Strategies for Neoglycan Conjugation to Human ... - ACS Publications

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Strategies for Neoglycan Conjugation to Human Acid r-Glucosidase Qun Zhou,* James E. Stefano, John Harrahy, Patrick Finn, Luis Avila, Josephine Kyazike, Ronnie Wei, Scott M. Van Patten, Russell Gotschall, Xiaoyang Zheng, Yunxiang Zhu, Tim Edmunds, and Clark Q. Pan Genzyme Corporation, Framingham, Massachusetts 01701, United States

bS Supporting Information ABSTRACT: Engineering proteins for selective tissue targeting can improve therapeutic efficacy and reduce undesired side effects. The relatively high dose of recombinant human acid R-glucosidase (rhGAA) required for enzyme replacement therapy of Pompe disease may be attributed to less than optimal muscle uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR). To improve muscle targeting, Zhu et al.1 conjugated periodate oxidized rhGAA with bis mannose 6-phosphate bearing synthetic glycans and achieved 5-fold greater potency in a murine Pompe efficacy model. In the current study, we systematically evaluated multiple strategies for conjugation based on a structural homology model of GAA. Glycan derivatives containing succinimide, hydrazide, and aminooxy linkers targeting free cysteine, lysines, and N-linked glycosylation sites on rhGAA were prepared and evaluated in vitro and in vivo. A novel conjugation method using enzymatic oxidation was developed to eliminate side oxidation of methionine. Conjugates derived from periodate oxidized rhGAA still displayed the greatest potency in the murine Pompe model. The efficiency of conjugation and its effect on catalytic activity were consistent with predictions based on the structural model and supported its use in guiding selection of appropriate chemistries.

’ INTRODUCTION Pompe disease, or glycogen storage disease type II, is an autosomal recessive disorder whose underlying pathology is an accumulation of glycogen in lysosomes in many tissues, principally cardiac and skeletal muscle.2 The accumulation of glycogen results from a deficiency in the glycogen-degrading lysosomal enzyme, acid R-glucosidase or GAA, leading to cardiac, skeletal, and respiratory muscle tissue damage and organ failure. The disease has a heterogeneous clinical presentation, from infants who have a rapidly progressive form of the disease resulting in early death, to older children and adults whose disease progresses at a slower rate but usually results in wheelchair use and the need for a ventilator as a consequence of the severe muscle weakness. Like most lysosomal enzymes, GAA contains mannose 6-phosphate (Man-6-P) in its oligomannose-type N-glycans, which directs its transport inside the cell from the Golgi to the lysosome via Man6-P receptors.3 GAA is initially synthesized as a 110 kDa protein in ER and Golgi of the cells, followed by proteolytically processing in the lysosome to mature forms.4,5 The enzyme replacement therapy with alglucosidase alfa (recombinant human R-glucosidase, rhGAA, Myozyme, Genzyme Corp) is used successfully for treating Pompe patients resulting in reduced cardiac hypertrophy, reduced the need for ventilation support, improved motor function, and increased life expectancy in infants.6
11 However, even at doses which were high relative to other enzyme replacement therapeutics, the extent of glycogen clearance in skeletal muscle cells was found to vary among the infants treated with the enzyme.12 The need r 2011 American Chemical Society

for high dose has also been observed in clearing skeletal muscle glycogen of GAA knockout mouse, a murine Pompe model.12,13 These findings could be caused by inefficient targeting of the recombinant enzyme to lysosomes through cation-independent Man-6-P receptor (CI-MPR) expressed on the surface of muscle cells, as rhGAA contains on average only a single Man-6-P and even lower amounts of bis Man-6-P, the preferred ligand for the receptor.14 This hypothesis is supported by the finding that conjugation of bis Man-6-P bearing glycans to the terminal sialic acids of rhGAA (neoGAA) resulted in a ∼5-fold greater potency for clearing lysosomal glycogen in muscle of GAA knockout mice than the unmodified enzyme.1,15,16 Although the sialic acid-mediated conjugation strategy targeting the 6
7 copies of sialic acids in rhGAA13 yielded a conjugate with greatly improved functional outcome in the murine Pompe model,1 there are multiple approaches to produce such neoglycoproteins.17 At one extreme, a single glycan can be conjugated to the single free cysteine on rhGAA via thiol-based chemistry to generate a defined product profile. At the other extreme, 10
20 copies of neoglycans can be conjugated to the 15 lysines, N-terminal amine, and possibly histidines on rhGAA through amine-based chemistry. Finally, the carbohydrate on rhGAA can be oxidized with an enzyme specific to galactose instead of periodate, resulting in galactose-mediated conjugation. Received: December 1, 2010 Revised: February 14, 2011 Published: March 18, 2011 741

