycoprotein from the sample, enzymatically releasing a glycan containing fragment from the immobilized glycoprotein, adding a reference standard containing isotopically labeled glycans, fluorescently label the glycans and analyzing the glycans using LC-MS. The present invention also relates to a method further comprising analyzing the glycans of the immobilized recombinant glycoprotein fragment, further comprising a pre-clearing step of the liquid sample, and releasing the glycans from the immobilized recombinant glycoprotein fragment.

Abstract: The use of anthranilic acid (2-AA) to label N-glycans prior to separation using a reversed-phase liquid chromatography (RP-LC) column under acidic conditions using formic acid is described herein. Negatively charged 2-AA offers stronger retention on the reversed phase column than 2-aminobenzamide (2-AB) in RP-LC and allows efficient ionization and detection of 2-AA labeled N-glycans. The acidic conditions used for the RP-LC leads to an efficient separation of acidic 2-AA N-glycans carrying terminal sialylation without the need for an ion-pairing reagent. The method and compositions described herein may be used with RP-nano-LC-MS and a 96-well plate sample preparation, which allows attomolar sensitivity and high throughput.