Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Filing 547

DECLARATION of Craig H. Casebeer in Support of Motion for Summary Judgment of No Inequitable Conduct by Amgen Inc.. (Attachments: #1 Exhibit Ex 1#2 Exhibit Ex 2#3 Exhibit Ex 3#4 Exhibit Ex 4#5 Exhibit Ex. 5#6 Exhibit Ex 6#7 Exhibit Ex 7#8 Exhibit Ex 8#9 Exhibit Ex 9#10 Exhibit Ex 10#11 Exhibit Ex 11#12 Exhibit Ex 12#13 Exhibit Ex 13#14 Errata Ex 14#15 Exhibit Ex 15#16 Exhibit Ex. 16#17 Exhibit Ex 17#18 Exhibit Ex 18#19 Errata Ex 19#20 Exhibit Ex 20#21 Exhibit Ex 21-1#22 Exhibit Ex 21-2#23 Exhibit Ex 22#24 Exhibit Ex 23#25 Exhibit Ex 24#26 Exhibit Ex 25#27 Exhibit Ex 26#28 Exhibit Ex 27#29 Exhibit Ex 28#30 Exhibit Ex 29#31 Exhibit Ex 30#32 Errata 31#33 Errata Ex 32#34 Exhibit Ex 33#35 Exhibit Ex 34#36 Exhibit Ex 35#37 Exhibit Ex 36#38 Exhibit Ex 37#39 Exhibit Ex 38-1#40 Errata Ex 38-2#41 Exhibit Ex 39#42 Exhibit Ex 40#43 Exhibit Ex 41)(Gottfried, Michael)

Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

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EXHIBIT 34

Casebeer Decl to Motion for SJ re IC - Public

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THE JOURNAL BIOLOGICAL OF CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263,No. 8, Issue of March 15, pp. 365-3663, 1988 Printed in U.S.A.

Comparative Study of Asparagine-linked Sugar Chains of Human the Erythropoietins Purified from Urine and the Culture Medium of Cells* Recombinant Chinese Hamster Ovary
(Received for publication, June 9, 1987)

Makoto TakeuchiSQ,Seiichi Takasaki$, Hiroshi MiyazakiS, Takashi KatoS, Sakuo HoshiS, Naohisa Kochibel, and Akira Kobata$((
From the §Departmentof Biochemistry, Institute of Medical Science, University of Tokyo, Minuto-ku, Tokyo 108 and the $Pharmaceutical Laboratory, KIRIN BreweryCo. Ltd. and the llDepartment of Biology, Faculty of Education, Gunma University, Maebashi371, Japan

be by the payment of page charges. This article must therefore hereby marked ``Oduertisemnt" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom all correspondence should be addressed. The abbreviations used are: urinary HuEPO, human erythropoietin purified from the urine of patients with aplastic anemia; CHO, Chinese hamster ovary; rHuEPO, human erythropoietin produced in recombinant CHO cells; ConA, concanavalin A DSA, Daura stramonium agglutinin; AAL, Aleuria aurantia lectin; Fuc, fucose.

The asparagine-linked sugar chainshuman of eryth- and biological properties and for the clinical application. ropoietin produced by recombinant Chinese hamster In order to overcome this problem, a new approach has ovary cells and naturally occurring human urinary been developed by using recombinant DNA techniques. So erythropoietin were liberated by hydrazinolysis and far, two groups reported the cloning of human erythropoietin fractionated by paper electrophoresis, lectin affinity gene and its nucleotide sequence analysis (7, 8).Lin et al. (8) chromatography, and Bio-Gel P-4 column chromatog- has succeeded in the expression of the erythropoietin gene in raphy.Botherythropoietins had threeasparaginelinked sugar chains in one molecule, of which were Chinese hamster ovary (CHO) cells by recombinant techall acidic complex type. Structural analysis of them re- niques. The rHuEPO has three possible sites for N-glycosylation, vealed that the sugar chains from both erythropoietins Asn-X-Ser/Thr (7, 8), and is actually sensitive to Nare quite similar except for sialyl linkage. All sugar glycanase digestion (9). Recently, Lai et al. (43) estimated on chains oferythropoietinproduced by recombinant the basis of amino acid sequencing data thaturinary HuEPO Chinese hamster ovary cells contain only the also has three N-linked sugar chains and one 0-linked sugar NeuAca243Gal linkage, while those of human urinary chain. Analysis of the monosaccharide composition of HuEPO erythropoietin contain the NeuAca24Gal linkage to- performed in our laboratory confirmed the occurrence of one gether with the NeuAca2+3Gal linkage. Themajor N-acetylgalactosamine residue, indicating that one 0-linked sugar chainswere of fucosylated tetraantennary complex type with and without N-acetyllactosamine re- sugar chain is included in recombinant HuEPO (44). Sugar peating units in their outer chain moieties common, moiety of urinary HuEPO has been suggested to affect bioin and small amounts of 2,4- and 2,g-branched trianten- logical properties such as turnover rate, antigenicity, and so nary and biantennary sugar chains detected. This on (10-14). Therefore, it is important to elucidate the sugar were chain structures of erythropoietin. paper proved, for the time, that recombinant techfirst Several bioactive glycoproteins have been produced in renique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar combinant mammalian cells (15-17). Among them, the sugar chains of the native one. chain structure of human y-interferon produced in CHO cells has been elucidated; however, its natural counterparthas not been analyzed (17). In this paper, we will describe the comErythropoietin is a glycoprotein hormone involved in the parative analysis of the asparagine-linked sugar chain strucregulation of the level of peripheral erythrocytes (1)by stim- tures of rHuEPO produced in CHO cellsand naturally occurulating the differentiation of the erythroid progenitor cells ring urinary HuEPO. into mature erythrocytes (2). The hormone is primarily proEXPERIMENTAL PROCEDURES RESULTS' AND duced in the kidney of adults (3). Therefore, the decrease of the erythropoietin production by the destruction of kidney Paper Electrophoresis of Oligosaccharides Released from mass (from chronic renal failure (4) or some other reasons) Erythropoietin by Hydrazinolysis-Radioactive oligosacchacauses anemia. Highly purified erythropoietin is expected to rides obtained from rHuEPO and urinary HuEPO by hydrabe useful in the therapeutic treatmentof such a type anemia zinolysis were subjected to paper electrophoresis at pH 5.4. of (5). Erythropoietin has been purified from urine of patients As shown in Fig. 1,3 both samples contained acidic oligosacwith severe aplastic anemia (6); however, it is quite difficult charides composed of at least seven components (A-1 to A-7) to obtain the sufficient amount of human urinary erythropo- but no neutral oligosaccharide. When digested with sialidase ietin (urinary HuEPO)' for the investigation of its chemical from Arthrobacter ureafaciens, approximately 90% of the total *This workwas supported by the Grant-in-Aid for Scientific acidic oligosaccharides from rHuEPO and 80% of those from Research from the Ministry of Education, Science and Culture of urinary HuEPO were converted to neutral oligosaccharides (data not shown). The residual oligosaccharides migrated Japan. The costs of publication of this article were defrayed in part
Portions of this paper (including "Experimental Procedures," part of "Results," and Figs. 3-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of astandard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. Subscript OT is used to indicate NaB[3H]r-reducedoligosaccharides. All sugars mentioned in this paper wereof D-configurations except for fucose, which was L-configuration. of