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In the current study, we explored these three alternative conjugation strategies and compared the resulting conjugates with sialic acid-mediated conjugates both in vitro and in vivo.

Galactose Aldehyde-Mediated (GAM) Conjugation. rhGAA was desialylated with 20 mU/mg Clostridium perfringens neuraminidase for 6 h at 37 °C in 25 mM sodium phosphate, 2% mannitol, and 0.005% Tween-80 (pH 6.25) and then treated with GAO (2
10 μg/mg) in the presence of catalase (2 U/mg) overnight at 37 °C. rhGAA was purified away from neuraminidase and GAO by anion-exchange chromatography. The reaction mixture was diluted with an equal volume of water and applied to a Poros 50D column equilibrated with 10 mM sodium phosphate (pH 6.9). Following a wash with 10 mM sodium acetate (pH 5.0), the protein was step eluted with 150 mM sodium acetate (pH 5.0). A 16.6-fold molar excess of bis Man-6-P neoglycan (aminooxy) was added directly to the eluate and the mixture incubated for 6 h at 37 °C. The product was then bufferexchanged into 25 mM sodium phosphate, 2% mannitol, and 0.005% Tween-80 (pH 6.25) either by diafiltration with Pellicon XL 50 or by centrifugal ultrafiltration over Amicon filters (Millipore, MA) to yield GAM6. Quantitation of Monosaccharides Including Man-6-P and Galacturonic Acid (GalA). The Man-6-P content was determined as described previously.20 Hydrolysates were filtered through S Mini H cartridges (Sartorius, NY) and ribose5-phosphate used as an internal standard. The number of glycans conjugated and conjugation efficiencies were calculated assuming 2 mol Man-6-P per mole neoglycan and correcting for the Man-6-P content of the starting rhGAA. Galactose and GalA were also quantified using HPAEC-PAD on a Dionex HPLC.21,22 The GalA was identified using LC/MS/MS of glycans released with PNGase F and resolved by normal-phase HPLC (TSK gel amide-80 column) in an acetonitrile
water gradient with 10 mM ammonium formate (pH 4.0). Mass spectrometry detection of glycans was performed in-line, in negative ion mode, using QStar or LCT time-of-flight mass spectrometers. HPLC-Based Cation-Independent Man-6-P Receptor (CI-MPR) Binding. Binding of rhGAA or neoGAA conjugates to CI-MPR was determined by HPLC affinity chromatography with soluble bovine CI-MPR (purified from fetal bovine serum) immobilized on Poros EP resin using step elution with Man-6-P. Protein was detected by intrinsic protein fluorescence (290 nm excitation/340 nm emission), and the relative amount of protein eluting at each step was determined by integration of the peak areas. GAA Activity Assay. Enzymatic activity was determined using p-nitrophenyl-R-D-glucopyranoside as substrate as described.13 One unit of activity is defined as the hydrolysis of 1 μmol of substrate per min at 37 °C. Protein Structure Modeling. Two secondary structure prediction algorithms PSIPRED23 and SSPRO24 were used to align the GAA sequence with the structure of human intestinal maltase-glycoamylase (MGAM, PDB code 2QMJ),25 followed by manual adjustment and model building in Prime 2.1 (Schr€odinger, OR). Loops of the resulting GAA model were refined using the default sampling for loops of 5 residues or shorter and the extended protocol for loops longer than 5 residues. Side chains were unfrozen within 7.5 Å of the corresponding loop and the energy cutoff was set to 10 kcal. Animal Studies. For the determination of serum clearance, 3 groups of 5 male and 5 female GAA knockout mice at 3
6 months of age (Charles River Laboratories, Wilmington, MA) received a single intravenous administration of rhGAA, GAM, and SAM conjugates (GAM6 and SAM6) at 20 mg/kg. Blood samples for analysis were collected via the retro-

