Genentech, Inc. v. Boehringer Mannheim GmbH (47 F. Supp. 2d 91, 1999)

MEMORANDUM AND ORDER

SARIS, District Judge.

INTRODUCTION

Defendants Boehringer Mannheim GmbH and Boehringer Mannheim Corporation (“collectively BM”) have moved for summary judgment on the claims of plaintiff Genentech, Inc. (“Genentech”) that BM infringes three patents involving Genen-tech’s tissue plasminogen activator (“t-PA”) product. The three patents are United States Patent Nos. 4,342,832 (“the ’832 patent”); 5,034,225 (“the ’225 patent”); and 4,511,502 (“the ’502 patent”). Genentech has filed a cross-motion for summary judgment with respect to the ’502 patent. After hearings held on June 17-18, 1997 and July 23, 1997, which included the testimony and report of an impartial court-appointed expert, Dr. Connie Cepko, this Court issued a memoran- • dum and order, dated December 30, 1997, addressing issues of claim construction. See Genentech, Inc. v. Boehringer Mannheim, 989 F.Supp. 359 (D.Mass.1997); see generally Markman v. Westview Instruments, Inc., 517 U.S. 370, 391, 116 S.Ct. 1384, 134 L.Ed.2d 577 (1996) (holding that issues of claim construction are matters of law to be determined by judge, not jury). Genentech has filed a cross-motions for summary judgment with respect to the ’502 patent.

After extensive supplemental submissions on the pending motions for summary judgment to take into account the Mark-man decision, and after hearing, BM’s motion for summary judgment is ALLOWED with respect to the ’832 patent and the ’502 patent, but DENIED with respect to the ’225 patent.

UNDISPUTED FACTS

1. Tissue Plasminogen Activators

Serious heart attacks can be caused by the presence of a thrombus, .which is a blood clot in the coronary blood vessels or coronary artery. The process that dissolves a thrombus is called thrombolysis, and the chemicals in the body that induce thrombolysis are called plasminogen activators. Two plasminogen activators, which are a natural part of a body’s defenses, are urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).

T-PA is a chemical that makes the body’s natural plasminogen become plas-min, an enzyme. Plasmin cuts through fibrin, the substance which makes up blood clots. Although the body naturally produces small amounts of t-PA, this quantity does not activate sufficient plasminogen to cut through the large clots involved in heart attacks.

In the late 1970s and early 1980s, scientists, whose research was sponsored by Genentech, successfully reproduced t-PA through recombinant DNA technology by identifying the DNA encoding t-PA — 527 amino acids (“aa”) with glycosylation (the attachment of sugars) at three sites — and inserting the DNA into bacteria. The five domains (or regions) of the t-PA protein are:

—The Fibrin Finger (“F”) domain (aa 4-49);

—The Epidermal Growth Factor (“E”) domain (aa 50-87);

—The Kringle 1 (“Kl”) domain (aa 88-176) glycosylated;

—The Kringle 2 (“K2”) domain (aa 177-262) glycosylated;

—The Serine Protease (“P”) domain (aa 263-527) glycosylated.

In July, 1979, Genentech applied for the ’832 patent for its particular method of reproducing t-PA and subsequently obtained approval from the Food and Drug Administration (“FDA”) to sell t-PA under the name Activase. Genentech also obtained a patent for purifying proteins (’502 patent) in 1985 and one for increasing the solubility of t-PA in a pharmaceutical composition by incorporating arginine (’225 patent) in 1991.

2. Basic Molecular Genetics

Analysis of the current dispute necessitates an understanding of basic molecular genetics and recombinant DNA technology. I borrow liberally from the excellent tutorials of Andrew C. Webb, a Professor of Biological Sciences at Wellesley College, BM’s expert, and Dr. Joseph Oliver Fal-kinham III, a Professor of Microbiology at Virginia Polytechnic Institute, Genentech’s expert. (See Dockets 224, 227.)

a. Proteins

The human body is comprised of tissues and organs constructed of microscopic cells which carry out their specific functions through the creation of molecules called proteins. Proteins are chains of repeating molecules called amino acids. There are twenty different amino acids. The order of amino acids in a protein is unique to the protein. Some proteins called enzymes serve as the engines of the cell by driving chemical reactions.