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Sugars of Recombinant and Native Erythropoietins

IB
0 L
1-4 A d A-E

A

AN'

/IANr

A-7

t

-10

distance f r o m origin (cm)

0

1 0

20

30

40

0

A

AN 2

FIG. 1. Paper electrophoresis of the radioactive oligosaccharides obtained fromrHuEPO and urinary HuEPO. The (panel A ) and urinary oligosaccharides released rHuEPO from HuEPO (panel B ) by hydrazinolysis were subjected topaper electrophoresis at a potential of 73 V/cm for 90 min. The arrows indicate the position of authentic oligosaccharides: 0 1, 2,3, and 4 indicate ,
GlcNAc .GlcNAcm, respectively

non-, mono-, di-, tri-, andtetrasialylatedGal3.GlcNAc3.Man3.
TABLE I

80

elution volume (n rl )

120

160

200

I

Fractionation of asialo-oligosaccharidesreleased from rHuEP0 and urinary HuEPO by serial lectin affinitycolumn chromatography
Fractions"
Molar percent of the total asialo-oligosaccharide fraction Urinary

rHuEP0

ConA+AAL+ ConA'AAL-

4.1 1.9

HuEPO 5.3 3.7
2.4 1.2

ConA-DSA'AAL+ ConA-DSA'AAL-

48 . 4.1

FIG. 2. Bio-Gel P-4 column chromatography of asialo-oligosaccharides. Asialo-oligosaccharides of rHuEPO,fractionated with lectin columns as shown in Table I, were analyzed by Bio-Gel P-4 columnchromatography. Panel A, the ConA+AAL+ fraction; panel B, the ConA+AAL- fraction; panel C, the Cod-DSA'AAL+ fraction; panel D, the ConA-DSA'AAL- fraction; panel E, the Cod-DSA+AAL+ fraction; panel F, the ConA-DSA+AAL- fraction. Arrows a, b, and c indicate the elution positions of authentic Gal,. GlcNAc,. Man3. GlcNAc .Fuc. G~cNAcoT, 2,4-branched Gal3.GlcNAc3, Man3.GlcNAc .Fuc .GlcNAco~, and Galp.GlcNAc2.Man3. GlcNAc .Fuc .GlcNAcm, respectively. B h k triangles indicate the elutionpositions of glucose oligomers (the numbersindicatethe glucose units).

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of the total asialo-oligosaccharides for rHuEPO and 87.4% for urinary HuEPO. The AAL-bound (AAL') fraction was also predominant in both samples: 85.6% for rHuEPO and (-), 82.2% for urinaryHuEPO, indicating that large parts of oligosaccharides are fucosylated at their core. around the area corresponding to monosialyl oligosaccharides Each fraction was then analyzed by a column of Bio-Gel Pand were then converted to neutral oligosaccharides by mild 4. Since no qualitative difference in elution profile was demethanolysis (0.05 N HC1-methanol, 37 "C, 4 h). From the tected between the two samples, only the results obtained analysis of each acidic fraction the oligosaccharides, which from rHuEPO were shown in Fig. 2. Four fractions except for were partially sensitive to sialidase and susceptible to the the DSA+ fraction were eluted as single components (Fig. 2, methanolysis, were shown to be derived from the small parts A-D). The ConA-DSA'AAL' and AAL- fractions were comof A-3 to A-6 (6-10% of each fraction) and from the major posed of four and two components, respectively (Fig. 2, E and part of A-7 (95% of this fraction) (data not shown). Thus, the F). The components thus separated were termed as AN1 to result suggested the presence of small amounts of oligosac- AN6 for the AAL' fractions and as AN1' to AN4' for the charides containingboth sialic acid residues and possibly AAL- fractions as shown in Fig. 2. Structural Analysis of the Sugar Chains Obtained from sulfate group. The structures of their neutral portions were common to those of sialidase-sensitive acidic oligosaccharides rHuEP0 and Urinary HuEPO-Structures of the oligosac(data not shown). However, no detailed analysis of them to charides were studied by exoglycosidase digestions, Smith assign the location of sialic acid and sulfate residues was degradation, and methylation analysis as described in detail in the Miniprint Section of this paper and proposed as listed performed because of the limited amounts of samples. Fractionation of Asiulo-oligosaccharides-The neutral oligo- in Table 11. saccharide fractions obtained by sialidase digestion were fracThere was no qualitative difference in the structures of tionated by a serial lectin affinity column chromatography. asialo-oligosaccharides between rHuEPO urinary and The sample was first applied to a column of ConA-Sepharose. HuEPO. Both samples contain tetraantennary sugar chains The fraction which passed through the column was then (AN4 and AN4') as major components: 46% for rHuEPO and separated into a retarded fraction and a bound fraction by 59.9% for urinary HuEPO. The tetraantennary sugar chains passing through a DSA-Sepharose column. Each of these with N-acetyllactosamine repeating units (AN5 and AN6) three fractions, thus obtained, was finally fractionated by a were also detected in both samples, but their contents were column of AAL-Sepharose. The proportion of each fraction much higher in rHuEPO (34.5%) than in urinary HuEPO obtained from rHuEPO and urinary HuEPO summarized (7.5%). The smaller portion of oligosaccharides (17.7% for was in Table I. There were quite similar points between two rHuEPO and 32.6% of urinary HuEPO) had biantennary and samples. The first is that the major parts of oligosaccharides 2,4-branched and 2,6-branched triantennary sugar chain were recoveredin theConA-DSA' fraction in common: 85.1% structures.