’ EXPERIMENTAL PROCEDURES Materials. rhGAA and neuraminidase from Clostridium perfringens were produced by Genzyme Corp. Bis Man-6-P neoglycans were synthesized as described previously.1,16 SFB (or S-4FB, succinimidyl 4-formylbenzoate) and pentafluorobenzamide poly(ethylene glycol) 4 (PEG4/PFB) were purchased from Solulink Corp (San Diego, CA). Galactose oxidase (GAO) was obtained from Worthington Biochemicals (NJ). 1,4-Di-(30 -[2-pyridyldithiol]-propionamido) butane (DPDPB) was obtained from Pierce (Rockford, IL). Other reagents, including catalase and sodium periodate, were obtained from Sigma (St. Louis, MO). Sialic Acid-Mediated (SAM) Conjugation. Glycan conjugation to sialic acids was performed according to a method described by Zhu et al.1 except that buffer exchanges were performed by diafiltration on Pellicon XL 50 or Amicon ultra-4 (Millipore, MA). Thiol-Based Conjugation. A thiol-reactive form of bis Man6-P neoglycan was prepared by a condensation of the acid form with a previously described thiol-reactive linker (3-nitro-2pyridinesulfenylethylamine, nipsylethylamine, NEA).18 A crude fraction of the free-acid form of bis Man-6-P neoglycan was converted to the triethylammonium (TEA) salt by neutralization with TEA followed by chromatography on a Superdex Peptide column (GE Healthcare, NJ) using 30% acetonitrile and 0.1% TEA bicarbonate as a mobile phase. Pooled fractions were lyophilized and conjugated with NEA in a reaction containing glycan:NEA:EDAC:NHS:HOBt:TEA (1:1:1.5:1:1:1 mol:mol) incubated overnight at 25 °C with gentle shaking. The product was purified on a Superdex Peptide column as described above and lyophilized. The NEA-glycan was reconstituted in water and incubated with rhGAA at 15-fold molar excess over protein in 50 mM sodium phosphate and 50 mM hydroxylamine (pH 7.2) for 2 h at 25 °C. The pH was adjusted to 6.2 with 50 mM sodium phosphate (pH 4.1) and the incubation continued overnight. The product was buffer-exchanged into 25 mM sodium phosphate (pH 6.2) using Amicon (Millipore, MA). rhGAA (∼3.5 mg/mL) was also modified with a thiol-reactive cross-linker by reaction with 60to 108-fold molar excess of DPDPB in 25 mM sodium phosphate (pH 6.2) with 10
20% organic solvent overnight at 25 °C. The extent of the reaction was determined by monitoring the absorbance at 343 nm of released 2-thiopyridone observed in an ultrafiltrate (Microcon YM-30, Millipore, MA) on a Beckman DU640 spectrophotometer or the spectrum of unfiltered material corrected for light scattering using the spectral data above 400 nm and instrument software.19 Lysine-Mediated Conjugation. Lysine residues in rhGAA were modified with SFB to introduce aldehydes, followed by reaction with modified neoglycans to form either hydrazone or oxime linked conjugates. rhGAA was reacted with a 20-fold molar excess of SFB in 150 mM sodium chloride and 50 mM sodium phosphate (pH 7.2) for 30 min, followed by buffer exchange into 100 mM sodium acetate (pH 5.5). The SFB modified protein was then coupled with bis Man-6-P neoglycan (hydrazide) for 2 h at room temperature. Alternatively, it was reacted with bis Man-6-P neoglycan (aminooxy) in 100 mM sodium acetate (pH 5.6) for 6 h at 37 °C. The final products were buffer-exchanged into 25 mM sodium phosphate, 2% mannitol, and 0.005% Tween-80 (pH 6.25). 742

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Figure 1. Structures for rhGAA, its conjugate, and bis Man-6-P neoglycan. (A) Linear structure of rhGAA. The positions of the lysine, cysteine, sialic acid, and galactose sites for neoglycan conjugation are shown. (B) A GAA structural model based on a crystal structure of human intestinal MGAM (see text). The N-linked glycosylation sites are shown in green (sites occupied by complex-type glycans as light green and those occupied by oligomannosetype glycans as dark green). Lysines are shown in light blue. Red ball: sulfur atom in the single free cysteine. Dark blue: acarbose substrate present in the MGAM structure. (C) Structure of the bis Man-6-P hexasaccharide glycan with different linkers (R groups): R1, aminooxy; R2, hydrazide; R3, thiolreactive NEA group. (D) Potential structure for neoGAA SAM conjugate. Models for the predominant N-glycans identified at each glycosylation site in rhGAA were added to the protein structure shown in B, followed by linkage of a hypothetical model for the synthetic neoglycan with aminooxy linker (R1) as shown in C. Light green: complex-type N-glycans and bis Man-6-P neoglycans. Light blue: aminooxy linker. Red: terminal glycan phosphate. Other labels are similar to those in B.

orbital plexus in conscious mice. The concentrations of rhGAA, GAM6, and SAM6 in serum were determined by GAA activity assay. 13 For the determination of efficacy in glycogen clearance from mouse, GAA knockout mice (3
6 months) were divided into 10 dose groups of 3 males and 3 females. Each group received vehicle; 20, 60, or 100 mg/kg rhGAA; and 4, 12, or 20 mg/kg of the GAM6 or SAM6 once a week for a total of 4 weeks. In order to prevent hypersensitivity induced by test articles, all groups received 5 mg/kg diphenhydramine (Baxter Deerfield, IL) intraperitoneally prior to each dose after the first administration. In the event that animals displayed clinical signs of hypersensitivity, an additional dose of 5 mg/kg diphenhydramine was administered intraperitoneally. Animals were euthanized 7 days after the last dose by CO2 asphyxiation. Heart, quadriceps, triceps, diaphragm, and psoas were harvested and analyzed for glycogen content as described previously.1,13,15,16 Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by a Newman-Keuls test. A probability value of p < 0.05 was considered statistically significant. Animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory

Animals (U.S. Department of Health and Human Services, NIH Publication No. 86
23) and were approved by Genzyme’s Animal Care and Use Committee. Other Methods. MALDI-TOF MS analysis was performed using a Voyager DE-PRO mass spectrometer as described in the previous report.5 Protein aggregation was determined by HPLC size exclusion chromatography.26 Identification of oxidized methionine residues was performed as described previously.27 Uptake of proteins into rat L6 myoblasts was performed as described.1,15,16

’ RESULTS neoGAA Conjugation Strategies. The rhGAA sequence contains a single free cysteine residue, 15 lysines, and 7 N-linked glycosylation sites (Figure 1A). In order to better design alternative conjugation strategies, the GAA structure was investigated. A homology model of GAA based on the structure of an R-glucosidase (maltase, MalA) from S. solfataricus has been published previously.28 However, only half of the GAA could be modeled based on the 30% sequence identity with MalA over the 452 amino acid residues. To obtain structural information for 743