In the late 1970s recombinant DNA technology was developed to allow individual proteins to be readily mass produced. The basic principle behind this technology is to give bacteria the DNA for a particular protein and have the bacteria make large quantities of it. The recipe for a protein in the DNA database is known as a gene. The genetic language of DNA consists entirely of three letter words called codons that are spelled using a four letter alphabet. These four nucleotides are the building blocks of DNA: A (for adenine), C (for cytosine), G (for guanine) and T (for thymine). Each three letter combination is known as a “triplet codon.” For example, the three nucleotides AAA code for the amino acid lysine. The order of nucleotides spells out the order of amino acids in the protein.

DNA is generally found as two strands wrapped around one another like a spiral staircase, in a form known as the double helix. The two strands stick to one another, through pairing of the bases on opposite strands. A always pairs with T and G always pairs with C. Any non-conventional base pairing, such as A with G, is said to be mismatched. The ends of DNA single strands are chemically distinct. The front is referred to as the 5' end, and the back as the 3' end. The two complementary DNA single strands are base paired such that the 5' end of one strand is positioned opposite to the 3' end of the other. This 5' to 3' orientation allows the base sequence of genes to be “read” in the correct direction.

In its normal state DNA is folded in bundles called chromosomes which are sequestered in the nucleus of the cell. The nucleus is surrounded by the cytoplasm which is where the proteins are made. Bacterial cells do not contain a nucleus and are called prokaryotic cells. The genetic material inside a bacterial cell consists of the large, circular chromosome and sometimes bacterial plasmids, which are small circles of DNA. Bacterial plasmids often contain coding information for antibiotic resistance together with noncoding sequences such as promoters and ribosome binding sites. These bacterial plasmids are essential to recombinant DNA technology-

b. DNA replication

When cells reproduce or divide, the DNA copies itself in a process called DNA replication. During replication, the original DNA serves as a template for the newly synthesized version. DNA replication or multiplication is carried out by the enzyme DNA polymerase which takes advantage of the specific chemical attraction between bases as a foundation for faithful duplication.

Genes are organized in DNA as transcriptional units. Both DNA and RNA polymerase will only recognize the correct position in the DNA to start copying while the DNA molecule is still in its double stranded configuration. These positions are the origin of replication in the case of DNA polymerase and the promoter in the case of RNA polymerase. However, polymerase copying of the DNA template can only occur when the DNA is rendered single stranded by severing of the AT and GC base pairs holding the double helix together. This separation of complementary strands in a DNA double helix is called denaturation. In its double-stranded form the DNA does not provide a template since the bases are already paired. For polymerase enzymes to function, it is imperative that the bases be exposed over short distances, so that new base pairing can occur.

The order of events for replication and transcription of DNA is as follows: (i) the polymerase enzyme recognizes and binds to a specific region of double-stranded DNA; (ii) the double helix denatures in this region to become two antiparallel, single strands; (iii) the polymerase enzyme initiates copying of the template strand, adding the next complementary base to the growing chain only in a 5' to 3' direction; and (iv) as the polymerase moves down the template strand, it progressively opens the double helix to reveal more single-stranded template to be copied.

c. Expression

Expression refers to the process by which the machinery of the cell reads the code recorded in the DNA and assembles protein following that code. The expression of DNA to produce proteins is a process involving steps of transcription and translation.

Information stored in DNA is transferred from DNA in order to make proteins by a courier molecule called messenger RNA or mRNA. RNA is single stranded. The DNA sequence is copied into a mRNA by the enzyme RNA polymerase during a process known as transcription. The mRNA carries the DNA sequence to the ribosomes (located in the cytoplasm) that are responsible for assembling amino acids into proteins by the process of translation. Translation, then, results in protein synthesis; the protein recipe contained in the mRNA is read by the ribosomes, one codon at a time into a sequence of amino acids.

Once bound to the promoter, RNA polymerase separates the two complementary DNA strands at the initiation site, joins the first two ribonucleotide bases of the gene based on their complementarity to the template DNA sequence, and terminates transcription at another specific DNA base sequence. At this termination site, the polymerase falls off the template DNA and releases the completed mRNA copy of the gene. Control of gene expression is influenced by proteins which can increase, decrease or inhibit the binding of RNA polymerase to its promoter.