76.6 ConA-DSA+AAL+ 14.5 ConA-DSA+AAL8.5 12.9 "The symbols represent the bound (+I, the passed-through and the retarded (r) fractions, respectively.

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Sugars of Recombinant and Native Erythropoietins
TABLE I1
Structures of the asparagine-linked sugar chains of r H u E P 0 and urinary HuEPO Fucal R = GlcNAcj314GlcNAcm. R' = GlcNAcj314GlcNAcoT. *, urinary HuEPO had a2-3 and a 2 4 linkages; the locations of the N-acetyllactosamine repeating units in the sugar chains of urinary HuEPO were not determined.
6

1

**,

Asialooligo y rHuEPO saccharide Structures fraction R

Percent molar ratio

R'

R

R'

Gal,914GlcNAcj31+2Manal\6 (NeuA~a2-*3)~-~ * { Galj314Gl~NAcj31+2Manal/1~ Galj314GlcNAcj3l+2Manal\6 Galj314GlcNAc@l\4 Mana14R/R' (Ne~Aca2+3)~-~ Manal /13 Galj314GlcNAcj31 Y2

AN1/1'

4.1 M a n a l 4 R / R ' 3.7 1.9 5.3

*

i

AN2/2'

2.4 4.8

4.1

1.2

AN3/3' Gal@14GlcNA~j31+2Mannal7'~ Galj314GlcNAcj31\6 2Manal\6 Galfi14GlcNAcj3lP ManP14RIR' (Ne~Aca2-*3)~-, GalS14GlcNAcj3114 Manal P3 Galfi1+4GlcNAcplP2

2.1 2.4

15.1

4.9

(NeuA~a2+3)~-~

`1

Galj31+4GlcNAcj31-3

*i
**{
**

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AN4/4'

39.6

6.4

51.9

8.0

Galj314GlcNAcj31 I 6Mana1\6 Galj314GlcNAcj31P Manj314R Galj314GlcNAcB114 Manal/13 P ~alal-A~lc~~cfi1

AN5

30.2

0

6.9

0

Gal@14GlcNAcj31+ 3 Galj314GlcNAcj31 I ** 6Mana1\6 Galj314GlcNAcpl+3 Galj31+4GlcNAcj31/12 Manj314R Gal/314GlcNAc@l I4 Manal/f3 Gal614GlcNAcS1 F2

4.3 AN6

0

0.6

0

The number of sialic acid residues in each acidic component was analyzed as follows. When sialidase digests of the acidic fractions as shown in Fig. 1 were separately subjected to BioGel P-4 column chromatography, it was shown that the tetraantennary oligosaccharide AN4 is included in the asialooligosaccharides of A-2, -3, -5, and -6, respectively (data not shown). Thus, the result and the mobility of each acidic fraction relative to authentic oligosaccharide standards indicated that one to four sites of AN4 are sialylated. Other oligosaccharides were also analyzed in the same way (data not shown). Methylation analysis of the oligosaccharide fraction of rHuEPO before (A) and after (AN) sialidase treatment indicated that almost all sialyl residues are linked at theC-3 position of galactosyl residues (Table 111). This is in agreement with the fact that all the sialic acid residues of rHuEPO were susceptible to Newcastle disease virus sialidase (data not shown), which specifically cleaves the Siaa2-3Gal linkage. In contrast, approximately 40% of sialyl linkages were resistant to this viral sialidase in thecase of urinary HuEPO (data not shown), indicating that other linkages are involved. Methylation analysis of oligosaccharide mixtures obtained from urinary HuEPO before and aftersialidase treatment revealed that sialic acids in these oligosaccharides are linked at theC-

3 and C-6 positions of galactose residues (data not shown). Therefore, the sialyl linkages resistant to the viral sialidase are considered to occur as the NeuAca24Galgroup.
DISCUSSION

Several glycoproteins have recently been produced by using recombinant techniques (15-17). To our knowledge, however, this is the first case to have analyzed comparatively the naturally occurring and the biotechnologically produced glycoproteins on thefine structural basis. Both rHuEPO and urinary HuEPOcontained tetraantennary oligosaccharides as major components. The residual oligosaccharides hadbiantennaryandtriantennary sugar chain structures. Both samples were also rich in fucosylated oligosaccharides. In addition to these similarities, some differences were also found in the sugar chains of the two erythropoietin samples. The total amount of complex-type oligosaccharides with N-acetyllactosamine repeating units in their outer chain moieties accounted for 34.5% in the case of rHuEPO, which was approximately five times higher than that of urinary HuEPO (7.5%).All sialic acid residues in the sugar chains of rHuEPO occur as theNeuAca2-3Gal group, while about 60% of those of urinary HuEPO occur as the

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TABLE I11

Methylation analysis of acidic (A) and asialo-ol&osaccharide(AN) fractions derived from rHuEP0
Molar ratio"