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Man-6-P contents consistent with up to ∼4 mol neoglycan per mole rhGAA, in agreement with the conjugate molecular weights obtained by MALDI-TOF MS (Figure 2A and B). However, an accelerated stability study performed in pH 6.2 buffer at 40 °C resulted in more than 50% loss of neoglycan over 14 days (Figure 2A and B). These results indicate that the hydrazone bond between neoglycan hydrazide and the benzaldehyde linker was not stable for extended periods. To increase the stability, the hydrazone linkage was replaced with a more stable oxime by use of an aminooxy form of the bis Man-6-P neoglycan (Figure 1C). Oxime linkages are known for their high stability over a broad range of pH including the mildly acidic conditions for storage buffer used here.30 As shown in Figure 2C, a 16.6-fold molar excess of glycan was saturating for the Man-6-P content with the introduction of 5 mol neoglycan per mole rhGAA (10 mol Man-6-P per mole protein). Higher molar ratios of neoglycan failed to further increase the Man-6-P content. The number of neoglycans coupled was also significantly lower than the incorporated SFB benzaldehyde content determined by absorbance (∼8
9 mol/mol). A high level of aggregation was also frequently observed (Figure 2D). In an attempt to overcome this problem, conjugation was also performed using a PEGylated version of SFB (PEG4/PFB, Solulink), in which the lysine-reactive pentafluoroester group is separated from the aromatic formylbenzoate by a hydrophilic PEG4 spacer. This failed to produce significant decrease in aggregation (data not shown), suggesting a very limited tolerance of the protein for hydrophobic substitutions. The susceptibility of the protein to aggregation in response to hydrophobic modifications suggests the importance of maintaining a hydrophilic surface environment. Carbohydrate-mediated conjugation chemistries were therefore evaluated since the aldehydes required for conjugation could be generated directly by an oxidation step without the need for linkers containing hydrophobic functional groups. Aldehydes on terminal carbohydrate residues would be expected to be well-separated from the protein surface. Seven N-linked glycosylation sites are present on rhGAA including oligomannose, phosphorylated oligomannose, and complex-type glycans (Figure 1A and B).13 Among these sites, Asn-390, Asn-652, Asn-882, and Asn-925 are occupied mainly by sialylated complex-type glycans, while a significant amount of oligomannose-type glycans are present on Asn-140, Asn-233, and Asn-470, with a higher abundance on Asn-233 and Asn-470. A fraction of the oligomannose-type glycans bear either a single (mono Man-6-P) or two phosphates (bis Man-6-P), resulting in an average Man-6-P content of 1 mol per mole in rhGAA. The complex-type glycans contain approximately 6
7 sialic acid and 10 galactose residues as potential sites of conjugation. Two methods for coupling these glycans were compared: a chemical oxidation process directed at sialic acids and an enzymatic approach targeting galactose. Conjugation using sialic acid-mediated (SAM) chemistry was performed using a method described previously.1 As expected, after periodate treatment, sialic acid was undetectable by HPAEC-PAD analysis, consistent with the complete oxidation to the aldehyde form (data not shown). Conjugation of the neoglycan with this intermediate was found to be highly reproducible, with yields of 13
17 mol Man-6-P per mole GAA consistent with an overall process efficiency approaching 100%. Strikingly different than the amino acid directed chemistries, the level of protein aggregation was consistently less than 3%. However, treatment by periodate also resulted in the nearly

the remainder to identify other potential sites of conjugation, we developed a model (Figure 1B) based on human intestinal maltase-glycoamylase (MGAM). Both GAA and MGAM belong to the glycosyl hydrolases family 31,25 and the same enzyme class 3.2.1.20. GAA and the N-terminal catalytic subunit of MGAM (PDB codes 2QLY and 2QMJ) also share 44% sequence identities over 860 residues. The result shows the same overall fold of the glycosyl hydrolase family 31 enzymes with 5 structural domains: a trefoil type-P domain, a β-sandwich domain, N-terminal to the catalytic (β/R)8 TIM barrel domain, followed by two additional C-terminal β-sandwich domains. As expected from the strong homology to MGAM, the position of residues within the active site, including Trp-516 and Asp-518 within the WIDMNE element, which were previously identified in GAA by crosslinking of a substrate analogue and site-directed mutagenesis,29 were preserved in the model and near the acarbose binding site. On the basis of this structural model, the lysines and N-linked glycosylation sites are found in solvent-exposed positions distributed relatively evenly over the entire structure without any notable clustering, suggesting those sites as potential alternative conjugation sites. Conjugation of a synthetic bis Man-6-P neoglycan (Figure 1C) to sialic acids on rhGAA N-glycans provides a substantial increase in the efficacy in GAA knockout mice.1,16 A structural model of such a conjugate was generated by linking in silico models of the dominant glycan forms at each site13 extended in solution consistent with their hydrophilicity and addition of a model of the synthetic glycan assuming charge repulsion of the bis-phosphates (Figure 1D). This model suggested potentially rather large extensions of the existing GAA structure were possible. This observation suggested that conjugation of bis Man-6-P neoglycans to alternative sites might provide advantages in preserving the activity of the protein against its natural substrate glycogen, a complex polysaccharide which may be sterically hindered from interacting at the active site by large glycans nearby. A number of different conjugation chemistries targeting different functional groups were then explored. Bis Man-6-P hexasaccharide neoglycans were synthesized with different appended functionalities (Figure 1C), including terminal aminooxy (R1), hydrazide (R2), and thiolreactive NEA groups (R3), for conjugating to either aldehydes or thiols. A particularly interesting target was the single free thiol group (Cys-374) in rhGAA, since exclusive coupling to this site would provide a homogeneous conjugate. The structural model however suggested relatively limited access to this residue, which was confirmed by negligible conjugation of thiol-reactive NEAneoglycan (data not shown). Quantitative modification of the thiol group was successful using a hydrophobic homobifunctional cross-linking reagent with spacer arm of 19.9 Å, 1,4butane (DPDPB, di-(30 -[2-pyridyldithiol]propionamido) Pierce) but only in the presence of organic cosolvents, which resulted in very strong light scattering and formation of a precipitate (data not shown). These results suggest that modification of Cys-374 may be achieved, but only under harsh conditions unlikely to preserve the structural integrity of the protein and creating undesirable aggregation. Conjugation to lysines was attractive because of the higher lysine content of rhGAA than sialic acids employed in previous conjugates1,15,16 could allow a higher degree of modification. Conjugates containing neoglycans hydrazone-linked to lysines modified with an aromatic aldehyde (SFB) were obtained with 744

