Liquid Chromatography

LC methods can distinguish individual oligosaccharides and quantify the individual oligosaccharides. There are two approaches for preparing the glycoprotein prior to the chromatography step. In one approach, the glycoprotein is cleaved into glyco-peptides with a peptidase, and in the other, the oligosaccharides are cleaved from the glycoprotein. For the first method, the glycopeptides are separated by reversephase high-performance liquid chromatography (RPHPLC). Peptidases are selected such that only one oligosaccharide is associated with any peptide. This analysis technique requires that the amino acid sequence is known in order to predict the resulting peptides. Trypsin is the most common peptidase used due its broad specificity (120). Researchers who initially characterized recombinant b-trace protein used trypsin gly-copeptides (52). The effect of different culture conditions on glycosylation have also been investigated using trypsin digested-glycopeptides to characterize the N-oligo-saccharides (75).

Another very sensitive LC method uses Biogel 4 and radiolabeled glycoproteins that have been degraded by either hydrozinolysis or PNGaseF. Biogel 4 and hydro-zinolysis were used to characterize the glycosylation of IgM obtained from cells with different physiological states: ascites, airlift fermenters, and human blood. The IgM obtained from the different culturing techniquess had unique glycosylation patterns (68).

LC coupled to MS (LC-MS) can provide detailed glycosylation by analyzing the glycopeptides or oligosaccharides (149,150). For glycopeptides, LC is used to separate the glycopeptides in a proteolytic digestion, and the glycosylation state at each site is determined by MS. Ohta et al. (149) were able to detect site-specific glycosylation differences in EPO obtained from three different sources. Additionally, LC-MS detected acetylation, sialylation, and sulfation differences in the three EPO sources. Oligosaccharide mapping by LC-MS can also determine carbohydrate heterogeneity. Kawasaki et al. (150) used LC-MS to compare the carbohydrate characteristic of EPO obtained from different sources.

HPLC Methods

HPLC methods are commonly used to characterize the glycosylation of a glycoprotein. Usually, the glycopeptides, oligosaccharides, or monosaccharides are analyzed. Anion-exchange HPLC separates charged groups, such as sialic acid; however, this method requires labeling and often the resolution is poor (129). Weak anion-exchange HPLC was used to demonstrate that the sialic acid content of EPO produced in CHO was reduced in the presence of elevated ammonia concentration (137). Labeled monosaccharide can be separated by RPHPLC with excellent resolution. Labeled oligosaccharides can also be detected by RPHPLC. One advantage of RPHPLC is that the buffers are salt-free, which is more compatible with MS systems. Typical labeling agents are anthranilic acid (ABA) for monosaccharides, phe-nylisothiocyanate (PITC) for amino sugar alcohols, and o-phenylene diamine (OPD) for sialic acids. ABA and OPD are detected by fluorescence, whereas PITC is detected by UV absorption. A monosaccharide sample is labeled separately with each dye to detect the different sugars. Typically, 5-50 mg of glycoprotein are hydro-lyzed in 20% TFA at 100°C for 6-7 hr to obtain the monosaccharides (129,142).

Xu and Cacia (130) developed an RPHPLC method to monitor the manufacturing process development of TNK-tPA (a genetically engineered variant of tPA). The RPHPLC method was able to identity and quantify the tPA glycoforms. Wu (141) used RPHPLC to separate tPA glycopeptides and peptides prior to CE. Thus, macroheterogeneity and microheterogeneity were determined. Wan et al. (143) developed an HPLC-ES-MS method for the direct analysis of the terminal Gal content of a glycoprotein. Cell culture media were directly injected into the HPLC followed by the ES-MS. The results obtained were comparable with the CE methods used to analyze the purified glycoprotein.

High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection

Oligosaccharides and monosaccharides can be analyzed by HPAEC-PAD. Mono-saccharides are obtained from the glycoprotein or glycopeptide by acid hydrolysis. Oligosaccharides are cleaved from the glycoprotein or a glycopeptide pool either enzymatically or chemically (18). The enzyme PNGaseF cleaves N-oligosaccharides from the glycoprotein or glycopeptide at the N-glycosidic bond (52,74,120). Hydra-zinolysis can also be used to cleave both N- and O-oligosaccharides from the glyco-protein or glycopeptide (18,120). Monosaccharides may also be obtained by acid hydrolysis of the oligosaccharides.

Once the oligosaccharides have been liberated from either the glycoprotein or the glycopeptide, HPAEC-PAD can be used to rapidly and sensitively determine and quantify the glycosylation pattern of the glycoproteins, often termed the oligosaccharide profile (18,87,120,144). HPAEC-PAD exploits the electrochemical properties of carbohydrate; however, every sugar electrochemical response is different. To quantify oligosaccharides by HPAEC-PAD, an identical external standard must be used to calibrate the peak area. This method has been used detect both macrohe-terogeneity and microheterogeneity. Monosaccharides can be separated and quantified by HPAEC-PAD. Monosaccharide standards are required to quantify the monosaccharides for all anticipated sugars, since each monosaccharide peak height has a different proportionality based on mass.

Gawlitzek et al. (74,75) used HPAEC-PAD to demonstrate the effect of different culture conditions on the N-glycosylation of recombinant IL-2. Robinson et al. (69) and Patel et al. (66) both observed N-glycosylation pattern changes for an MAb production with different culture methods and/or conditions. Andersen et al. (87) demonstrated that HPAEC-PAD was also a sensitive method for determining O-gly-cosylation. They also were able to correlate increased ammonium ion concentration with a decrease in the proportion of a(2,6)-linked sialic acid, compared to terminal GalNAc for GM-CSF secreted by CHO cells (87). Kunkel et al. (104) demonstrated decreased the galactose content of the oligosaccharides by HPAEC-PAD.

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