A

AN

Fucitols 2,3,4-Tri-O-methyl (1,5-di-Oacetyl) Galactitols 2,3,4,6-Tetra-O-methyl (1,5-di0-acetyl) 2,4,6-Tri-O-methyl (1,3,5-tri-Oacetyl) Mannitols 3,4,6-Tri-O-methyl (1,2,5-tri-0acetyl) 3,6-Di-0-methyl (1,2,4,5-tetra0-acetyl) 3.4-Di-0-methyl (1,2,5,6-tetra0-acetyl) 1.0 2,4-Di-O-methyl (1,3,5,6-tetra0-acetyl) 2-N-Methylacetamido-2-deoxyglucitols 1,3,5,6-Tetra-O-methyl (4mono-0-acetyl) 3,6-Di-O-methyl (1,4,5-tri-0acetyl) 1,3,5-Tri-O-methyl (4,6-di-Oacetyl)

0.8 0.8
3.0

0.9 3.3 0.6
0.3

0.2
0.9

0.9 0.8

0.8
1.0

0.2
5.0

0.2 5.0 1.0

1.0

a Numbers were calculated by taking the value for 2,4-di-O-methylmannitol as 1.00.

Glycosylation of proteins primarily depends on the level of glycosyltransferases in the cells and is also affected by their primary amino acid sequences. The difference in sialyl linkages detected in the two erythropoietin samples may be the result of the former situation. In accordance with rHuEPO, G protein of vesicular stomatitis virus grown in CHO cells (44,45) and recombinant y-interferon produced in CHO cells (17) have been shown to contain only the NeuAcaBj3Gal linkage. Therefore, the exclusive expression of this sialyl linkage may indicate that CHO cells lack CMPNeuAc:Gal@l-t4GlcNAca2+6sialyltransferase. All of the oligosaccharides detected in rHuEPO were also found in urinary HuEPO. This results also suggests the similarity of biosynthetic background between CHO cells and erythropoietinproducing cells in the human kidney. Alternatively, large parts of sugar chain structures may be regulated by the primary amino acid sequence of peptide. The major oligosaccharides of rHuEPO have tetraantennary structure, and considerable amounts of N-acetyllactosamine repeating structures were detected. However, such structures have been detected neither in y-interferon produced in CHO cells (17) nor in vesicular stomatitis virus grown in CHO cells (40, 41). Instead, yinterferon exclusively expressed biantennary structure, and vesicular stomatitis virus expressed biantennary and 2,6branched triantennary structures. On the basis of these results, it seems likely that theprimary amino acid sequence of polypeptide moiety is another important factor controlling the synthesis of sugar chains.
Acknowledgments-We would like to express our gratitude toYu-

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miko Kimizuka for her skillful secretarial assistance and to N. Inoue NeuAca2-+3Galgroup and the others as the occur NeuAcaZ6Gal linkages. Despite these dissimilarities, the most impor- for his technical aids. tant evidence is that the oligosaccharides found in rHuEPO all REFERENCES were included in urinary HuEPO. The absence of unusual 1. Graber, S. E., and Krantz, S. B. (1978) Annu. Rev. Med. 29,51sugar chains in rHuEPO is favorable for the clinical applica66 tion of this hormone, since we do not need to take any account 2. Krantz, S. B., and Goldwasser, E. (1984) Proc. Natl. Acad. Sci. of antigenicity on its sugar moiety. It has been shown that U. S. A, 81. 7574-7578 sialidase digestion of urinary HuEPO results in theloss of its 3. Koeffler, H. P., and Goldwasser, E. (1981) Ann. Intern. Med. 97, biological activity in vivo because of hepatic clearance (10, 44-47 4. Brown, R. (1965) Br. Med. J. 2, 1036-1038 11). Therefore, the fact that rHuEPO contained no neutral 5. Eschbach, J., Mladenovic, J., Garcia, J., Wahl, P., and Adamson, oligosaccharides might also be important for its clinical apJ. (1984) J. Clin. Invest. 74, 434-441 plication. 6. Miyake, T., Kung, C. K.-H., and Goldwasser, E. (1977) J. Biol. The functional role of the sugar moiety of erythropoietin Chem. 262,5558-5564 has not been resolved well, although its physiological signifi7. Jacob, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, cance was suggested by several studies. Sialic acid residues of R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fritsch, erythropoietin are important not only for escaping from the E. F., Kawakita, M., Shimizu, T., and Miyake, T (1985) Nature . 313,806-810 hepatic clearance system of asialoglycoproteins but also may 8. Lin, F. K., Suggs, S., Lin, C. H., Browne, 3. K., Smalling, R., contribute to theconformational stabilization, since asialoerEgrie, J. C., Chen, K. K., Fox, G. M., Martin, F., Stabinsky, Z., ythropoietin becomes sensitive to heat denaturation and trypBadrawi, S. M., Lai, P. H., and Goldwasser, E. (1985) Proc. sin digestion (12). Desialylation does not decrease the i vitro n Natl. Acad. Sci. U. S. A. 82, 7580-7584 activity of EPO but rather stimulates it when assayed at low 9. Egrie, J . C., Strickland, T. W., Aoki, K., Cohen, A. M., Smalling, concentration (12, 13). Thus, it is possible that the degree of R., Trail, G., Lin, F. K., Browne, J. K., and Hines, D. K. (1986) Immunobiology 172, 213-224 sialylation affects the physical and biological properties of this glycoprotein. Recently, Dordal et al. (14) have shown that 10. Lowy, P. H., Keighley, G., and Borsook, H. (1960) Nature 186. 102-103 digestion of erythropoietin with endoglycosidase F or mixed 1 . Goldwasser, E., Kung, C. K.-H., and Eliason, J. (1974) J . Biol. 1 glycosidases from Diplococcus pneumniae results in the comChem. 249,4202-4206 n plete loss of its i vivo activity, but approximately 50% of its 12. Briggs, D. W., Fisher, J. W., and George, W. J. (1974) Am. J . n activity i vitro and immunoreactivity still remain. It has Physiol. 227,1385-1388 been suggested that the sugar chains are located at or near 13. Schooley, J. C. (1985) Ezp. Hematol. (N. Y.)13,994-998 the binding domain of erythropoietin for its target cells (39). 14. Dordal, M. S., Wang, F. F., and Goldwasser, E. (1985) Endocrinology 116,2293-2299 Therefore, it is likely that thesugar moiety of erythropoietin contributes to its biological function. Availability of the suf- 15. McCormick, F., Trahey, M., Innis, M., Dieckmann, B., and R h gold, G. (1984) Mol. Cell. Biol. 4, 166-172 ficient amount of rHuEPO and the structural information of 16. Kaetzel, D. M., Browne J. K., Wondisford, F., Nett, T. M., its sugar moiety as obtained in this study will help us to Thomason, A. R., and Nilson, J . H. (1985) Proc. Natl. Acad. resolve the functional roles of the sugar moiety of erythropoSei. U. S. A. 82, 7280-7283 17. Mutsaers, J. H. G. M., Kamerling, J. P., Devos, R., Guisez, Y., ietin in the future.

