N
-Glycosylation is a common post-translational modification of monoclonal antibodies with a potential effect on the efficacy and safety of the drugs; detailed knowledge about this glycosylation is ...therefore crucial. We have developed a reversed-phase liquid chromatographic–mass spectrometric method, with different fluorescent labels, for analysis of
N
-glycosylation, and compared the sensitivity and selectivity of the methods. Our work demonstrates that anthranilic acid as fluorescent label in combination with reversed-phase liquid chromatography–mass spectrometry is an advantageous method for identification and quantification of neutral and acidic
N
-glycans. Our results show that mass spectrometry-based quantification correlates with quantification by fluorescence. Chromatographic discrimination between several structural glycan isomers was achieved. The sharp peaks of the eluting anthranilic acid-labeled
N
-glycans enabled on-line mass spectrometric analysis of even low-abundance glycan species. The method is broadly applicable to
N
-glycan analysis and is an orthogonal analytical method to the widely established hydrophilic-interaction liquid chromatography of 2-aminobenzamide-labeled
N
-glycans for characterization of
N
-glycans derived from biopharmaceuticals.
Glycosylation of recombinant human erythropoietin (rHuEPO) is a post‐translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends ...on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE‐ESI‐TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE‐ESI‐MS separation method for enzymatically released underivatized N‐glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH2 are successfully characterized. Information of the protein‐backbone molecular mass has been combined with results from peptide analysis (revealing information about O‐glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.
The paper presents data on the primary structure of the glycan variants present in human antithrombin (AT) isoforms obtained from a plasma pool. The analysis is conducted on the level of ...glycopeptides gained by tryptic digestion. The glycopeptides were pre-separated by lectin-affinity chromatography and analyzed by means of electrospray ionization–tandem mass spectrometry involving collision-induced dissociation. Variations of the canonical biantennary complex-type structure were present with relative abundances of about 1–5% and most of them were found site-specifically. Core fucosylation was observed at one single glycopeptide only (peptide containing N
155), triantennary glycan structures with two glycopeptides (containing N
155 and N
135). Deficiency of one terminal sialic acid was observed as not site-specific. Fucosylation was not yet reported to be present in human AT from plasma, opposite to recombinant human AT from baby hamster kidney cells, which was reported as fully core fucosylated. In total, the variability in the carbohydrate structure of plasma derived AT appears as being quite limited. This might be of significance in the context of the reported correlation between glycosylation and physiological activity.
The carbohydrate structures of five isoforms of alpha-AT and two isoforms of beta-AT were determined by applying capillary zone electrophoresis (CZE) on-line coupled to electrospray ionization-mass ...spectrometry (ESI-MS) using an ion-trap analyzer. For the AT preparations gained from a plasma pool at least semiquantitative information on the isoform-distributions could be gained. Unlike to the commonly used approaches starting from enzymatically treated glycoproteins, this approach deals with intact proteins. The high accuracy of the molecular mass determination obtained by the ion-trap analyzer allows one to calculate and ascertain the carbohydrate composition assuming no variations in the protein moiety of AT and to exclude or confirm the presence of the potential post-translational or other modifications. Therefore, the direct coupling of CZE with ESI-MS does not only represent a fast alternative technique to two-dimensional electrophoresis (2-DE) but serves as a method which provides structural information complementary to that gained from peptide mapping methods.
The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the ...gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP‐HPLC‐ESI‐MS equipped with a quadrupole ion‐trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of α‐AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF‐band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main‐isoform of α‐AT but missing one terminal SA. There was no site specificity found for the mono‐sialo structure. The isoform at pI 5.31 is the major isoform of β‐AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than β‐AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance.
Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass ...determination of intact isomeric glycoproteins based on capillary electrophoresis‐electrospray‐time of flight‐mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on‐line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.
ABSTRACT
Purpose
In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during ...pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.
Methods
Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program.
Results
The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate.
Conclusion
The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.
N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of ...biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.
The carbohydrate structures of five isoforms of α‐AT and two isoforms of β‐AT were determined by applying capillary zone electrophoresis (CZE) on‐line coupled to electrospray ionization‐mass ...spectrometry (ESI‐MS) using an ion‐trap analyzer. For the AT preparations gained from a plasma pool at least semiquantitative information on the isoform‐distributions could be gained. Unlike to the commonly used approaches starting from enzymatically treated glycoproteins, this approach deals with intact proteins. The high accuracy of the molecular mass determination obtained by the ion‐trap analyzer allows one to calculate and ascertain the carbohydrate composition assuming no variations in the protein moiety of AT and to exclude or confirm the presence of the potential post‐translational or other modifications. Therefore, the direct coupling of CZE with ESI‐MS does not only represent a fast alternative technique to two‐dimensional electrophoresis (2‐DE) but serves as a method which provides structural information complementary to that gained from peptide mapping methods.
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