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Figure 2. Properties of neoGAA conjugates prepared by introducing aldehyde groups at lysines. Man-6-P content (A) and MALDI-TOF MS analysis of the molecular weight (B) of rhGAA or SFB-hydrazone conjugate incubated at pH 6.2 at 40 °C. (C) Conjugation of SFB modified rhGAA with increasing amounts of aminooxy neoglycan. (D) Aggregation of the same conjugates in C as determined using size exclusion chromatography.

complete oxidation (>95%) of two methionine residues, Met172 and Met-173, in the final conjugate (Figure 3A). The generation of aldehyde groups on the glycans by enzymatic oxidation of galactose residues (GAM) with galactose oxidase (GAO) was explored as a means to avoid undesirable side products of chemical oxidation. However, since rhGAA is highly sialylated and GAO reacts only with galactose at the terminal position on glycans,13,31 the protein was first treated with neuraminidase to remove the terminal sialic acids and expose the galactose residues. Following oxidation with GAO, the aldehydes were then coupled with the bis Man-6-P neoglycan (aminooxy) to yield a conjugate (GAM6) with an oxime-linked glycan. Initial evaluation showed that desialylation of rhGAA was complete after treatment with neuraminidase for 6 h at 20 mU/ mg rhGAA. Following GAO treatment (10 μg/mg), conjugation was saturated with a 16.6-fold molar excess of the neoglycan over rhGAA, yielding active conjugates with high neoglycan contents (11
12 mol Man-6-P per mole protein) and low aggregation (1 mol/mol), which increased with further decreases in GAO. Above the optimum, a reduction in the amount of residual galactose was accompanied by a higher GalA content and a decrease in the final Man-6-P content, consistent with the loss of aldehyde groups available for conjugation. Even at the optimum ratio, a significant amount of both GalA and residual galactose were present. In Vitro Comparison of SAM and GAM Conjugates. The enzyme activity of a panel of SAM and GAM conjugates containing a wide distribution of neoglycan content was determined. All of the conjugates showed some reduction in specific activity relative to the rhGAA control (Figure 4A), and the loss in activity inversely correlated with the Man-6-P content of the conjugate (r2 = 0.87, p < 0.0001). There was no significant difference between the activity of conjugates with comparable Man-6-P contents prepared by either the SAM or GAM process. Binding of rhGAA and neoGAA to the cation-independent Man6-P receptor (CI-MPR) was followed by HPLC-based affinity chromatography using immobilized bovine CI-MPR (Figure 4B). To assess receptor affinity, protein was applied to the column and the bound fraction eluted with steps of increasing Man-6-P concentration. Representative profiles from rhGAA, SAM, and GAM conjugates are shown in Figure 4B. The majority of unmodified rhGAA either failed to bind or eluted with a low concentration of Man-6-P (0.25 mM) in the mobile phase. In contrast, more than 95% of both GAM and SAM conjugates were bound and could be eluted only with 5 mM Man-6-P or above. SAM and GAM conjugates showed a similar profile by this method. The uptake of rhGAA and neoGAA by rat L6 myoblast cells in vitro was followed to determine if differences in the conjugation method had an effect on the efficiency of internalization through CI-MPR. As shown in Figure 4C, more than 10-fold higher GAA activity was observed in cells incubated with neoGAA conjugates than with rhGAA lacking neoglycan over a wide range of protein concentrations. GAM and SAM conjugates showed a comparable saturable uptake with EC50 of ∼5 nM (0.5 μg/mL) protein. The EC50 for rhGAA could not be determined in this experiment due to its low level of uptake. Serum Half-Life and Glycogen Clearance in the GAA Knockout Mouse. The serum clearance of rhGAA and neoGAA was determined following tail vein injection at single dose (20 mg protein/kg) to GAA knockout mice. All proteins including rhGAA and neoGAA conjugates were rapidly cleared from circulation with serum half-lives (T1/2) between 0.5 and 2.3 h (Figure 5). Both GAM and SAM conjugates demonstrated faster clearance than rhGAA (T1/2 of 24% and 37% of rhGAA, respectively). The GAM conjugate was cleared most rapidly with a serum half-life of 33 min, and its serum concentration was below the limit of quantitation at both 240 and 480 min. The SAM conjugate was cleared at a relatively slower rate than GAM conjugate and it was below the limit of quantitation at 480 min with a serum half-life of 51 min. The impact of bis Man-6-P neoglycan conjugation on glycogen clearance by rhGAA and both neoGAA conjugates (GAM6