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18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Fiers, W., and Vliegenthart, J. F. G. (1986) Eur. J. Biochem. 166,651-654 Uchida, Y., Tsukada, Y., and Sugimoto, T. (1974) Biochem. Bwphys. Acta 360,425-431 Li, Y.-T., and Li, S.-C. (1982) Methods Enzymol. 2 8 , 702-713 Glasgow, L. R., Paulson, J. C., and Hill, R. L. (1977) J. Biol. Chem. 262,8615-8623 Kitamikado, M., Ito, M., and Li, Y.-T. (1982) Methods Enzymol. 83,619-625 Amano, J., and Kobata, A. (1986) J. Biochem. (Tokyo) 99,16451654 Paulson, J. C., Weinstein, J., Dorland, L., van Halbeek, H., and Vliegenthart, J. F. G. (1982) J.Bwl. Chem. 2 5 7 , 12734-12738 Kochibe, N., and Furukawa, K. (1982) MethodsEnzymol. 8 3 , 373-377 Yamashita, K., Totani, K., Ohkura, T., Takasaki, S., Goldstein, I. J., and Kobata, A. (1987) J. Biol. Chem. 2 6 2 , 1602-1607 March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anal. Biochem. 60,149-152 Yoshima, H., Takasaki, S., and Kobata, A. (1980) J. Biol. Chem. 256,10793-10804 Mizoguchi, A., Takasaki, S., Maeda, S., and Kobata, A. (1984) J. Bwl. Chem. 259,11949-11957 Takasaki, S., Mizuochi, T., and Kobata, A. (1982) Methods Enzym01. 83,263-268 Takasaki, S., Murray, G. J., Furbish, F. S., Brady, R. O., Barranger, J. A., and Kobata, A. (1984) J. Biol. Chem. 2 5 9 , 1011210117

31. Yamashita, K., Mizuochi, T., and Kobata, A. (1982) Methods Enzymol. 83,105-126 32. Yamashita, K., Kochibe, N., Ohkura, T., Ueda, I., and Kobata, A. (1985) J. Biol. Chem. 260,4688-4693 33. Ogata, S., Muramatsu, T., and Kobata, A. (1975) J. Biochem. (Tokyo) 78,687-696 34. Endo, Y.,Yamashita, K., Tachibana, Y., Tojo, S., and Kobata, A. (1979) J. Biochem. (Tokyo) 85,669-679 35. Ohkura, T., Yamamshita, K., Mishima, Y., and Kobata, A. (1984) Arch. Bwchem. Biophys. 236,63-77 36. Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., and Kobata, A. (1981) J. Biol. Chem. 256,8476-8484 37. Takasaki, S., and Kobata, A. (1986) Biochemistry 25,5709-5715 38. Yamashita, K., Ohkura, T., Yoshima, H., and Kobata, A. (1981) Biochem. Bwphys. Res. Commun. 100,226-232 39. McDonald, J. D., Lin, F. K., and Goldwasser, E.(1986) Mol. Cell. Biol. 6,842-848 40. Stanley, P., Vivona, G., and Atkinson, P. H. (1984) Arch. Bwchem. Biophys. 2 3 0 , 363-374 41. Campbell, C., and Stanley, P. (1984) J.Biol. C h m . 2 6 1 , 1337013378 42. Yamashita, K., Ueda, I., and Kobata, A. (1983) J. Biol. Chem. 2 5 8 , 14144-14147 43. Lai, P.-H., Everett, R., Wang, F.-F., Arakawa, T., and Goldwasser, E. (1986) J. Biol. Chem. 2 6 1 , 3116-3121 44. Takeuchi, M., Takasaki, S., Inoue, N., and Kobata, A. (1987) J. Chromatogr. 400,207-213 45. Davis, J. M., Arakawa, T., Strickland, T. W., and Yphantis, D. A. (1987) Biochemistry 26,2633-2638
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l l n k e d S u g a r C h a i n s of Human E r y t h r o p o i e t i n s P u r l f i e d f r o m tirlne a n d the Culture Medium ofRecombinantChinese Hamster ovary Cells

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EXPERIMENTAL PROCEDURES

RESCLTS

Pepuvuliun OlEPO-rHUEPO w a s p u r l l i e df r o mt h em e d i a o f r e c o m b i n a n t CHO c e l l r c u l t u r e by the method Davis of 1 4 5 1 . U r i n a r y HuEPO W B E c o n c e n t r a t e d f r o mu r i n e o f a p l a s c i c anemla patlencs by t h ep h e n o l p-ammo s a l i c y l a t e t r e a t m e n t followed by e t h a n o lp r e c i p i t a c i o na c c o r d i n g to t h e meLhod of Mlyakc er 01. ( 6 ) , and p u r z i i e d by a i f i n i c yc h r o m a t o g r a p h yu s i n g an inmobillred column. Each p r e p a r a t i o n showed s l n g l e band on s o d l v n monoclonal ilbody m dodecyl ulfate-polyacrylamide S gel eleccropharesks.