The DNA sequence encoding the protein carried in the newly synthesized mRNA bases is interpreted by another type of RNA called transfer RNA (tRNA) with the help of the cell’s ribosomes. tRNA transfers amino acids from the cytoplasm to the site of the protein synthesis on the ribosome. There are different tRNAs for each of the twenty amino acids. The initiation of protein synthesis starts with a ribosome “scanning” down the mRNA. At the start codon (ATG), specifying methionine, the initiator tRNA attaches methionine and the elongation of the protein chain begins, by stepwise codon recognition. Once the ribosome reaches a stop codon, it falls off the mRNA and ejects the completed protein. The protein has an amino (“N”)-Terminus end and a Carboxyl (“C”)-Terminus end.

The order and composition of amino acids in a protein will determine the shape into which the protein spontaneously folds. Folding of the protein chain is crucial to the production of a functional protein, and failure to fold correctly or subsequent unfolding (denaturation) is accompanied by a partial or total loss of functional activity. Discrete units within folded proteins are known as domains. In addition to the folding of the protein, there are other modifications that have to take place before the protein can function. One example is the attachment of sugars (glycosylation) to specific amino acids in the chain. ■ It is also extremely common for proteins to be made initially as large, often inactive “precursor” proteins. Such precursors have leader sequences of amino acids that are cleaved by specific enzymes to yield the smaller “mature” proteins with full activity. Some precursor proteins include a leader or signal sequence which is a string of amino acids which are found at the amino (“N”) terminus of the protein sequence. Tissue-type plasminogen activator (t-PA) is initially made with a leader sequence which is removed to yield mature t-PA.

d. Primers

Unlike RNA polymerase, DNA polymerase requires a “starter” or primer before it can add bases one to another. Whenever DNA is replicated, either in a cell (in vivo) or in a test tube (in vitro), primers consisting of small lengths of single-stranded DNA or RNA, called oligonucleotides, must first base pair with their complementary sequence on the DNA template before DNA polymerase activity can begin. Oli-gonucleotides are short (approximately 15-50 nucleotides long) stretches of single-stranded DNA or RNA that are chemically synthesized on programmable instruments. These primers precisely define the starting point of DNA replication and are then added to by the polymerase, one base at a time, in a reaction referred to as a “primer extension.” The stretches of newly synthesized DNA are glued together by another enzyme called DNA ligase.

The synthesis of copy or complementary DNA (cDNA) involves the single-stranded template of mRNA and the DNA polymerase called reverse transcriptase. Reverse transcriptase also requires a primer and moves in a 5' to 8' direction along a single-stranded mRNA.

3. DNA Recombinant Technology or Genetic Cloning

In gene cloning, the DNA to be cloned is inserted into a cloning vector, such as a plasmid, to form hybrid or recombinant DNA molecules that are then introduced into host cells like bacteria. There they are replicated as the bacterial cells divide. Plasmids are circular DNA molecules which replicate themselves when inserted into bacteria.

The basic technology for gene cloning relies on the use of specific enzymes to “cut” and then “paste” together fragments of DNA to form recombinant DNA. “Cutting” is accomplished by use of enzymes called restriction enzymes which recognize and cut DNA at specific nucleotide sequences called restriction sites. The cloning site into which DNA is inserted contains recognition sites for multiple restriction enzymes. Restriction enzymes cut the foreign DNA at certain specific DNA nucleotide sequences. The DNA of the vector is also cut with the same enzyme so -that the ends of the fragments match. Cleavage with one of these restriction enzymes linearizes the plasmid circle, allowing insertion of foreign DNA. The “pasting” or rejoining at DNA fragments (also called ligation) is accomplished by the use of an enzyme called DNA ligase. If the recombinant DNA (vector and foreign DNA) is fused to a promoter, the gene can be expressed by the host cell to make the protein.

An example of the process of cloning genes using bacterial plasmid vectors is as follows:

First, isolate the plasmid vector DNA from bacteria and the foreign DNA containing the gene of interest. The source of foreign DNA to be cloned can either be chromosomal or cDNA.

Second, digest both the vector and foreign DNA with compatible restriction enzymes which are the molecular scissors of recombinant DNA technology. They allow large stretches of double-stranded DNA to be cut into manageably sized pieces or fragments. Some cut the two strands of DNA opposite each other, creating a “blunt” end. Others cut asymmetrically to yield a staggered or “sticky” end, where a short two or four base sequence protrudes as a single stranded extension to the end of the DNA. Because blunt ends can join only inefficiently to other blunt ends, one solution is to add synthetic linkers or adaptors onto the blunt ends of DNA to create the sticky end of choice. They are chemically synthesized on a “gene machine” as short, complementary, single-stranded oligonucleotides that contain the recognition sequence for one or more restriction enzymes. Following synthesis, the complementary strands are allowed to base pair, creating the synthetic double stranded fragment.

Third, the vector and foreign DNA cut with restriction enzymes (possibly modified with adapters and linkers) are joined together permanently by another enzyme called DNA ligase.

Fourth, when a plasmid with the desired DNA and a properly positioned promoter is introduced into bacterial cells, like E. Coli, the cells will express the DNA carried by the plasmid to encode the protein. This process is called cloning.

4. Genentech’s 'patented, method

The direct testimony of Dr. Jeffrey V. Ravetch, a Professor at the Rockefeller University and Head of the Laboratory of Molecular Genetics and Immunology, provides a useful framework for understanding the pioneering nature of the ’832 patent.

In the early days of biotechnology, scientists used as their models various hormones to make recombinant human proteins. The DNA for the hormones was made chemically by hand. Because this process was time consuming, it was difficult to make DNA for large proteins. Genentech scientists David Goeddel and Herbert Heyneker, the named co-inventors of the ’832 patent, resolved this problem. They isolated the desired gene from biological sources and then cloned it by using the “cDNA” technique.

However, making proteins from sources such as cDNA resulted in having cDNA which was either “too long or too short.” This is because cDNA genes typically coded for not only the desired protein itself but also for the leader sequence attached at the beginning of the protein. The leader sequence in nature helps the protein emerge from the human cell and is clipped off automatically as the protein leaves the cell. But when a mammalian protein is produced in a bacterial (prokaryotic) cell, no mechanism may be present in the bacterial cell to clip off the mammalian leader sequence. Cutting off the leader sequence sometimes resulted in cDNA that was too short in that it did not code for the mature enzyme. In other situations, the cDNA obtained by reverse transcription from the mRNA was too short in that it coded for a protein which was less than the desired protein.

The ’832 patent provides a method of making a cloning vehicle capable of expressing a gene for a particular polypeptide by combining cDNA and synthetic DNA so that the DNA (in Goldilocks’ words) is neither too long, nor too short, but “just right.” In Dr. Ravetch’s words, “[i]t provides the method allowing a researcher to work backwards, starting with the idea of a particular polypeptide sequence he desires to make and instructing him on how to create a DNA plasmid capable of expressing that polypeptide sequence.” (Ravetch Dir. Test. ¶ II.A.5) (Docket 226.)

5. Reteplase

a. Background

Reteplase is a new thrombolytic for the treatment of heart attack victims developed by BM. On October 30, 1996 Rete-plase was approved by the FDA for use in the United States. Reteplase is a member of a class of protein based drugs used to dissolve the clots which block coronary arteries during the onset of a heart attack. It is not a naturally occurring protein. The DNA encoding Reteplase was created by changing the DNA of human t-PA. BM’s scientific director Dr. Stephan Fischer refers to it as a “designer gene” because “it is not simply a recombinant version of a natural protein but is an entirely new molecule with new properties.” (Fischer Decl. II ¶ 11.)

Reteplase has 355 amino acids and consists only of the Kringle 2 (“K2”) and Serine Protease (“P”) domains. Hence, it is called K2P. In contrast to t-PA, none of the amino acids is modified with glycosidic groups. (See id. at ¶ 13.) A side-by-side comparison (in Attachment A hereto) reveals the differences between the two molecules.

Reteplase was developed as a result of a research program to search for new plas-minogen activators which had improved pharmacological activity as compared to t-PA. BM sought to make a non-glycosylat-ed plasminogen activator which would have a better half-life, and therefore be a better therapeutic agent than t-PA which is a glycosylated molecule. Thus, BM chose to use E. Coli as a host cell to express recom-binantly a new plasminogen activator. Bacterial cells, like E. Coli, differ from eukaryotic cells in that they do not have the cellular machinery to attach glycosidic (sugar) molecules to the recombinantly made polypeptide. The development of Reteplase involved the use and modification of many different plasmids, until reaching the production plasmid PA27-T2L which contains the DNA encoding Reteplase.