Figure 3. Characterization of SAM and GAM conjugates. (A) Oxidation of Met-172 and Met-173 determined by LC/MS. GAM6 was prepared with or without catalase. (B) SDS-PAGE of the SAM and GAM conjugates, followed by silver stain. The 110 kDa is precursor form, while 76 kDa and 70 kDa are processed forms of rhGAA. (C) Effect of GAO:rhGAA ratio on Man-6-P, GalA, and residual galactose levels in GAM conjugate.

GAO treatment at 10 μg/mg (data not shown). However, following GAO treatment, galacturonic acid (GalA), in which the C6 position of galactose is fully oxidized to the carboxylic acid form, was also identified by LC/MS/MS of protein glycans released by PNGase F. This product likely arose by further oxidation of the desired galactose aldehyde to the carboxylic acid form by the action of GAO during the reaction,32,33 suggesting that the lower efficiency observed with GAM conjugation under these conditions may have resulted from the conversion of the aldehyde to the unconjugatable acid form. To improve on the Man-6-P content, the GAO:rhGAA ratio was varied to limit 746

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Figure 5. Serum clearance of neoGAA SAM and GAM conjugates in the GAA knockout mice. Animals were administered with rhGAA or neoGAA conjugates via the tail vein as a single bolus injection. The amount of proteins was determined using the GAA activity assay of blood collected at 5, 15, 30, 60, 120, 240, and 480 min post injection. The Man-6-P contents were 0.9, 13.3, and 11.3 mol per mole for rhGAA, SAM6, and GAM6, respectively.

administration. The dose ranges tested were 5-fold higher for rhGAA than for the neoGAA conjugates in order to obtain comparable glycogen clearance. At the lowest dose level, rhGAA (20 mg/kg) reduced glycogen in the heart by only 17% (Figure 6A). By comparison, the SAM conjugate produced a greater reduction (61%) in glycogen at a 5-fold lower dose (4 mg/kg), while the GAM conjugate at 4 mg/kg was less effective, showing a modest clearance comparable to rhGAA administered at 20 mg/kg (Figure 6A). At the intermediate dose level (60 mg/kg rhGAA and 12 mg/kg for neoGAAs), rhGAA or the SAM conjugate achieved 90% and 94% glycogen clearance, respectively, while the GAM conjugate reduced glycogen by 51%. Finally, at the highest dose level (100 mg/kg rhGAA and 20 mg/kg for neoGAAs), essentially complete clearance of glycogen was obtained with rhGAA or a 5-fold lower dose of the SAM conjugate but not the GAM conjugate, which showed a somewhat lower efficacy (79%). The dose required to achieve 50% reduction in glycogen in the heart was estimated as ∼40, 12, and