Releure O r lhc Aspa,.uqine-Ltnked Sugar Cholns os OligDsocchorides ftmm HuEPOIen mllllgrams a i ~ H u E P O a n d t h r e e m i l l i g r a m so fu r i n a r y HuEPO w e r e s u b j e c t e d t o h y d r a z i n o l y s i s a s d e s c r i b e dp r e v l s u s l y (29). The a l i g o s a c c h a . r i d e f r a c t i o n s were f r e e d Cram t h e mucln-type o l q o s a c c h a r i d e s by paper c h r a m s c a g r a p h v using s o l v e n t 11. O n e - f i f t h of the o l l g a s a c c h a r i d ef r a c t i o n from rHuEPG and a h a l f a i c h a tf r o m u r i n a r y HuEPO were l a b e l l e d w i t h t r i t i u m by NaBI'H!. r e d u c t i o n . The r e m a i n i n go l i g o s a c c h a r i d e f r a c t i o nf r o m rHuEPO v a s r e d u c e d by NaBI'HI. i no r d e r t o o b t a i ns a m p l e sf a r m e ~ h y l a t l o na n a l y s i s .

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Case 1:05-cv-12237-WGY
3662

Document 547-36

Filed 06/22/2007

Page 7 of 8

Sugars of Recombinant andErythropoietins Native
SITUCIUPUI Anolysts of Olzgorclcchor'Lde tn FmCfmn AN6-By sequential digestion w i r hj a c kb e a n$ - g a l a c t o r i d a r ea n d jack bean 8-N-acetylhexosaminidase. four r e s i d u e se a c ho fg a l a c r o s ea n dN - a c r r y l g l u c o s a m i n e were r e l e a s e d f r o m r a d i o a c c i v e AN6 ( d a t a nor shown). The p r o d u c t was t h e n o n v e r r e d c t o che f v c o s y l a c e dc r i m a n n o s y l core bythesecondcycleof sequential d i g e s t i o n w i t h of g a l a c t o s ea n dN - a c e c y l g l u c o t h e two enzymes releasing t w o r e s i d u e se a c h samine ( d a t a not shown). These reiutls I n d i c a t e dt h a tt h el r a c r i a n AN6 c o n t a i n s t e r r a a n f e n n a r yo l i g o s a c c h a r i d e sw i c h two Ga181*4GlcNAcB1-.3Ca18114 G ~ C N A ~ B ! . outer When AN6 was d i g e s t e d e h d o - e - g a l a c t o s i d a sic .s witn c size d e c r e a s e d by a p p r o x i m a t e l y 9 glucose u n i t i n d l c a t i n gh a t s t two G a l o l r 4 G l c N A c e l - ~ 3 G ~ 1g ~ o u were r e l e a s e d by t h i s treatment !Fig. 7 A ) . Two ps o f w w l y exposed acetylgl glucosamine were t h e n removed by d i g e s t l o " w i ~ h bean jack e - ~ - a c e ~ y l h e x o s ~ m i n l d a( F i g . 8 ) . se 7 The r a d i o a c t i v e r o d u c t p a t t h i s stage showed t h e same size a s a u t h e n r l c Gal:.GlcNAc2.Nanl.GlcNAc. F ~ ~ . G ~ c N~ Ac a i i s cone mannose r e s i d u e was removed e~~~. from t h er a d i o a c t i v e pIOducc by i n c u b a t i o n w i r h 2 u n i t s o f jackbean,>-mannosidase(Fig.7C).but no^ byincubaLionwitho-mannosidase I 1 from A s p e r g ~ l l u s1 m 1 0 1 which c l e a v e s n o n - s u b s t i r u t e d Mano!+3 l i n k g e b u r n o t " o n - s u b s t i t u t e d Manol-6 l i n k a g e ( 2 2 ) . t h e structure o ft h er a d i o a c r i v ep e a ki n F i g . 7B was eSCimaced a s f o l l o w :

P i - a c e t y l g l u c o s a m i n er e s i d u e s by d i p l o c o c c a l O-N-acetylhexolaminidasc digestion ( F i g .3 8 )a n d chhen one N - a c e t y l g l u c o s r m l n er e s i d u e by jackbean ~ - N - a c e L y l h E x o s a m i n i d a s ed i g e s t i o n F i g 3 C ) . ( . On che b a s i s o l t h e u b s t r a c e s s p e c i i i c i i yt h a td i p l o c o c c a l j-N-acerylhenosaminidase c l e a v e s GlcNAcSl-2 l i n k a g e s i n t h e GlcNAcH1+2Man g r o u pa n dr h e GlcNAcB1-.2(61cNAc31*4)Man group b u t nor i n t h e GlcNAc~1.2!GlcNAc~lt6)Man g r o u p 1 3 8 1 , AN2 is supposed to have the .4-branched riancennaiy 2 c sugar c h a i nS t w c t w e . That A N 1 was r e c o v e r e d a s t h e r e t a r d e df r a c t i o nf r o m a DSA-Sfpharose column a l s o s u p p o r t e d t h i s s t r u c ~ u r e .b e c a u s e2 , 4 - b r a n c h e d rridncennary o l i g o s a c c h a r i d er e c a r d si nt h e DSA-Sepharosccolumnwhile2.6-branchedcrianrennaryoligosaccharldcstrongly b i n d st ot h e column (25). The I a c a L i o n o ft h e2 , i - b r a n c h was d e t e r m i n e d by LWO c y c l e s o f Smith d e g r a d a t i o n . By t h e first c y c l eo f S m i t hd e g r a d a t m n , r a d i o a c t i v e A N > was c o n v e r t e d t o r a d i o a c t i v e GlcNAcaltZ(ClcNAciil-4)Mani~l-3 or 6Maniil-4GlcNAcS1-4 GIcNAcoT. I L was t h e n converred r o Man~.GlcNAc.XylNAc0~ by Lhe s e c o n d y c l e c of S m i t h e g r a d a t i o n d a t a d ( n o r s h o w n ) h e s e e s u l t sn d i c a t e dh a fh e T r i tt 2 . 4 - b r a n c h i s l o c a r e d on t h e Manol-3 s i d e . Therefore. t h e structure of AN2 s h o u l db e d s shown m T a b l e I:
Slr'ucturol Anolys18 or Oilgosaccharide I WFmctton A N 3 - A 5 i d l o - 0 1 i g ~ ~ d ~ ~ h d ~ i d e AN3 was Supposed to have2.6-branchedCriantennary sugar c h a i n S L ~ U C C U T ~ on t h e b a s i s of i t s elution p o s i t i o n upon Bio-Gcl P - 4 column chromatography of i t s strong a f f i n i r y c o a DSA-Sepharose column. Thi. ( F i g . 2E) and a s s u m p t i o n was c o n f i r m e d by c h ef o l l o w i n g experiments. When AN3 was d i g e s t e d w l c hj a c kb e a na - g a l a c r o s l d a s e .t h r e eg a l a c t o s er e s i d v e s were r e l e a s e d ( F i g . 4 A ) . The r a d i o a c t i v ep r o d u c t was t h e nd i g e s t e d w i c h d i p l o c o c c a l - N - a c e t y l E h e x o s a m i n i d a s e . A s shown ~n F l g . 4 8 , one N - a c e t y l g l u c o s a m i n e e s i d u e r was r e l e a s e d by c h i s treatment The r e s u l t i n g a d i o a c t i v e l i g o s a c c h a r i d e r o was f i n a l l y c o n v e r t e d co t h ef u c o s y l a i e dt r i m a n n o s y l c o r e a f c c r d i g e s t i o nw i c h j a c kb e a n B-N-acerylhexosaminidase by r e l e a s i n g two N - a c e t y l g l u c o s a m i n e r e s i d u e s( F i g . 40). These r e s u l t s c o i n c i d ew i r ht h ed i g e s t i o np a t f e z " of 2 . 6 - b r a n c h e d trlantennary sugar chains a s a l r e a d y r e p o r t e d ( 3 8 1 . When t h e r a d i o a c t i v eD r o d u c fi nF i e . 4 8 w a s d i e e s c f dw i t ho - m a n n u s i d a s e I: from

Manl.GlcNXc.Fuc.GlcNAcO~ upon Bio-Gel P - 4 column chromatography (Fig. 2C). When AN2 was d i g e s t e dw l t hj a c kb e a nO - g a l a c t o s i d a s e .r h r e eg d l a c t o s y l r e s i d u e s wcrc removed ( F i g . A ) . 3 The r e s u l t i n go l i g o s a c c h a r i d e was f i n a l l y c o n v e r t e d L O t h ef u c o s y l a t e dr r i m u n n o s y l core w i t h the r e l e a s eo f LWO

Structural Analysis of Ollyosocehartde I" Fr-aCL~onAN2-A~ialo-oligasacchrride AN2 was e l u t e d a t che same p o s i ~ ~ o n authentic2.4-branchedGa13.GlcNAcl. as

Fucsl

This estimation was f v r t h c r c o n f i r m e d by r h ef o l l o w i n ge x p e r i m e n t a l e v i d e n c e s .D i g e s t i o n o f t h er a d i a a c c i v ep r o d u c ti nF i g . 7B w i t h a mixcure o f d i p l o c o c c a l B - g a l a c t a s i d a s e a n dd i p l o c o c c a l B-N-ace~ylhexosaminidasc r e l e a s e d t w o g a l a c t o s e r e s i d v e sa n d one N - a c e t y l g l u c o s a m i n er e s i d u e( F i g .7 0 ) .a n dt h e r e m a i n i n g m e N - a c e t y l g l u c o s a m i n er e s i d u e w a s removed by inCubatic..withjack core bean 8 - N - a c e t y l h e x 0 ~ a m i n i d a s r c o p r o d u c et h ef u c o s y l a t e dc r i m a n n o s y l

on t h e Mannl-6 arm of t h ec r i m a n n o s y l

core.

Slrucluml Anolysrs or Oligoaoccharide m F#ucfmn A N 4 "Jack bean 6-galactosid a s ed i g e s t i o no f AN4 r e s u l t e di nt h e release of f o u r g a l a c t o s y l residues [ r i g . 5A). The r a d i o a c t i v e r o d u c c p was t h e n o n v e r t e d c m t h ef u c o s y l d t e d r r i m a n n o s y l core w i t h r e l e a s e of m e 2nd t h r e eN - a c e r y l g l u c o s a m i n er e s i d u e s b yJ e q u c n r i a l digestion w i t hd i p l o c o c c a le n d j a c k beanB-N-acecylhenosaminid a s e , respectively ( F i g . 58 and These C). results indicated hat he c S L ~ U C C U W of AN4 i n G a l d l - 4 G l ~ N A c 6 1 - 6 I G a l U 1 - 6 G l c N A c Q ! - Z ~ M ~ n u l or 3lGallil-4 ~6 C l c N A c l l l t 4 ~ G l c N A c l i 1 1 4 G i i N A c l l 1 . 2 ~ M a n n l - 01 6)ManOltLGlcNAcBl+i(Fucal+b) 3 GIcNAcoT. Two c y c l e s of S m i t hd e g r a d a t i o n of A 4 gave c h r same r a d i o a c t i v e N p r o d u c t s a s i nt h e c a s e o f AN2 ( d a t a n o t s h o w n ) ,i n d i c a t i n g that t h e 1 . 4 - b r a n c h i s e x c l u s i v e l yl o c a t e d on che Man.il-3 arm of t h et r i m a n n o s y l core. T h e r e f o r e , A 4 s h o u l dh a v et h e N structure a s shown i n T a b l e 11.
SI,.UCIUIUI y s t s of 01qoracchot'lde.s in Fmctio!l AiVS--Sequen~ial Anal digrstxon of AN5 w i t hj a c kb e a n3 - g a l 2 c c 0 s i d a s ea n dj a c k bean 6-N-acrtylhea0saminidase r e l e a s e d f o u r r r s l d u r sc e c h of galactose and N-acecylglucosamlne ( d a t a not shown). The r a d i u e c t i v e r a d u c c p a t t h i s s t a g e showed t h e Same m o b i l i t y m Biu-Gel P - 4 column a s a u r h e n c l c C a l . G l c N A c . M a n , . G l c N A c . P u c . G l c N A ~ ~ ~ I d a t & not shown). I t was t h e n o n v e r t e d c to t h ef u c o s y l r r e df r i m n n 0 S y l core by che s e c u n dc y c l e uf s e q u e n t i a ld i g o s t i o nw i t ht h e two e n z y m e sr e l e a s i n g one r e s i d u e each of g a l a c t o s e and Pi-acetylglucosamine ( d a t a not shown). These resulcs i n d i c a t e d c h a t AN5 i s a t e t r a a n t e n n a r yc o m p l e x - t y p eo l i g u r a c c h a r z d e t r m a n n o s y l core, an o u c e r c h a m of which i s i h e Gal: w i c ht h ef u c o s y l a t e d l~PGlcNAc,!1*3Gal~il~4GlcNAcDl-. r r i s a c c h a r i dree s i d u e . a l , ~ l + 4 G l c N A c ~ l * 3 A C G a l was r e l e a s e d f r o m c h i s d i - N - a c e ~ y l l a c t 0 S a m i n e m o i e t y by e n d o - 8 - g a l a c c o s i 6A). The newly e x p o s e d - a c e t y l g l u c o s a m i n e e s i d u e N r was d a s ed i g e s t i o n( F i g . t h e n removed by j a c k e a n b 3 - N - a c e t y l h r x o s d m i n i d a s e d i g e s t i o n( F i g . dB). The f u l l o w i n g four p o a ~ z b l e s t r u c t u r e s were considered f o r t h er a d l o a c r l v r F i g . 6B. producc in

S~PUCLY~~S o i Actd Oligosoechnrlder-In o r d e r to o b t a i n t h e l n f o m a t i o n a s L O t h es i a l y ll i n k a g e sm e c h y l a r i o na n a l y s i so ft h et o t a la c i d i co l i g o s a c c h a r i d ef r a c t i o n ( f r a c k A) was p e r f o r m e d . As shown m T a b l e I l l . t h e f r a c t i o n g a v e a l m o s tt h e same results a s a . i a l a - 0 l i g o s a c c h a r i d ef r a c t i o n (AN) e x c e p tf o rg a l a c t i c 0 1d e r i v a t i v e s .A p p r o x ~ m a c e l y2 0 1o ft h eg a l a c i o s co f f r a c t i o n A occur a s " o n - r e d u c i n gt e r m i n ia n dc h er e m a i n d e r a s - 3 G a l l + . The amount of r h e 2 , 4 . 6 - r r i - O - m e c h y l g a l a c c i t o l was prominently d e c r e a s e d a f t e r d e s i a l y l a t i o n . The d e c r c a s e was, compensated the by increase o f 2 . 3 , 4 . 6 t e r r a - 0 - m e t h y lg a l a c f i t o l .T h e s er e s u l t si n d i c a t e dc h a t almost a l l s i a l i c a c i d a r e l m k e d ac t h ec - 3p o s i t i o no ft h et e r m i n a lg a l a c t o s er e s i d u e so ft h e neutral o l i g o s a c c h a r i d e s . t h e S C ~ Y C ~ U I -of S which were e l u c i d a t e d by t h e ~ 11). s t u d i e sd e s c r i b e da l r e a d y( T a b l e Mcrhylacion analysis of the o l i g o s a c c h a r i d e f r a c t i o n o b t a i n e d from u r m a r y HuEPO b e f o r e a n d a f t e r s i a l i d a s r d i g e s t i o n r e v e a l e d c h a t d i f f e r e n c e was dececfrd nly o i n galactose d e r i v e r i v e s . 3 . 4 - n d , 4 , 6 - t r i - O - m c r h y l 2 a2 g a l a c t i i o l s were c o n v e r t e d t o 2 , 1 , 4 , 6 - t e r r a - O - m e r h y l g a l a c t i t a l r ( d a t a not shown). These r e s u l t s i n d i c a c r dt h a ts i a l i ca c i d s i n t h i s sample are l i n k e d b o t h at t h e C - I and C-6 p o s i t i o n s of the t e r m i n a l g a l a c t o s e .

Downloaded from www.jbc.org by on April 25, 2007

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Galil-.4GlcNAc~<l~ D i g e s t ~ o nof c h er a d m a c c i v ep r o d u c t m F L ~ .68With a m i x t u r e of d i p l o c o c c a l ~ - g e l a c r o s i d a s ea n dd i p l o c o c c a l 8 - N - a c c f y l h e x a s a m i n i d a s e produced t w o radioactive ompanrntand c s 8 i n a m a l a r r a t i o o f 1 - 2 ( F i g6 C ) . . Both o f core by j a c k t h e s e componencs were c o n v e r t e d t o t h ef u t o s y l a t e dr r i m a n n o s y l bean ? - ~ - a c e c y I h e x o s a m i n i d a s e d i g e s t i o n r e l e a s i n g two and one N - a c e t y l g l u c a samine r e s i d u e respectively I F l g6 0 ) . . Based on ;he s u b s t r a t e S p e c i f i c i t y o f diploCOCCa1 3 - N - a c e ~ y l h e x 0 5 a m i n i d a s e L I B ) t h er a d i o a c c i v e componcnr u i nF i k . 6C s h P v l db ee i t h e r one or b o t h of t i e f o l l o w i n s t w o o l i g o s a c c h a r i d e s I a n d :V , which were C O b e d e r i v e d from o l i g o s a c c h a r i d e s I a n dI V ,r e s p c c t i v e l y . Fucol

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Case 1:05-cv-12237-WGY

Document 547-36

Filed 06/22/2007

Page 8 of 8
3663

Sugars of Recombinant and Natiue Erythropoietins

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