This Application Note demonstrates the potential of comprehensive two-dimensional liquid chromatography using the Agilent 1290 Infinity 2D-LC Solution with DAD detection for the analysis of monoclonal antibody tryptic digests.
In this study, hen egg white lysozyme was used as a model protein for PEGylation using N-terminal specific mPEG propionaldehyde (PEG aldehyde) in presence of cyanoborohydrate. A method for the analytical-scale purification of PEG lysozyme was developed using an Agilent 1260 Infinity Bio-Inert LC System and an Agilent Poroshell 120 SB-C18 column. Analytical-scale Fraction Collector with peak-based fraction collection was employed to collect the purified PEG lysozyme. The fractions were then re-analyzed by RP HPLC and SEC to demonstrate the homogeneity of the purified PEG conjugate.
Tween 20 has various beneficial properties for biological analysis, such as stabilizing proteins, solubilizing protein membranes, and preventing nonspecific binding of primary and secondary antibodies. Therefore, Tween 20 is often present in samples of monoclonal antibodies that require analysis using techniques such as SEC. This app note investigates whether the presence of Tween 20 detergent directly in the sample and in the eluent during multiple injections of an IgG sample onto an Agilent Bio SEC column affects the analysis or the columns.
Analytical-scale purification of PEG lysozyme was developed using an Agilent Bio-LC and Poroshell column. PEGylated lysozymes were re-analyzed by SEC and RP HPLC. The results indicated that purified PEG lysozyme was homogenous.
This Application Note shows the determination of molecular weight, size, and relative concentration of protein samples after SEC with the Agilent 1260 Infinity Bio-inert Quaternary LC System with Multi-Detector GPC/SEC.
The authors describe pH-based separation of acidic and basic charge variants for mAbs using the Agilent 1260 Infinity Bio-inert Quaternary LC System and an Agilent BioMAb PEEK 4.6 x 250 mm IEX column, featuring a special particle coating designed for charge-based separation of MAbs.
In this note, we describe a fast and efficient liquid chromatography-TOF mass spectrometry approach to characterize an IgG derived from CHO cells. An Agilent Poroshell 300SB-C3, 5 um superficially porous IgG1 column was used to obtain accurate glycoform masses of an intact IgG1, with a subsequent analysis of papain-digeste3d IgG1 to obtain site-specific glycosylation profile information of the Fc region for further characterization of IgG1 heterogeneity. Additionally, a subsequent mAb-glyco chip LC/MS analysis was used to build a glycan accurate mass database for additional validation of the glycans identified in the TOF mass analysis.
A method using LC/MS is described for improving the analysis PEGylated peptides and proteins using the post-column addition of a charge stripping agent to obtain simpler mass spectra for detailed structural characterization.
Reversed-phase UHPLC/HPLC is routinely used for peptide mapping, but if the digest contains hydrophilic peptides, such as glycopeptides, valuable information can be missed. This note demonstrates peptide mapping of digested glycoprotein erythropoietin (EPO) protein using an Agilent ZORBAX Rapid Resolution High Definition 300-HILIC 1.8 ?m LC column and a TOF MS. Taking advantage of the high organic solvent system of the mobile phase for HILIC (hydrophilic interaction chromatography), the glycopeptides are effectively dissolved, retained and separated by the HILIC column. This app note includes the separation, sequence coverage and comparison of reversed-phase and HILIC data to demonstrate the utility of HILIC as an orthogonal and complementary approach to RPLC/MS for peptide analysis.