Using Glycosidases to Remove, Trim, Or Modify Glycans on Therapeutic Proteins

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 FOCUS ON  TECHNICAL ARTICLES 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 6 monitor and characterize the 4 glycosylation patterns of their 19.41 2 11.34 25.02 recombinant therapeutic proteins (3, 4). × 10 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. Lane 1 is the untreated etanercept control. Lane 2 is etanercept incubated in the presence of buffer method is that the protein is destroyed containing SDS and DTT. Lane 3 is etanercept incubated with PNGase F in the presence of a buffer and can no longer be analyzed for its containing SDS and DTT. Lane 4 is etanercept incubated with a deglycosylation mix, which is a structure or activity. By contrast with mixture of PNGase F, O-glycosidase (from Enterococcus faecalis), an α2–3,6,8 neuraminidase, a β1–4 intact chemical release of O-glycans, galactosidase, and a broad specificity β N-acetylglucosaminidase in the presence of SDS and DTT. the O-glycans can be trimmed using a Lane 5 is etanercept incubated with Rapid PNGase F, O-glycosidase, and an α2–3,6,8 neuraminidase in a proprietary Rapid PNGase F buffer. Complete removal of all glycans indicated by a complete combination of exoglycosidases and an shift of etanercept only occurs with conditions in lane 5. (b) ESI-TOF profile of etanercept before O-glycosidase (Figure 2). Although treatment (average molecular weight = 64 KDa); (c) after treatment (51 kDa) with Rapid PNGase F, this method can preserve a protein’s O-glycosidase and an α2–3,6,8 neuraminidase in a proprietary Rapid PNGase F buffer for 1 hour at structure and activity, it degrades the 37 °C, demonstrating that all N- and O-glycans have been removed. glycans so they cannot be A S 1 2 3 4 5 B characterized. In addition, some O-glycans contain sugar modifications 4 (e.g., sulfation or acetylation) that ) 2 3 render modified sugars resistant to 2 cleavage by exoglycosidases. ( × 10 1 trimminG Glycans For sequencinG 0 52 54 56 58 60 62 64 66 68 70 Counts vs. Deconvoluted Mass (amu ×1,000) Exoglycosidases are important C enzymes that can trim glycans one 51,112.35 2 sugar residue at a time from the

) nonreducing end of a glycan. A large

4 1.5 1 number of different exoglycosidases ( × 10 0.5 are commercially available. 0 Exoglycosidases are specific for 52 54 56 58 60 62 64 66 68 70 both the type of sugar and its anomer Counts vs. Deconvoluted Mass (amu ×1,000) (alpha or beta). Some exoglycosidases are more general and can hydrolyze a Figure 3: Sequencing of Enbrel glycans; the glycans have been released from Enbrel using PNGase number of different linkages. For F, labeled with procainamide, treated with different glycosidases and analyzed using LC–MS; (a) LC example, a broad-specificity trace of glycans release from Enbrel with no exoglycosidase treatment; (b) LC trace of release neuraminidase can remove sialic acid glycans treated with α2–3,6,8,9 Neuraminidase A; (c) LC trace of release glycans treated with residues linked α2–3, α2–6, α2–8, or α2–3,6,8,9 neuraminidase A and β1–4 galactosidase S; (d) LC trace of release glycans treated with α α2–3,6,8,9 neuraminidase A, β1–4 galactosidase S and α1–2,4,6 fucosidase; (e) LC trace of release 2–9 to a glycan. Other glycans treated with α2–3,6,8,9 neuraminidase A, β1–4 galactosidase S, α1–2,4,6 fucosidase and β-N- exoglycosidases are more specific for a acetylglucosaminidase S. Glycan structures were assigned to specific major peaks deduced from particular linkage. For example, a the molecular weight of the labeled glycans and confirmed by exoglycosidase digestion. b1–4 galactosidase will remove only b1–4 linked galactose. Because of the innate specificity of these enzymes, )

A 6 4 Untreated exoglycosidases are useful for both 2 ( × 10

Counts trimming glycans and determining the sequence of the glycans. Basic LC-MS

) or matrix-assisted laser desorption/ B 6 4 Treated with ionization (MALDI) analysis can 2 sialidase ( × 10 Counts determine only the size of a glycan. However, to confirm the linkage or )

C 6 10 Treated with sialidase and type of monosaccharide present on a β galactosidase

( × 10 glycan, it is necessary to perform Counts 5 tandem mass spectrometry (MS-MS), ) D 6 15 such as collision-induced dissociation Treated with sialidase,

