Carbohydrate composition analysis of glycoproteins by HPLC using highly fluorescent anthranilic acid (AA) tag.

Oligosaccharides in glycoproteins by their very nature influence many aspects of protein function, e.g., half-life and activity/potency. Recombinant IgGs constitute a major portion of therapeutic proteins. Though the glycans in IgGs account for about 2% of the total weight, they influence biologic activity apart from antigen binding. Characterization of the carbohydrates is not only a regulatory requirement but it may allow understanding of structure-function of proteins. Current advances in analytical techniques permit structural elucidation of small quantities of glycoproteins. At a fi rst glance monosaccharide analysis may provide insight into the types of glycosylation similar to information afforded by amino acid composition. It is the only stand-alone technique by which individual sugar residues can be identifi ed and quantitated (mol/mol). Fluorescent anthranilic acid (AA) has been extensively used as a high sensitivity detection tag for carbohydrates. HPLC methods with fl uorescence detection described in this chapter are suitable for the analysis of monosaccharides (including sialic acids) on a routine basis. AA is used for the determination of hexoses and hexosamines, and o-phenylenediamine for sialic acids. These methods were validated and found to be highly reproducible compared to HPAEC-PAD and CE methods.

Monitoring glycosylation of therapeutic glycoproteins for consistency by HPLC using highly fluorescent anthranilic acid (AA) tag.

Majority of protein drugs in development today are glycoproteins e.g. recombinant antibodies expressed in various cell lines. Oligosaccharides through conformational changes can modulate therapeutic value (potency) of glycoproteins e.g. complement dependent cell cytotoxicity (CDCC) and antibody-dependent cell cytotoxicity (ADCC) activities of MAbs. Carbohydrate structure analysis in detail is an integral part of protein drug characterization. This not only allows understanding of carbohydrates, but may allow deeper insight into the structure-function of the whole protein molecule. Oligosaccharide mapping by HPLC with fluorescence detection is a powerful technique that sheds considerable light into understanding of glycan structures with minimal effort. Oligosaccharide analysis using pulsed amperometric and/or chromophore detection methods lack resolution, sensitivity and ease of operations. In addition, these older methods are not highly reproducible. Simple labeling chemistry of anthranilic acid (AA) described here provide robust methods with the highest sensitivity and resolution for oligosaccharide analysis. Further, post-chromatography techniques such as mass spectrometry and NMR are amenable to this AA technology for detailed structure analysis.

Comparability and monitoring immunogenic N-linked oligosaccharides from recombinant monoclonal antibodies from two different cell lines using HPLC with fluorescence detection and mass spectrometry.

One of the most important structural features of recombinant monoclonal antibodies produced in mammalian cells is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a multitude of ways affecting distribution, efficacy, and immunogenicity. High mannose, alpha-gal and other oligosaccharide species are highly immunogenic and in most cases should be minimized during manufacturing. A recombinant monoclonal antibody, h5G1.1, was produced in NS0 and CHO cell lines and tested to identify changes in the N-linked oligosaccharide profiles caused from a change in cell line. Traditional peak analysis using HPLC with fluorescence detection was augmented by mass spectrometric analysis. Nano LC-MS following tryptic digestion corroborated HPLC findings of the presence of several alpha-gal oligosaccharide species in the recombinant IgG (rIgG) from NS0 cell line. Both cell lines possessed rIgGs with complex and small amounts of high mannose glycans.

Mass spectrometry and HPLC with fluorescent detection-based orthogonal approaches to characterize N-linked oligosaccharides of recombinant monoclonal antibodies.

A number of HPLC and mass spectrometric techniques are used to characterize post-translational modification in recombinant monoclonal antibodies (MAbs) using the intact glycoprotein and free glycans. LC separation utilizing fluorescent detection technique allows tentative structural assignment of MAb oligosaccharides. Intact molecular weight analysis via electrospray allows for an accurate mass determination and observation of the native glycoform mass envelope. N-linked oligosaccharides are then analyzed by MALDI-ToF. Their structures are further confirmed by analyzing the fragmentation patterns formed by MS/MS. All these techniques provide useful information when performed in isolation. However, the combined information allows for definitive and robust characterization of the N-linked glycans from recombinant MAbs.

Prospects and limitations of the rational engineering of fibrillar collagens.

Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large-scale production of nonmodified recombinant collagens; however, engineering of novel collagen-like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen-like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi-D4 collagens defined as collagen-like proteins, consisting primarily of a tandem of the collagen II D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase 1 cleavage site present in the D4 period. We demonstrated that the recombinant collagen alpha-chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N-proteinase and procollagen C-proteinase. The nonmutated multi-D4 collagen had a normal melting point of 41 degrees C and a similar carbohydrate content as that of control. In contrast, the mutant multi-D4 collagen had a markedly lower thermostability of 36 degrees C and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi-D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences.