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

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DECLARATION re #1162 Brief of Harvey F. Lodish, Ph.D. in Support of Bench Memorandum and Offer of Proof Regarding No Obviousness-Type Double Patenting by Amgen Inc.. (Attachments: #1 Exhibit A#2 Exhibit B#3 Exhibit C#4 Exhibit D#5 Exhibit E#6 Exhibit F#7 Exhibit G#8 Exhibit H#9 Exhibit I#10 Exhibit J#11 Exhibit K#12 Exhibit L#13 Exhibit M#14 Exhibit N#15 Exhibit O)(Rich, Patricia)

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UNITED STATES DISTRICT COURT DISTRICT OF MASSACHUSETTS ) ) ) Plaint iff, ) ) v. ) ) ) F. HOFFMANN-LAROCHE ) LTD., a Swiss Company, ROCHE ) DIAGNOSTICS GmbH, a German ) Company and HOFFMANN LAROCHE ) INC., a New Jersey Corporation, ) ) Defendants. ) __________________________________________) AMGEN INC.,

Civil Action No.: 05-12237 WGY

DECLARATION OF HARVEY F. LODISH, PH.D. IN SUPPORT OF AMGEN'S BENCH MEMORANDUM AND OFFER OF PROOF REGARDING NO OBVIOUSNESS-TYPE DOUBLE PATENTING

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TABLE OF CONTENTS PAGE NO. I. BACKGROUND ..................................................................................................... 1 A. B. Level o f Skill in the Art ................................................................................ 5 Before Dr. Lin's inventions, a person of ordinary skill in the art attempting to produce in vivo bio logically active recombinant EPO would have been confronted with many possible approaches, great uncertainty as to each, and no reasonable expectation that any particular approach would succeed ............................... 6 1. 2. Little was known about the structure of erythropoietin ...................... 7 In 1983-84, the glycosylation of erythropoietin was known to be important for biological function, but its structure was unknown.................................................................... 10 The cellular source of erythropoietin was unknown......................... 13 Reco mbinant expression of glycoproteins was in its infancy in 1983-84 .......................................................................... 14

3. 4. C.

A person of ordinary skill in the art in possession of a DNA sequence encoding EPO in 1983-84 would still have lacked a reasonable expectation of success in producing in vivo bio logically active EPO outside the human body absent proof of successful prior production of such EPO..................................................... 21 The references cited by Roche's experts during discovery do not establish that an ordinarily skilled artisan in 1983-84 would have had a reasonable expectation of success in expressing in vivo bio logically active EPO .............................................................................. 28 1. 2. Art icles relating to general tools or methods for protein expression in mammalian cells........................................................ 28 Art icles discussing recombinant expression of some mammalian glycoproteins in mammalian cells ................................ 30

D.

II.

THE INVENTIONS CLAIMED IN DR. LIN'S `868 AND `698 PATENTS ARE PATENTABLY DISTINCT FROM THE INVENTIONS CLAIMED IN DR. LIN'S `008 PATENT...................................... 56 A. B. Legal Standard ........................................................................................... 56 Summary o f non-obviousness of `868 and `698 patent claims over `008 patent claims ...................................................................................... 57 i

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C.

The claims of Dr. Lin's `008 patent are directed to EPO DNA and host cells transfected with EPO DNA that have certain desired capabilities, but the `008 claims do not describe EPO with in vivo bio logical funct ion, or processes for making same ...................................... 60 `868 Claims 1 and 2 are patentably distinct from `008 Claims 2, 4, 6, 7, 25, and 27 ....................................................................................... 71 `698 Claims 6-9 are patentably distinct from `008 Claims 2, 4, 6, 7, 25, and 27............................................................................................... 72 My opinio ns in the In re Columbia University Patent Litigation case are consistent with my opinions in this case ........................................ 74

D. E. F. III.

THE CLAIMS IN DR. LIN'S `933, `349 AND `422 PATENTS FALL WITHIN GROUPS I, IV AND V OF THE 1986 RESTRICTION REQUIREMENT IN DR. LIN'S `298 APPLICATION ......................................... 77

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I. BACKGROUND 1. I am a Professor of Biology and Professor of Bio Engineering at the

Massachusetts Institute of Technology (MIT) and a Member of the Whitehead Institute for Biomedical Research. I am submitting this declaration in support of Amgen's Bench Memorandum and Offer of Proof Regarding Obviousness-Type Double Patenting. If called to testify as to the truth of the matters stated herein, I could and would do so competently. 2. A copy of my curriculum vitae, reflecting my professional experience,

affiliations, and work has previously been filed as Docket Item ("D.I.") 502, Ex. A. 3. I received an A.B. degree summa cum laude from Kenyon College in 1962, and a

Ph.D. from the Rockefeller University in 1966. I was a post-doctoral Fellow at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England from 1966 to 1968. I held the positions of Assistant Professor and Associate Professor at the Massachusetts Institute of Techno logy (MIT) during the years 1968-71 and 1971-76, respectively. Since 1976, I have been a full Professor of Biology at MIT and since 1999 Professor of Bioengineering. In 1982, I became a Founding Member of the Whitehead Institute for Biomedical Research. 4. Since 1961, I have authored or co-authored more than 500 scientific publications,

in a variety of peer-reviewed scientific journals, as detailed in D.I. 502, Ex. A. 5. I was elected to the National Academy of Sciences in 1987. In 2004, I was

President of the American Society for Cell Biology, an international organization of more than 10,000 scientists. I have also served on a variety of external advisory boards and grant review panels. A complete list is provided in D.I. 502, Ex. A. 6. As described in detail in my curriculum vitae, I have been a researcher, a teacher,

a writer, and an editor in the fields of molecular and cellular biology for over 35 years. Adherence to the scientific method is the common thread that runs through all the aspects of my 1

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career. Excellence is achieved in the field of science through application of the logical principles and philosophies shared by the scientific community. Like other scientists, through study and practice I have collected a set of analytical tools that I use to address all scientific problems. For example, I apply these tools when I evaluate others' work. If others do not rigorously apply scientific methodology, I properly discount their assertions. 7. In the course of my career, I have taught many M.I.T. undergraduates, Ph.D.

students, and post-doctoral fellows. Imparting an understanding of proper scientific method is one of my major goals. More specifically, I teach students how to formulate testable hypotheses, how to design and perform well-controlled, repeatable experiments to validate hypotheses, and to evaluate experimental outcomes objectively. It is only by understanding and applying the scientific method rigorously that students can develop into scientists whose work will withstand the scrutiny of the scientific community and advance scientific knowledge. 8. I have served on the Editorial Boards for many peer-reviewed scientific journals.

For example, I was a member of the Board of Reviewing Editors of the journal Science fro m 1991 to 1999, and a Member of the Editorial Board of the journal Proceedings of the National Academy of Sciences fro m 1996 to 2000. Furthermore, I have reviewed hundreds of articles for publication in many different journals. When I review papers for potential publication, I must consider critically whether the work is well conceived, controlled, and performed in order to establish whether its scientific conclusions are correct. Additionally, I consider whether the work is sufficiently described such that other workers in the field can repeat, confirm, and extend the reported findings. 9. I am the principal editor and author of the textbook MOLECULAR CELL

BIOLOGY, now in its Fifth Edition.1 The Sixth Edition has just been published. In addition to

1

See Lodish et al., Molecular Cell Biology, 5th Ed. W.H. Freeman Co., New York. 2

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my seven co-authors, literally dozens of our scientific colleagues from around the world have contributed to chapters, and reviewed and commented on the manuscripts. This textbook has been relied upon by scientific researchers, undergraduate students, and graduate and medical students all around the world since the publication of our First Edition in 1986. The Fifth Edition has been translated into six languages. It is considered one of the most authoritative resources in the fields of molecular and cellular biology. The textbook presents a comprehensive, authoritative review of the fields of molecular and cellular biology, and is intended for advanced undergraduates and graduate and medical students. In the course of preparing my book over the past 20 years, I have comprehensively studied, in detail, the published literature to determine what experimental work is new, significant, and sufficiently credible to merit reliance by the scientific community at large. 10. In the course of my career as a researcher, I have personally applied the scientific

method to many different avenues of research, including cell signaling, protein synthesis, cell membranes and their formation, cell death, fat cell biology, and, most relevant here, blood cell differentiation. One example of my experience in blood cell differentiation is my work concerning the characterization of the murine erythropoietin ("EPO") receptor, the protein on the surface of red blood cell precursors that binds to EPO and that mediates the activity of EPO in cells and in vivo (in the body). 11. I have been studying glycoprotein synthesis and function in mammalian cells

since about 1976. My laboratory has made several significant contributions to the understanding of the glycosylation process. Prominent examples of our work include first establishing that the addition of oligosaccharides (or "glycans") to asparagines on glycoproteins occurs during the synthesis of the polypeptide and its translocation into the endoplasmic reticulum, and purifying

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and characterizing the hepatocyte asialoglycoprotein receptor, a major component of the system of clearance of glycoproteins from the circulation. 12. Representative examples of my pre-1983 publications in the field of glycosylation

include: Lodish, H.F., et al., "Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein," Birth Defects Orig Artic Ser. 14(2):155-75 (1978); Rothman, J.E., and Lodish, H.F., "Synchronised transmembrane insertion and glycosylation of a nascent membrane protein," Nature 269(5631):775-80 (1977); Lingappa, V.R. et al., A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function," J Biol Chem. 253(24):8667-70 (1978); Rothman, J.E. et al., "Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein," Cell 15(4):1447-54 (1978); Schwartz, A.L. et al., "Difficulties in the quantification of asialoglycoprotein receptors on the rat hepatocyte," J Biol Chem. 255(19):9033-6 (1980); Schwartz, A.L., et al., "Identification and quantification of the rat hepatocyte asialoglycoprotein receptor," Proc Natl Acad Sci U S A. 78(6):3348-52(1981); and Lodish, H.F., and Kong, N., "Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex," J Cell Biol. 98(5):1720-9. I have continued to research and publish in this field to the present day. 13. I was also very involved in cloning genes from several eukaryotic cells, including

human and other mammalian cells, beginning in 1980 and continuing throughout the 1980s to the present day. 14. Moreover, in the early 1980s, I was also particularly interested in the production

of recombinant proteins for therapeutic and industrial purposes. In particular, I was interested in how it would be possible to recapitulate the complex processing of mammalian proteins in

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heterologous expression systems. In 1981, I published a review article on this subject. Lodish, H.F., "Post-translational modification of proteins," Enzyme Microb Technol. 3(3):177-188 (1981). This article demonstrates that I am uniquely qualified to opine on the knowledge and understanding of an ordinarily skilled artisan in the fields pertinent to the claims-at-issue during the 1983-84 time period. 15. During the course of prior litigation involving the patents-in-suit between Amgen

and Transkaryotic Therapies and Hoechst Marion Roussel, I reviewed in detail the patents-insuit, portions of the prosecution histories, and related scientific publications. I testified at trial in connection with that action and prepared several expert reports. 16. Earlier in the present litigation between Amgen and Roche, I submitted two

declarations explaining my opinions regarding certain obviousness-type double patenting issues. (See D.I. 502, D.I. 578.) My prior declarations included as exhibits a number of prior art references, patent documents, and other publications. To the extent I rely on those same documents in this declaration, I have cited the versions previously filed with the Court. In other instances herein, I have cited the trial exhibit ("TX") versions of documents. Documents that have not previously been filed with the Court, either in my prior declarations or as trial exhibits, are attached to this declaration and identified as follows: "9/26/07 Lodish Decl., Ex. __." A. 17. LEVEL OF SKILL IN THE ART I have been asked to consider whether a person of ordinary skill in the art in 1983-

84 (i.e., at the time just before the inventions taught and claimed in Dr. Lin's patents-in-suit) would have found claims 1 and 2 of the `868 patent and claims 6-9 of the `698 patent to be obvious over claims 2, 4, 6, 7, 25 and/or 27 of the `008 patent. A "person of ordinary skill" or "ordinarily skilled artisan" in the field relevant to Dr. Lin's claims would have been a research scientist with a Ph.D. or M.D. and at least two years of postdoctoral research experience in the

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field of molecular biology, cellular biology, or protein expression. As discussed below, it is my opinion that a person of ordinary skill in the art in 1983-84 would have found each of claims 1 and 2 of the `868 patent and claims 6-9 of the `698 patent to be not obvious over each of claims 2, 4, 6, 7, 25 and 27 of the `008 patent. B. BEFORE DR. LIN'S INVENTIONS, A PERSON OF ORDINARY SKILL IN THE
ART ATTEMPTING TO PRODUCE IN VIVO BIOLOGICALLY ACTIVE RECOMBINANT EPO WOULD HAVE BEEN CONFRONTED WITH MANY POSSIBLE APPROACHES, GREAT UNCERTAINTY AS TO EACH, AND NO REASONABLE EXPECTATION THAT ANY PARTICULAR APPROACH WOULD SUCCEED

18.

When Dr. Lin began his efforts to produce in vivo bio logically active EPO using

recombinant DNA techniques, he faced a daunting array of competing choices and difficult problems. The amino acid sequence of EPO was unknown. The DNA sequence of EPO was unknown. The particular cell type(s) in the human body that naturally produce EPO was unknown. The cellular receptor(s) with which human EPO interacts in the human body to produce red blood cells was unknown, and consequently what, if any, recombinant EPO products would interact effectively with the EPO receptor(s) in vivo was unknown. Because the human cell type(s) that naturally produce EPO was unknown, the set of post-translational modifications that are made to EPO polypeptides by those cells was also unknown. Whether any such posttranslat ional modifications were needed to produce a man-made product that would perform the desired function of human EPO in vivo and, if so, which modifications were needed, which if any cell types would in fact produce those modifications -- and only those modifications -- correctly, and, if so, how to identify cells that would reliably do so, were all unknown and unknowable until empirically tested and proven. To the extent that minute amounts of human EPO protein had been isolated from urine, the available product was insufficient to characterize the complete amino acid sequence and carbohydrate structures of the purified product. Even then, such excreted urinary products were necessarily exposed to conditions that would be 6

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expected to alter their composition from natural, biologically act ive EPO protein found in the bloodstream, and therefore could not be relied upon to predict with confidence the actual structure and composition of EPO products needed to achieve EPO's in vivo funct ion in the body. 19. Reco mbinant expression of biologically active human glycoproteins in cultured,

mammalian cells was still in its infancy. In fact, prior to 1984, I am not aware of and Roche has not cited any report of any in vivo bio logically active recombinant human glycoprotein successfully produced in cultured, mammalian cells. While scient ists did understand that glycosylation potentially played an important role in the function of glycoproteins like EPO, they did not know or understand what function(s) it performed, how naturally occurring EPO was glycosylated when it was produced and circulated in the body, or whether differences in glycosylation caused by production in different cell types would affect the biological activit y of EPO and, if so, how. Only as a result of Dr. Lin's successful production of an in vivo bio logically active recombinant human EPO glycoprotein in CHO and COS cells, were scientists then able to explore and begin to resolve these uncertainties. 1. 20. Little was known about the structure of erythropoietin

Erythropoiet in as it is produced in the body is a glycoprotein hormone that

stimulates progenitor cells in the bone marrow to multiply and to differentiate into reticulocytes (immature red blood cells) and then mature red blood cells. This in vivo bio logical function had been established by the work of many researchers over the course of almost 100 years. 21. Before Dr. Lin's ground-breaking inventions provided an abundant source of

high-quality EPO glycoprotein, only minute amounts of human urinary EPO were available. Much of the pre-1984 research on erythropoietin was performed with crude, unpurified material. The primary source of purified EPO before Dr. Lin was from the urine of aplastic anemia

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patients. Miyake, et al., "Purificat ion of human erythropoietin," J. Biol. Chem. 252(15):6538-64. (1977) (D.I. 578, Ex. A). Because there is so little EPO in urine, even from these patients who have much higher levels than normal, very little urinary EPO was available. Moreover, because EPO could not be isolated directly from the blood, there was no way to know whether EPO purified from urine accurately reflected the structure of naturally produced EPO prior to its removal from circulat ion and excretion in the urine. Because excreted urinary EPO is exposed to a different environment than EPO in the bloodstream, one skilled in the art at the time would have understood that urinary EPO is exposed to different enzymes that could either remove or damage structures normally present on naturally occurring plasma EPO, or could impart structures to the excreted urinary EPO molecule that are not present on plasma EPO. 22. Because only vanishingly small amounts of EPO could be obtained from urine,

researchers before Lin were actively searching for other sources of EPO, but failed to find any adequate source. For example, extensive searches by Goldwasser and others for tumor cells or other cultured cells that produced EPO were largely unsuccessful. Potential cells produced crude extracts that showed infinitesimal erythropoietic activity in biological assays. However, the ability to sustain such activity quickly declined over time, and no one ever succeeded in isolating EPO from these extracts. Consequently, it was simply unknown whether the faint erythropoietic activity detected in the biological assays of these extracts was attributable to the presence of human EPO in the extract, or to some other agent or combination of agents. 23. Given the minute amounts of EPO that were available, very little was known

about the structure and function of erythropoietin prior to Lin's inventions. Prior to 1983, a partial amino acid sequence was reported for the N-terminus of the human EPO protein, but that reported sequence subsequently proved to be not only incomplete but incorrect in several important respects. While it was known that EPO was a glycoprotein, the specific number,

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locat ion and structures of its glycans had not been investigated. Nor did skilled artisans in 1983 have any insight or knowledge about whether or the extent to which a biologically act ive EPO glycoprotein would require any number of post-translational cellular modifications, such as (1) proteolytic cleavage; (2) formation of disulfide bonds; (3) particular glycosylation; or (4) covalent addition of other molecules such as sulfate, phosphate, carboxyl or acetyl groups. Because the actual structure of EPO was unknown, it was impossible to know which, if any, such post-translational modifications would prove to be necessary to produce a recombinant EPO product that would perform the biological function of human EPO in vivo. Indeed, since it was known that naturally occurring EPO was apparently produced by very few, highly specialized cells in the kidney, the likelihood that such cells used special or unique enzymes to process and modify the final, secreted structure and composition of the naturally occurring EPO glycoprotein was very real. 24. It was known by 1983 that mammalian cells perform many post-translational

modifications that impact biological function in a species, cell-type, and protein specific manner. Moreover, an ordinarily skilled artisan would have appreciated that any of these potential modifications could have been critical for function. And, the ordinarily skilled artisan would have understood that every cultured cell had its own particular properties and capacity to impart any or all of these post-translational modifications to an expressed protein. Whether EPO had any such modifications was unknown. Thus, expression of EPO in a mammalian cell that did not normally produce EPO could easily result in different post-translat ional modifications of the EPO protein in ways that would disrupt or destroy the intended biological function of the protein. A worker at the time would have been doubtful that cells that did not normally produce human EPO would properly make any of these modifications, and would therefore expect that EPO

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expressed in such heterologous cells could be non-functional, absent proof of successful expression of in vivo bio logically active EPO from any mammalian host cell. 2. In 1983-84, the glycosylation of erythropoietin was known to be important for biological function, but its structure was unknown

25.

Some of the studies performed before Dr. Lin's inventions indicated that certain

carbohydrate structures appeared to be necessary for the in vivo bio logical function of EPO. For example, Dr. Goldwasser's 1974 article described how the sialic acids on sheep plasma EPO are necessary for in vivo but not in vitro bio logical function: "Desialation (decrease in sialic acids) results in complete loss of biological act ivit y when it is assayed in vivo. When the assay is done in vitro asialoeryt hropoiet in has full activity, or when assayed at low levels of hormones is about three times more active that the native hormone. The loss of activity can be explained by the hepatic removal of asialogylcoproteins from the circulation." Goldwasser, et al., "On the mechanism of erythropoietin-induced differentiation. The role of sialic acid in erythropoietin action," J. Biol. Chem. 249:4202-6 (1974) (D.I. 578, Ex. B); see also Lowy, et al., "Inactivation of Erythropoiet in by Neuraminidase and by Mild Substitution Reactions," Nature 186:102 (1960) (D.I. 578, Ex. C); Briggs, et al., "Hepatic clearance of intact and desialylated erythropoietin," Amer. J. of Physiology 227:1385-1388 (1974) (D.I. 578, Ex. D) ("These results indicate that desialylation of ESF causes its rapid hepatic clearance from the circulation . . ."). 26. Dr. Goldwasser hypothesized that the higher in vitro activit y observed for

desialylated EPO was a result of relieving repulsion between the sialic acids on EPO and the target cell surface: "This increase [in vitro activit y] may reflect the fairly large reduction in negative charge that accompanies desialation. If the target cells are negatively charged, the presence of 16 to 18 strong anionic groups on the native hormone may retard interaction with the cells; the asialohormone might then have easier access to the cells. A similar situation obtains with human chorionic gonadotropin where the asialo form of the hormone has a higher affinity 10

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for receptor sites than for the native hormone." Goldwasser, et al., J. Biol. Chem. 249:4202-6 (1974) at 4205 (D.I. 578, Ex. B). 27. Moreover, Dordal demonstrated that complete deglycosylation of EPO had

similar effects to desialylation: Digestion of the hormone with S. pneumoniae mixed glycosidases reduces the apparent molecular weight from 39,000 to 28,500. The glycosidase-treated epo retains 50-70% of its activity in vitro but is inactive in vivo. . . . These results suggest that deglycosylated epo may retain its intrinsic ability to stimulate erythropoiesis but may lack the stability in vivo required for successful hormone replacement therapy. Dordal, M., "The Function and Composition of the Carbohydrate Portion of Human Urinary Erythropoietin." Thesis, University of Chicago, 7/27/82. (D.I. 578, Ex. E, at 984).2 28. Thus, it was known in 1983 that the presence of sialic acids on the termini of the

carbohydrates attached to EPO appeared to play an important role in the in vivo bio logical function of EPO. It was also known that the complete elimination of glycosylation from EPO protein apparently led to the loss of in vivo bio logical act ivit y. It was not known why these carbohydrates were required for in vivo funct ion, nor was it known whether changes or differences in the location, number or type of carbohydrate structures attached to an EPO polypeptide would affect or impair its in vivo activit y. 29. By the end of 1984, no specific analysis of the glycan structures of either urinary

EPO or recombinant EPO had been published. In the late 1980s, the glycosylation structures found on urinary and recombinant EPO were studied in depth. It was confirmed that both

2

Dr. Lin and colleagues confirmed the result found for urinary EPO using recombinant EPO in early 1984. See D.I. 578, Ex. F ("Determine the effect of deglycosylation of EPO on its in vivo and in vitro bio logical act ivit y. Deglycosylated EPO has full in vitro act ivit y but no in vivo activit y."). 11

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urinary EPO and recombinant EPO have three N-linked and one O-linked oligosaccharides. It was further found that the N-linked carbohydrate chains attached at positions 24, 38, and 83 of EPO are heterogeneous with respect to sugar composition and structure. Sasaki, H., et al., "Carbohydrate Structures of Erythropoietin Expressed in Chinese Hamster Ovary Cells by a Human Erythropoietin cDNA," J. Biol. Chem. 262:12059-12076 (1987) (D.I. 578, Ex. G); Sasaki, H., et al., "Site Specific Glycosylation of Recombinant Human Erythropoietin," Biochemistry 27, 8618-8626 (1988) (D.I. 578, Ex. H). As a consequence, different molecules of EPO will have different numbers of attached sialic acid residues. Egrie, J. and Browne, J., "Development and Characterization of Novel Erythropoiesis Stimulating Protein (NESP)," Nephrol. Dial. Transplant 16 [suppl]:3-13 (2001) (D.I. 578, Ex. I); Takeuchi, M., et al., "Relationship Between Sugar Chain Structure and Biological Activity of Recombinant Human Erythropoietin Produced in Chinese Hamster Ovary Cells," Proc. Natl. Acad. Sci. USA, 86:78 19-7822 (1989) (D.I. 578, Ex. J). 30. Further, it was found that the glycosylation of recombinant human EPO produced

by CHO cells differs from human urinary EPO. Sasaki, H. et al., "Carbohydrate Structures of Erythropoietin Expressed in Chinese Hamster Ovary Cells by a Human Erythropoiet in cDNA," J. Biol. Chem. 262: 12059-12076 (1987) (D.I. 578, Ex. G); Takeuchi, M., et al., "Comparative Study of the Asparagine-linked Sugar Chains of Human Erythropoietins Purified from Urine and the Culture Medium o f Reco mbinant Chinese Hamster Ovary Cells," J. Biol. Chem. 263(8):3657-3663 (1988) (D.I. 578, Ex. K). 31. In fact, it is only because the carbohydrates attached to urinary EPO

characteristically differ from those attached to recombinant EPO that sporting authorities, such as the International Olympic Committee and the Tour de France, are able to test for the illicit use of recombinant EPO by athletes. The differences in carbohydrate chains attached to naturally

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occurring urinary EPO and recombinant human EPO are detected using an assay called isoelectric focusing gel electrophoresis, which is used to perform the urine analyses for EPO "doping" in the Olympic games and the Tour de France. Lasne, F. and de Ceaurriz, J., "Recombinant Erythropoietin in Urine," Nature 405:635 (2000) (D.I. 578, Ex. L). 3. 32. The cellular source of erythropoietin was unknown

While the work of Goldwasser and others had demonstrated that the principal site

of erythropoietin production appeared to be the kidney, as of 1983-84 the specific cell type(s) within the kidney that naturally produce EPO were unknown. See, e.g., Erslev, A.J., and Caro, J., "Physiologic and molecular biology o f erythropoiet in," Med. Oncol. Tumor. Pharmacother. 3(3-4):159-64 (1986) (D.I. 578, Ex. M) ("The exact cellular source for erythropoietin production in the kidney is still unknown."). 33. Indeed, the cell type(s) that naturally produce human EPO is still subject to

debate. Some believe that tubular cells of the kidney are responsible for EPO production. Mujais SK, et al., "Erythropoietin is produced by tubular cells of the rat kidney," Cell Biochem Biophys. 30(1):153-66 (1999) (D.I. 578, Ex. N). Others, including Roche's expert Dr. Fisher, have stated that interstitial cells are the primary site of EPO production in the kidney. Fisher, J.W., et al., "Erythropoietin production by interstitial cells of hypoxic monkey kidneys," Br. J. Haematol. 9527-32 (1996) (D.I. 578, Ex. O) ("The present finding that interstitial cells produce Epo in hypoxic monkey kidneys suggests that interstitial cells in the kidneys of other primates such as human are likely to be the primary site of Epo productions as well."). 34. Because the specific cell type(s) that produce EPO in the human body were

unknown as of the date of Dr. Lin's inventions, it was not possible to identify the specific posttranslat ional modifications that such cells make to the EPO polypeptide before it is secreted from the cells for circulation in the bloodstream. Thus, there was no way to know what carbohydrate

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and other structure(s) would be required to replicate biologically functional EPO, and thus what cell type(s) could be transformed with DNA encoding human EPO and grown in culture to produce such glycoprotein products. 4. 35. Recombinant expression of glycoproteins was in its infancy in 1983-84

Early experiments in the field focused primarily on expression in bacterial cells

like E. coli, which were incapable of glycosylation. Prior to Dr. Lin's inventions, expression of heterologous proteins in mammalian cells was still in the earliest stages of development. Most importantly, there were no published reports of the successful production of a recombinant human glycoprotein in mammalian host cells that was biologically active in vivo. Even if there had been such a report, successful production of one particular in vivo bio logically active glycoprotein would not have led a person of ordinary skill in the art in 1983 to believe that production of biologically active EPO was predictable. While a person of ordinary skill in the art may have had a reasonable expectation of success in achieving some expression (i.e., production of a protein), they would not have had, prior to Dr. Lin's work, a reasonable expectation of success that the human glycoprotein produced in a mammalian host cell would be biologically active in vivo. 36. Techniques for the recombinant expression of proteins were first developed using

bacteria, principally E. coli, as host cells. These techniques were adequate for the production of some mammalian proteins in functional form. Examples of functional E. coli-produced recombinant proteins include human insulin, G-CSF, hGH, and certain interferons. 37. Such mammalian proteins made in bacterial cells, however, generally will not

undergo any of the post-translational modifications such as glycosylation that would normally

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occur if the protein were made in mammalian cells.3 This is because the enzymes that catalyze these modifications are generally not found in bacteria. 38. The bacterial expression approach proved adequate for some proteins including

the ones I mentioned above because they do not require mammalian-specific modifications such as glycosylation for in vivo bio logical funct ion in animals. 39. Some mammalian proteins were functionally expressed in E. coli even though the

native molecules are modified by glycosylation. Thus, not all glycoproteins need be glycosylated for function. One example is interferon ("leukocyte A interferon"). Gutterman, et al., "Recombinant Leukocyte A Interferon: Pharmacokinetics, Single Dose Tolerance, and Biological Effects in Cancer Patients," Annals of Internal Medicine 96:549-566 (1982) (D.I. 578, Ex. P). On the other hand, some glycoproteins do require glycosylation for their function in vitro or in vivo. "The retention of biological act ivit y by glycoproteins void of carbohydrate is variable and unpredictable. In some instances, the absence of carbohydrate results in no loss of functional activity as is the case of the antiviral activity associated with the - and -interferons (Kelker, et al., 1983; Knight & Fahey, 1982). In other cases, murine C4 loses hemolytic activity (Karp, et al., 1983) or the von Willebrand-VIIIC complex appears inactive upon partial deglycosylation (Gralnick, et al., 1983)." Little, S.P., et al., "Functional Properties of Carbohydrate Depleted Tissue Plasminogen Activator," Biochemistry 23, 6191-6195 (1984) (D.I. 578, Ex. Q). Proteins thus can require proper glycosylation for either in vitro or in vivo activit y or both. 40. In 1983-84, the field was just beginning to explore the use of mammalian host

cells for the expression of mammalian glycoproteins. At that time, an ordinarily skilled artisan would, in my opinion, have understood that the use of cells from different species, or the use of
3

With the possible exception of the disulfide bond formation, which can occur in bacterial cells 15

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different cell types from the same species, frequently resulted in differences in post-translational modification of an expressed protein, and that such differences could prevent expression of in vivo bio logically act ive glycoproteins. 41. In my opinion, my 1981 review article accurately captured the uncertainty that

ordinarily skilled artisans would have experienced prior to Lin's inventions when expressing secreted mammalian proteins in recombinant systems: "Most proteins, secreted proteins in particular, are extensively modified after their synthesis by proteolytic cleavages, S-S bond formation, and glycosylation. The roles of each of these modifications in the structure, function or stability of any particular protein must be determined directly as it is not yet possible to make any generalizations or predictions concerning the physiological importance of these posttranslat ional alterations of any specific glycoprotein or secreted protein."4 I know of no research between 1981 and 1984 that would have altered the uncertainty to express a secreted protein by recombinant techniques as I stated in my 1981 article. Therefore, while ordinarily skilled artisans were often motivated to express newly cloned genes for complex glycoproteins in cells other than those from which the proteins naturally originated, they would not reasonably expect to succeed in doing so until they had empirically demonstrated that the expressed glycoprotein protein exhibited the in vivo bio logical act ivit y of the native polypeptide. 42. By 1983, the field of recombinant expression of glycoproteins in mammalian host

cells was still in its infancy. Expression of only a handful of mammalian glycoproteins had been attempted. Of those attempted, some were proteins that were previously known not to require glycosylation for biological act ivit y. In many cases, the biological act ivit y of the recombinant proteins was not measured. Likewise, in no case had analysis of the specific oligosaccharide under certain conditions.
4

Lodish, "Post-Translational Modification of Proteins," Enzyme Microb Technol. 1981 Jul; 3(3):177-280, at 186 (D.I. 578, Ex. R). 16

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structures imparted by heterologous host cells onto any recombinant glycoprotein been performed. Clearly by 1983 -- or 1984 for that matter -- there was insufficient experience with the expression of recombinant glycoproteins in heterologous host cells for ordinarily skilled artisans to be able to generalize any principles that would enable one to predict whether any given glycoprotein could be successfully expressed in an in vivo bio logically act ive form, absent proof of successful prior production. 43. By 1983, it was well-recognized that different cell types and different species

could impart different structures to a single protein. Thus, an ordinarily skilled artisan would have been well aware that expressing a recombinant mammalian glycoprotein in a cultured cell line that was a different cell type or species than that from which the desired protein originated would likely result in a novel glycoprotein with different oligosaccharides than the native molecule expressed from its normal environment. 44. Also, by 1983, there was a great deal of scientific interest concerning the roles

specific glycan structures play in the function of the glycoprotein to which they are attached. Many different functions had been identified or postulated for the oligosaccharides on glycoproteins, including stability, protein-protein interaction, clearance rate, and selfrecognit ion. Nonetheless, the field's understanding of the function of glycosylation was rudimentary in 1983. Because of this limited knowledge base and the recognized importance of glycosylation, the next decade saw an enormous amount of research regarding the functions of glycosylation, which confirmed the complexity and importance of this biological pheno menon. Varki, A., "Biological roles of oligosaccharides: all of the theories are correct," Glycobiology 3:97-130 (1993) (D.I. 578, Ex. S). Thus, an ordinarily skilled artisan in 1983 would have been aware that the glycosylation structures found on any given glycoprotein could contribute one or more of a wide array o f different functions. However, the particular functions of glycosylation

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on a given glycoprotein and the tolerance for variation in oligosaccharide structure would have been highly unpredictable in the absence of proof that a particular glycoprotein had been successfully expressed in a heterologous host cell and demonstrated in vivo biological activity. 45. In 1983, Konrad and his colleagues noted that glycosylation of recombinant

glycoproteins would depend on the host cell chosen. Konrad, M. et al., "Applications of genetic engineering to the pharmaceutical industry," Ann N Y Acad Sci. 413:12-22 (1983) (D.I. 578, Ex. T). · "Hopefully one of the contributions of genetic engineering will be to make experiments possible that will more completely elucidate the role of the carbohydrate residues. IFNs are certainly not unique in being glycosylated. Of the major proteins in the blood, only serum albumin is not glycosylated." Id. at 17. "However even in its present form [expressed in CHO cells] this cell line produces levels that are an order of magnitude higher than that produced from regular fibroblasts. It is unlikely that the pattern of sugar residues will be exactly the same as that produced by human fibroblasts, although it may be quite close. It will enable us to proceed more rapidly in investigations of just what the sugar means to the biochemical properties of this kind of IFN." Id. at 21. Likewise, Goeddel's patent application filed in 1983 (U.S. Patent No. 4,766,075

·

46.

(D.I. 578, Ex. U)) also anticipated the dependence of glycosylation on the host cell: "depending upon the host cell, the human tissue plasminogen activator hereof may contain associated glycosylation to a greater or lesser extent compared with native material." (`075 Col. 4:10-14). "In addition, the location of and degree of glycosylation will depend on the nature of the host cellular environment." (`075 Col. 5:18-20). 47. Dr. Lin's patent specification also identified this issue. Lin's patent specification

explicitly recognized the differences between different species' glycosylation: "Depending upon the host employed, polypeptides of the invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated." (`933 Patent at 10:28-31).

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48.

The authors of the Colby paper noted that differences in glycosylation between

CHO cell material and native material have immunological consequences. Colby, C.B., et al., "Immunologic differentiation between E. coli and CHO cell-derived recombinant and natural human beta-interferons," J. Immunol. 133(6):3091-5 (1984) (D.I. 578, Ex. V). · "Because the HuIFN-b gene is expressed in a hamster cell environment, it is possible that the CHO cells glycosylated HuIFN-b different ly than do human fibroblast cells. Such differences in glycosylation may result in an unmasking of the anti-viral site on the CHO cell produced IFN molecule, with the site becoming form accessible to the anti HuIFN-b antibody while the overall conformation of the protein molecule remains unchanged. Alternatively, differences in glycosylation could produce overall conformational differences between the molecules such that the anti-viral site of the IFN molecule cross-reacts with higher affinity with the neutralizing antibody. In either case, CHO-rHuIFN-b would be preferentially neutralized by anti-HuIFN-b, as reported in Table I." Id. at 3094. "In view of the immunological non-identit y of the __ IFN, it is important to know whether these in vitro immuno logic differences are significant enough for the host's immune system to perceive the recombinant IFN as foreign. If so, the recombinant HuIFN-b could elicit an antigenic response in vivo. Recent ly it was reported that recombinant HuIFN-a was antigenic in several human cancer patients treated i.v. with recombinant HuIFN-a (27), whereas an antigenic response is rarely observed in human cancer patients treated with either natural HuIFN-a, HuIFN-b, or both." Id. at 3094. Furthermore, a 1984 review article by Roche's expert Dr. Gaylis is particularly

·

49.

revealing as to the uncertainty and confusion of the field in the 1983-84 timeframe as to whether recombinantly produced EPO would be biologically act ive in vivo. In his assessment of the state of the art as of 1984, Dr. Gaylis stated: "It is hoped that with new advances in genetic engineering, the Ep gene will be cloned and transferred to a different organism such as E. coli, as this would facilitate production of the hormone in quantities adequate for clinical use. Clearly, then, the production of EP by 1411H is of significant biological interest and may be of clinical value if the gene controlling Ep synthesis can be cloned and used for the manufacture of the hormone."5
5

Gaylis, F.D., et al., "In vitro models of human testicular germ-cell tumors." World J. of Urol. 2:2-5, 5 (1984) (D.I. 578, Ex. W). 19

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50.

Thus, in 1984, Dr. Gaylis proffered the hope that if EPO could be cloned it could

be inserted into E. coli cells for "production of the hormone in quantities adequate for clinical use." But, of course, as we know now, the EPO material produced from E. coli by Dr. Lin, although active in vitro, turned out to be inactive in vivo. That is because E. coli., a bacterial cell, is incapable of glycosylating the proteins it produces -- a fact known to the skilled artisan in 1984. That in 1984 Dr. Gaylis suggested to his peers that EPO for clinical use could be made from E. coli. reflects the truly unsettled state of affairs as to whether recombinantly produced EPO would in fact be in vivo bio logically act ive. In particular, Dr. Gaylis' suggestion that E. coli was the route for making recombinant EPO for clinical use shows that, it certainly was not obvious that recombinant EPO made from mammalian cells in culture would be in vivo active and therapeutically effective. 51. It would be incorrect to assume that any oligosaccharide structures added by host

cells to a protein requiring glycosylation would confer in vivo bio logical activity. Because there are many known, and even more unknown, in vivo interact ions between the carbohydrate chains on glycoproteins and other proteins (such as antibodies, and receptor proteins) and cells, it was simply not possible in 1983 (and today still is not possible) to successfully predict a priori how a differently glycosylated glycoprotein will behave and perform in vivo. It may interact with its intended receptor or it may not. It may be removed from the blood or from other body tissues faster or slower. It may prove antigenic and elicit an immune reaction or it may not. It may interact with a different and unintended receptor or it may not. These are just some of the uncertainties that result from changes made to the carbohydrate structure of a glycoprotein. Until one makes and empirically tests how a glycoprotein actually behaves in vivo, one cannot successfully predict whether it will behave as desired.

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52.

To summarize, by 1983 one could not have predicted which specific

oligosaccharides a host cell would add to a given protein. Moreover, the field could not predict the tolerance of a particular glycoprotein to changes in its oligosaccharide structure. Today, we know that certain recombinant glycoproteins will function despite significant changes in their oligosaccharide structure as compared to their native structure, but even this knowledge regarding specific glycoproteins does not allow those skilled in the art to successfully predict a priori how changes made to a different glycoprotein can and will affect its ability to perform its intended in vivo function. C. A PERSON OF ORDINARY SKILL IN THE ART IN POSSESSION OF A DNA SEQUENCE ENCODING EPO IN 1983-84 WOULD STILL HAVE LACKED A
REASONABLE EXPECTATION OF SUCCESS IN PRODUCING IN VIVO BIOLOGICALLY ACTIVE EPO OUTSIDE THE HUMAN BODY ABSENT PROOF OF SUCCESSFUL PRIOR PRODUCTION OF SUCH EPO

53.

A central premise of Roche's obviousness-type double patenting argument is that

once Dr. Lin was in possession of a DNA sequence encoding EPO, he could predictably expect that he would produce in vivo bio logically active EPO by merely inserting the DNA into mammalian cells using known techniques and waiting for production. I disagree. 54. While some of the tools and techniques for producing recombinant glycoproteins

in mammalian cells were known prior to October 1983, the field had not progressed to a state in which one of ordinary skill in the art could reasonably expect success, particularly where the protein of interest (EPO) had never been successfully produced in a recombinant cell.6 Moreover, in my opinion, a person of ordinary skill in the art at that time would have reasonably believed that it was just as likely that in vivo biologically active EPO could not be successfully produced. A person of ordinary skill in the art in October 1983 would have expected that differences in post-translational modifications like glycosylation between the cells in the human
6

My opinio n would be the same if the relevant date of analysis were prior to November 30, 1984. 21

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body that naturally produce EPO and the selected recombinant cells could prevent production of EPO glycoprotein in a form that was biologically active in vivo absent experimental validation. 55. The following facts are apparent from the literature published before (or, in some

instances, contemporaneous with) Lin's inventions: · · Erythropoiet in is a glycoprotein, and at least the sialic acids attached to the carbohydrate chains are important for biological activity. Oligosaccharide chains added to proteins by eukaryotic cells, specifically including mammalian and other vertebrate cells, have an extremely large variety of different carbohydrate substituents, structures, and properties. Specific oligosaccharide structures are required for the function of many glycoproteins. The host cell species and cell type can determine the oligosaccharide structures attached to a particular glycoprotein. Mammalian cells perform many post-translational modifications in addition to glycosylation that impact function in a species and cell-type and protein specific manner. Whether EPO had any such modifications was unknown. At the time of Lin's inventions only a handful of recombinant glycoproteins had been expressed in vertebrate cells, and prior to November 1984 only one (tPA) may have been shown to have in vivo bio logical activit y. It was assumed that recombinant proteins produced in host cells from the homologous cell types and species would be more likely to have in vivo bio logical activity and be useful than would recombinant proteins produced in cells of a different type or different species. Given the art of expression of recombinant proteins in mammalian cells in 198384, there was no reasonable expectation that any given glycoprotein could be produced in any specific mammalian or other vertebrate host cell in an amount sufficient to have an in vivo bio logical or therapeutically effective activity. In addition, the 1983-1984 time period was the very birth of the technology of

· · ·

·

·

·

56.

recombinant expression of glycoproteins in mammalian cells. As I discussed above, even by the end of 1984, there was insufficient experience with this technology to draw any conclusio ns about whether any particular recombinant glycoprotein could be expressed in an in vivo bio logically active form. Only a few proteins had been expressed, and in no case had reasonable 22

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fidelit y of glycosylation as compared to the native glycoprotein been established. Moreover, most of the earliest proteins produced did not require glycosylation to be biologically active in vivo, and in almost all instances, the researchers had not even tested whether their recombinant products had any in vivo bio logical activity. 57. Prior to Dr. Lin's successful expression of in vivo bio logically active recombinant

human EPO, there were few, if any, reports of glycoproteins that had been produced by recombinant means and demonstrated to possess in vivo bio logical activity. I understand that during prosecution of the patents-in-suit, Amgen's attorneys characterized EPO as an "obligate glycoprotein." 58. As discussed above, earlier experiments by Goldwasser and Dordal demonstrated

that naturally occurring EPO that lacked sialic acids or was deglycosylated lacked in vivo bio logical act ivit y. Therefore, a person skilled in the art in 1983 would have expected that EPO likely required some form of glycosylat ion in order to be in vivo bio logically active. 59. Two implications followed from this expectation. First, it meant that some

structure in addition to EPO's amino acid sequence was required and must be present on the protein in order for the protein to have in vivo bio logical activity. What those precise structures were for EPO and whether any recombinant cell would predictably produce such structures as were needed for EPO's in vivo biological activity was not known or obvious prior to Lin's work, although for EPO it evidently entailed some form of glycosylat ion. Whether differences in the type, amount, or structure of the required glycosylation would affect the protein's in vivo bio logical act ivit y was not known, and what if anything else in addition to glycosylation might also be needed, was not known. Second, it was not known whether a recombinant cell would add other unwanted or unneeded structures to the protein, or change the protein in some way that would impair bio logical activit y in vivo. In sum, until a particular protein was

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actually expressed in a cultured cell and tested for bioactivity in vivo, it was not possible to predict with reasonable confidence whether the recombinantly produced glycoprotein would have the desired in vivo bio logical activity. Once Lin demonstrated that EPO could be produced in vertebrate cells, e.g., CHO and COS, and have in vivo bioactivit y, efforts to produce recombinant EPO in other vertebrate cells became much more predictable. 60. These facts establish that at the time Lin's inventions were made, it was highly

unpredictable whether EPO could be produced in an in vivo bio logically active glycosylated form fro m recombinant host cells. 61. I understand that it is improper to use hindsight to determine whether a patent

claim would have been obvious at the time of invention. In my opinion, Roche's argument that the successful outcome of Lin's plan to produce in vivo bio logically act ive material in heterologous recombinant host cells was expected or predictable is an exercise in hindsight. 62. A priori, in 1983-84 an ordinarily skilled artisan would have had no way of

knowing whether CHO host cells would add appropriate glycans to human EPO and, if they did, would add them efficiently enough to produce a population of EPO glycoproteins of sufficient quality to provide detectable in vivo bio logical activity. Thus, in 1984 there was no reason to believe that a transformed CHO cell would modify EPO with the same or similar sugars as a human cell that naturally makes EPO, or that the sugars added by the non-human cell would impart the claimed biological effect. 63. Today we know that CHO cells are a good host for the production of recombinant

human glycoproteins. But in 1983, the field had no experience with expression of in vivo bioactive glycoproteins on which to draw. 64. Post-1984 publications concerning the glycosylation of recombinant

glycoproteins, including EPO, reinforce the surprising nature of Lin's successful expression of in

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vivo functional EPO from heterologous cells. For example, a 1991 review article emphasized that researchers were pleasantly surprised that when they examined the glycosylation of recombinant proteins expressed in CHO cells that the CHO oligosaccharides were as similar as they are to the native glycosylation of these human proteins: "Detailed N-linked and O-linked oligosaccharide structures have been determined for several glycoproteins produced using recombinant CHO cells, including EPO, t-PA, interferon-l and IL-2. A pleasant surprise from these recent analyses has been the remarkable degree to which the oligosaccharide structures from the CHO-produced glycoproteins correspond to the structures of those same proteins isolated from human urine or produced using normal human diploid cells. As a result, Chinese hamster ovary cells have emerged as the cell line of first choice for the synthesis of recombinant human therapeutic glycoproteins, although CHO cells do possess deficiencies that may limit their applicability in specific cases, such as limited capability for -carboxylation and inability for oligosaccharide sulfation." Gooche et al., "The Oligosaccharides Of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure And Their Effect On Glycoprotein Properties" BioTechnology 9:1347-1355 (1991) (emphasis added) (D.I. 578, Ex. X). 65. Similarly, a scientific 1988 publication analyzing the glycosylation patterns found

on EPO states that "[t]his paper proved, for the first time, that recombinant technique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar chains of the native one." Takeuchi et al., "Comparative Study of the Asparagine-linked Sugar Chains of Human Erythropoietins Purified from Urine and the Culture Medium of Recombinant Chinese Hamster Ovary Cells," J. Biol. Chem. 263(8):3657-3663 (1988) (emphasis added) (D.I. 578, Ex. K). 66. A 1993 review article by Lis and Sharon ("Protein glycosylation, structural and

functional aspects," Eur. J. Biochem. 218:1-27 (1993) (D.I. 578, Ex. Y)) is particularly compelling evidence of the inventive significance of Dr. Lin's process and EPO product inventions.

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Also, genetic engineering makes it possible to produce glycoproteins in heterologous systems on a large scale, both for research purposes and for therapeutic use (Table 1). We are indeed witnessing the emergence of glycotechnology [70], a branch of biotechnology that uses novel approaches to manipulate carbohydrates or related materials, with the aim of creating new products or new procedures for the betterment of our lives. An impressive example is erythropoietin, a circulating glycoprotein hormone that stimulates erythropoiesis, which has the distinction of being the first recombinant glycoprotein produced industrially for clinical use. It is being employed on a wide scale for the treatment of anemia in patients on haemodialysis [71]; its sales in 1991 reached $645 million. Another clinically important recombinant glycoprotein is the thrombolytic agent, tissue plasminogen activator (tPA), with sales of close to $200 million in the same year. Still, the manifold effects of carbohydrates on the stability and biological activities of glycoproteins are a source of much concern in the biotechnological production of pharmacologically useful glycoproteins [72-75]. (emphasis added). 67. This passage from Lis and Sharon is significant in a number of respects. First, it

acknowledges that "glycotechnology" was still an emerging field 10 years after Dr. Lin's inventions. This statement makes plain that the field of glycoprotein production was new and unpredictable in the 1983-84 time period. Second, Lis and Sharon rightly describe Lin's work as "an impressive example" of glycotechnology, given that it "has the distinction of being the first recombinant glycoprotein produced industrially fo r clinical use." Lastly, the authors note that "the manifold effects of carbohydrates on the stability and biological activit ies of glycoproteins" remained "a source of much concern in the biotechnological production of pharmacologically useful glycoproteins." 68. Successful heterologous expression of in vivo bio logically active EPO from

recombinant host cells was unexpected and surprising. Thus the claimed production of in vivo bio logically active EPO is not obvious in light of EPO DNA-containing cells. EPO was the one of the first two glycoproteins requiring glycosylation for in vivo funct ion to be successfully produced by recombinant means in mammalian cells. Therefore, ordinarily skilled artisans

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would not have expected recombinant EPO produced in non-natural cell types and species to have proper in vivo bio logical funct ion until after Dr. Lin's successful experiments. 69. Because of the uncertainties in the art I described above, in my opinion the

ordinarily skilled artisan could not have reasonably expected to have produced in vivo bio logically active EPO until he actually received the positive in vivo test results. Thus, Dr. Lin did not have possession of the inventions of the claims-in-suit until he actually successfully transformed and tested heterologous mammalian cells for the production of in vivo bio logically active EPO, which I understand to have occurred in early March 1984.7 70. Unt il Dr. Lin proved that in vivo bio logically active EPO could be made in cells

outside of the body, no one could predict whether it would ever work. Once Dr. Lin was successful, persons skilled in the art knew that EPO could be produced in an in vivo bio logically form outside the body. Future efforts to produce EPO under different conditions might require some additional experimentation, but the expectation of success changed dramatically. By proving in vivo bio logically act ive EPO could be produced in hamster cells and monkey cells in addition to the natural production from human cells in the body, Dr. Lin's teachings would have led one of ordinary skill in the art to believe that in vivo bio logically active EPO could be expressed in a broad range of different vertebrate or mammalian host cells, albeit with some additional experimentation required.

7

Amgen Inc. v. Chugai Pharm. Co. Ltd., 13 U.S.P.Q.2d 1737, 1748 (D. Mass. 1989). 27

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D.

THE REFERENCES CITED BY ROCHE'S EXPERTS DURING DISCOVERY DO NOT ESTABLISH THAT AN ORDINARILY SKILLED ARTISAN IN 1983-84
WOULD HAVE HAD A REASONABLE EXPECTATION OF SUCCESS IN EXPRESSING IN VIVO BIOLOGICALLY ACTIVE EPO

71.

Taken collectively, Roche's experts cited many articles from the pre-1985

time frame that relate to the recombinant expression of proteins in mammalian cells. Although many articles are cited, the sum total of the information known to ordinarily skilled artisans would not have lead to a reasonable expectation of success in expressing in vivo bio logically active EPO. 1. Articles relating to general tools or methods for protein expression in mammalian cells In their expert reports, Roche's experts cite many articles that relate to

72.

"tools" or methods for expressing proteins in mammalian cells. None of these articles actually show the expression of a recombinant glycoprotein in mammalian host cells: · · · · · · · Axel U.S. Pat. No. 4,399,216 Axel U.S. Pat. No. 5,149,636 Gluzman et al. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. (1981) Jan;23(1):175-82 Graf et al. Transformat ion of the gene for hypoxanthine phosphoribosyltransferase. Somatic Cell Genetics 5: 1031-1044 (1979) Graham et al. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. (1973) Apr;52(2):456-67 Kaufman et al. Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes. Mol Cel Bio. Sept (1983); 1598-1608 Kaufman and Sharp, "Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J.J. Mol Biol. 159: 601-621 (1982) McBride et al. Transfer