( × 10 10 β galactosidase, and α fucosidase (CID) or use exoglycosidases followed Counts 5 by CE or MS analysis. When using exoglycosidases, it is ) E 6 20 Treated with sialidase, β galactosidase, important to consider the quality of α fucosidase, and β N-acetyglucosaminidase ( × 10

Counts 10 enzyme preparation. Enzyme 0 preparations isolated from native 10 15 20 25 30 35 40 sources often can be contaminated with Time (minutes) other glycosidase activities, making glycan structure elucidation more Figure 4: Example of enzymatic transglycosylation for glycoengineering Figure 5: Example of the use of exoglycosidases and a sialyltransferase to of an antibody to create homogenous glycoforms create uniform glycosylation on an antibody

Digest with α 1–3, 4, 6 galactosidase and Deglycosylation with α 2–3, 6, 8, 9 neuraminidase Endo S or Endo S2

Transglycosylation with an Endo S mutant (D233Q) α 2–6 sialytransferase and CMP-sialic acid

Chemi-enzymatically synthesized glycan

difficult. Recombinant sequencing- An alternative method of drastically improved the speed of grade versions of most commonly used glycoengineering involves using N-glycan removal from antibodies and exoglycosidases are now commercially exoglycosidases to trim glycans many other therapeutic proteins from available for use in sensitive glycan down to a uniform size on a overnight to five minutes. analysis workflows. glycoprotein. Such glycans can then New antibody-specific proteases Figure 3 shows how exoglycosidases be reconstructed using specific such as IdeS and IdeZ are can sequence glycans released from the glycosyltransfereases, enzymes that improvements over typical trypsin/ Enbrel therapeutic protein transfer monosaccharides from a LysC or papain digestions of antibodies (etanercept). Exoglycosidases also can nucleotide sugar donor to a glycan. because they specifically cleave at the be used to sequence glycans still That creates a glycoprotein with a hinge region of IgG with no secondary attached to a glycoprotein. These more homogenous glycan structure site cleavage. Use of those proteases enzymes are especially useful for (Figure 5). with improved software allows MS analyzing and detecting potential analysis of the constant region of an antigenic structures on glycans. For Future aPPlications antibody. That allows determination of example, using a general alpha So far, these in vitro glycoengineering the types of glycans present on the Fc galactosidase such as the enzyme from methods have been used mostly at without the need to remove, purify, and green coffee bean to treat therapeutic small scales. The exception is for label N-glycans. proteins can help identify low levels of glycoprotein enzymes used to treat Other areas of improvement might Galα1–3Gal (α Gal epitopes) that are lysosomal storage diseases. Several of lead to the discovery of new enzyme not present in humans and can be these enzymes have been specificities. For example, an enzyme immunogenic (9). glycoengineered using exoglycosidases with a broad specificity for complex to expose mannose structures on a O-glycans would allow better analysis modiFication oF Glycans protein to improve the enzyme’s of these types of protein modification. In addition to removal of intact transport to the lysosome by receptor- And a PNGase that could remove all glycans and glycan sequencing, mediated endocytosis (13). In addition, N-glycans would be very useful as more glycosidases can be used to modify modification of glycosylation pathways therapeutic proteins are expressed in glycans on glycoproteins (termed in host cells (including Roche’s transgenic plants and insect cell lines. glycoengineering) (10). One method is GlycArt and Kiran’s Potelligent Finally, as methodologies for glycan to remove the undesired N-linked expression systems) are being used to analysis become more refined with glycans using endoglycosidases. Then produce therapeutic proteins with higher throughput and increased the desired uniform glycans created desired glycosylations. Therapeutic sensitivities, it should be possible to using chemoenzymatic synthesis can protein production in those cell lines select for clones during development or be attached to the glycoprotein is well under way, with several use selected glycoengineered expression through the GlcNAc residue left by therapeutic proteins in clinical trials. hosts that produce antibodies with the endoglycosidase cleavage. Scientists expect that the speed and desired glycosylation patterns. Such Attachment of the desired glycan can simplicity of glycan analysis will speed and sensitivity will also allow for be performed using endoglycosidases progress with further improvements in constant monitoring of fermentation (which naturally transglycosylate) such enzymes and in analytics as the need processes, which will improve as Endo-M (11) or using for higher-throughput methods production of therapeutic glycoproteins. endoglycosidase mutants that drive the increases. 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New England Biolabs, Inc. 240 County Road Ipswich, MA 01938 www.neb.com [email protected]

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