February 2016 | Volume 14 | Number 2 www.bioprocessintl.com www.bioprocessintl.com Covering the Whole Development Process for the Global Biotechnology Industry BioProcess International BioProcess FOCUS ON TECHNICAL ARTICLES Vol.14 No.2 February 2016 February No.2 Vol.14 Facing the 483: What to Do? Detecting Impurities: High- Throughput Method Evaluation BridgingUsing Analytical Glycosidases Methods for to Remove, Trim, or Release and Stability TestingEllen P. Guthrie andModifying Paula E. Magnelli Glycans Using Glycosidases Real EstateModify Challenges Glycans in on Therapeutic Proteins Developing Markets Development of a Plant HCP Assay SPECIAL REPORT: CMC Strategy Forum Tackles Process Impurities 14-2-Cover.indd 1 1/13/16 9:59 AM B IOP ROCESS TECHNICAL Using Glycosidases to Remove, Trim, or Modify Glycans on Therapeutic Proteins Ellen P. Guthrie and Paula E. Magnelli ne of the most common Figure 1: Glycoforms identified by LC/MS analysis of intact Erbitux (cetuximab) digested with PNGase F posttranslational modifications of eukaryotic 13.89 Fucose O proteins is glycosylation. 20 Mannose Glycosylation of proteins can affect 18 Sialic acid (NANA) many biological activities. For 16 GlcNAc 15.12 therapeutic glycoproteins, it can 14 Galactose modify biological activity, targeting, 12 Sialic acid (NGNA) trafficking, serum half life, clearance, 10 18.59 and recognition by receptors (1, 2). For (Fluorescence Trace) (Fluorescence 8 6 Or such reasons, biomanufacturers must 10 6 × monitor and characterize the 4 glycosylation patterns of their 19.41 2 11.34 25.02 recombinant therapeutic proteins (3, 4). Counts 9.33 16.37 24.23 27.50 30.99 34.46 37.87 two 0 Therapeutic proteins have 10 15 20 25 30 35 40 main types of glycosylation : N-linked Time (min) glycans and O-linked glycans (5). Attachment of an N-glycan starts in the endoplasmic reticulum (ER), and Golgi apparatus. Host cell and remove, trim, or modify glycans. where a core nascent glycan is linked cell culture conditions can alter the Glycosidases (glycoside hydrolases) through the side-chain amide nitrogen type of N-glycosylation present on the are enzymes that hydrolyze the on specific asparagine residues of a glycoprotein (from high mannose to glycosidic bonds of complex sugars. protein at sites that have the sequence complex and hybrid N-glycans). Such enzymes are applied in three NXS/T, where X can be any amino O-glycosylation occurs in the Golgi main analytical areas for therapeutic acid residue except proline. N-glycans apparatus. N-glycans have a common glycoproteins: removing glycans for are trimmed and further modified as a core consisting of two analysis, trimming glycans for glycoprotein transits through the ER N-acetylglucosamine (GlcNAc) sequencing, and modifying glycans residues and three mannose (Man) in glycoengineering. Herein we PRODUCT FOCUS: RECOMBINANT residues. But the only common core describe the different enzymes used PROTEINS, GLYCOPROTEINS for O-glycans is the N-acetyl- in each area with specific examples galactosamine (GalNAc) residue of their applications. PROCESS FOCUS: PRODUCTION AND attached through the oxygen atom on DOWNSTREAM PROCESSING the side chain of serine or threonine REMOVAL OF GLYCANS FOR ANALYSIS residues of a protein. The most commonly used enzyme for WHO SHOULD READ: PROCESS/PRODUCT Glycosidases: Glycosylation is the removal of N-glycans from a DEVELOPMENT AND ANALYTICS complex and heterogeneic, so multiple glycoprotein is peptide-N-glycosidase KEYWORDS: GLYCOSYLATION, analytical methods must be used to F (PNGase F). It is an amidase that CHROMATOGRAPHY, MASS SPECTROMETRY, determine the structure of glycans and cleaves between the innermost CE-LIF, LC-FLD, ENZYMES their location on a glycoprotein. GlcNAc residue of the N-glycan and Glycosidases are important tools often the asparagine residue that releases LEVEL: INTERMEDIATE used with other analytical methods to the N-glycan. That creates an aspartic Table 1: Substrate specificity of commercially available endoglycosidases ( sialic acid, galactose, mannose, GlcNAc, fucose) the N-glycan core) leaving a GlcNAc Endo- residue attached to the protein. So glycosidase Type of N-Glycans Cleaved Notes Reference they often are used to determine Endo H α 2 α 6 α 6 α α α 6 α 6 14 Endo F1 6 6 α 3 α 6 occupancy or presence of an N-glycan α3 α 2 α 3 α 3 β 4 β 4 α 3 β 4 β 4 α 3 4 β 4β at a specific site. That is particularly α 2 α 2 β 4 β 2 High mannose Hybrid useful for glycoproteins with multiple Endo F2 α 6 N-glycosylation sites. α 2 α 6 α 6 15 α 3 α 3 β 4 β 2 α 2 β 4 β 4 Typically a glycoprotein is digested α 3 β 4 β 4 α 3 α 3 β 4 β 2 α 2 α 2 with an endoglycosidase followed by High mannose Complex biantennary protease cleavage. The peptide Endo F3 β 4 β 2 α 3 fragments are then analyzed using α 6 α 6 β 4 β 2 α 6 15 α 3 β 4 β 2 α 3 MS. The additional mass of a single α 3 β 4 β 4 β 4 β 4 β 4 α 3 β 4 α 3 GlcNAc residue on any peptide α 3 β 2 Complex biantennary β 4 fragment confirms that the site was with core fucosylation Complex triantennary occupied by an N-glycan (6). Because Endo S α 3 Works only b α 3 β 2 16 the site of hydrolysis by endo- -N- β 4 β 2 α 6 α 6 β 4 on IgG Fc β 4 α 6 α 3 β 42 β 4 αβ 62 β 4 acetylglucosaminidases is away from β 4 βα 4 3 α 3 β 4 glycans β 2 α 3 α 3 α 3 β 4 α 3 β 4 the protein backbone, those enzymes β 4 β 4 β 2 often can cleave N-glycans under Complex biantennary with or without a bisecting GlcNAc nondenaturing conditions. That Endo S2 α 6 α 6 17 occurs even when these sites are α 6 Complex biantennary α 3 α 6 α 6 α 3 α 3 refractory to PNGase F cleavage α 3β 4 β 4 α 3 β 4 β 2 β 4 β 4 α 6 β 4 β 4 β 2 under similar native conditions. This High mannose α 3 β 4 β 2 α 3 β 4 Hybrid is especially true for Endo S, which is Endo D α 6 α 6 α 6 18 specific for hydrolysis of N-glycans on β 4 β 4 α 3 α 3 α 3 β 4 β 4 the Fc region of IgG and prefers Paucimannose nondenaturing conditions. The ability Endo M Can also α 6 α 3 19 of such endoglycosidases to hydrolyze α 3 α 6 do trans- β 4 β 4 β 4 β 2 α 3 glycans under native conditions is α 6 α 3 β 4 β 4 glyco- β 4 β 2 Paucimannose sylation especially useful when the goal is to Complex biantennary α 2 reactions α 6 α 6 preserve a protein’s structure. α 2 α 6 α 3 α 3 α 6 α 6 Currently, no known broad α 3 β α 2 β 4 β 4 α 3 β 4 β 4 α 2 β 4 β 2 specificity endoglycosidase can cleave High mannose without Hybrid with all O-glycans from glycoproteins. So core fucosylation bisecting GlcNAc analysis of O-glycans is far more difficult than for N-glycans. Two O-glycosidases are commercially acid residue in place of the asparagine and/or mass spectrometry (LC–MS) available. One is from Streptococcus residue on the protein. or capillary electrophoresis coupled to pneumoniae and can release Core 1 One main reason that glycosidases laser induced fluorescence detection disaccharides consisting of a galactose are so frequently used is that they have (CE–LIF) analysis. Figure 1 (Gal) linked b1–3 to a very broad specificity cleaving high illustrates the variety of N-glycans N-acetylgalactosamine (GalNAc), mannose, complex, and hybrid released from the Erbitux therapeutic where the GalNAc residue is alpha N-glycans. The only limitation for antibody (cetuximab) by PNGase F linked to either a serine or threonine such enzymes is that one cannot and detected by LC–MS analysis. residue of the protein (7). The other cleave if there is an α1–3 fucose linked In addition to PNGase F, many O-glycosidase is from Enterococcus to the core GlcNAc residue (the sugar different endo-b-N-acetylgluco- faecalis and has a slightly broader residue next to the protein). Such saminidases also release N-glycans specificity, cleaving both the Core 1 modification occurs only in plant, from glycoproteins. Most of those disaccharide and the Core 3 insects, mollusks, and parasitic worms. endoglycosidases are limited in the disaccharide that consists of GlcNAc b N-glycans harboring this modification type of N-glycans that they can linked 1–3 to GalNAc (8). can be removed by the enzyme recognize and cleave. Table 1 shows Neither enzyme can cleave if the PNGase A. the different specificities of most disaccharide is further modified by Once an N-glycan has been commercially available endo-b-N- sialic acids or other sugars. Intact released by an amidase, it can be acetylglucosaminidases. All of those O-glycans can be released chemically fluorescently labeled for analysis by endoglycosidases hydrolyze the using b-elimination, but care must be liquid chromatography coupled to N,N′diacetyochitobiose moiety taken with this method not to degrade fluorescence detection (LC–FLD) (between the two GlcNAc residues on the released glycans. Figure 2: Deglycosylation of the etanercept therapeutic protein, a fusion protein with three N-glycans and up to 13 O-glycans: (A) SDS-PAGE gel of etanercept treated with different Another major disadvantage to this combinations of PNGase F and exoglycosidases; lane S is the molecular weight standards.