All of the subsequent cloning steps utilized site directed mutagenesis (“SDM”) according to the method of Morinaga et al., Bio/Technology 636-639 (July 1984), or a technique called the polymerase chain reaction (“PCR”). Before describing the development of plasmid PA27-T2L in detail, I digress for a moment to describe these two Nobel Prize winning techniques generally.

b. Site-Directed Mutagenesis (SDM)

Mutations are changes in the sequence of a segment of DNA, known as a “parent” sequence. The changes can be single nucleotide changes, additions of nucleotides or deletions of nucleotides. The new sequence is called the “progeny” sequence. A technique known as site-directed muta-genesis allows scientists to insert a mutation at a specific preselected place in a piece of parent DNA. This process is done in a test tube. The technique of SDM uses an organically synthesized single-stranded oligonucleotide that is perfectly base-paired to the template parent DNA sequence, except that it is “mismatched” at the site of the base to be changed or mutated. This organically synthesized oli-gonucleotide acts as a primer for the reaction. DNA polymerase extends the primer during the enzymatic synthesis of new progeny DNA.

For example, the primer sequence might lack a specified region of the parent template DNA sequence. It is organically synthesized with a sequence that is complementary to the template DNA sequence on either side (flanking) of the DNA region to be deleted. On extension of this primer, the mismatched region would be absent from the progeny sequence, and would, in that sense, have been “deleted” from the parent DNA sequence. When the synthetic primer is hybridized to the template strand, the unwanted region forms a loop or is “looped out.” The DNA polymerase is added to extend the primer enzymatically in a clockwise 5' to 3' direction to make the second strand that is complementary to the template strand. Thus, a heterodu-plex DNA molecule (consisting of the mutated strand and the looped strand) is created. This heteroduplex is inserted into a bacterial cell. As the normal DNA replication process takes place, each strand is copied to obtain two populations of double stranded DNA: one population with the original sequence, and the other with the looped sequence. The population of progeny double stranded, homoduplex molecules is isolated and used to express a mutant protein (a “deletion mutant”) with a shorter amino acid sequence than that coded by the parental DNA sequence.

A modified SDM method is known as the Morinaga or “gapped duplex” method. A key difference is that it uses a partially double-stranded DNA molecule having a single-stranded gap as a template region instead of a completely single-stranded template. The primer containing the desired change hybridizes to a template site in the single-stranded gap again yielding a loop-out of the unwanted region. DNA polymerase then fills any gap that remains between the ends of the primer and the double-stranded DNA via enzymatic synthesis, and DNA ligase joins the ends. Thus, only the gap is filled by enzymatic sequences, rather than the whole progeny strand.

c. Polymerase chain reaction (PCR)

The polymerase chain reaction (“PCR”) is an in vitro reaction in which a specific region of DNA is amplified several million-fold by repeated synthesis using a DNA polymerase and specific oligonucleotide primers to define the ends of the DNA region to be amplified. To perform PCR, a scientist needs the sequence of DNA flanking the region to be amplified. A pair of primers is synthesized, usually 18-30 bases in length. One primer is complementary to the sequence at one end of the DNA region, and the other is complementary to the sequence on the opposite strand of the DNA at the other end of the region to be amplified. Primers initiate DNA synthesis by annealing or hybridizing to their complementary DNA base sequence and DNA polymerase then extends them only in a 5' to 3' direction, adding nucleotide bases according to the template sequence.

PCR reactions take place in the test tube where double-stranded DNA and oli-gonucleotide primers are mixed. The PCR cycle consists of repeated rounds of (i) separating template double DNA strand by heat denaturation; (ii) after cooling, annealing of primers to their complementary nucleotide sequence on the two separate, template DNA strands; and (iii) after reheating to the optimal temperature, synthesizing new DNA by polymerase extension of primers. By repeated heating to separate both original and newly synthesized strands, the net result of PCR after n cycles is 2n double stranded copies of the original DNA sequence between the primers.

What results are two double-stranded DNA molecules, one strand of which is the parental template and the other strand (the progeny) which incorporates the organically synthesized primer and enzymat-ically synthesized DNA. The amplification produces a piece of DNA that is a hybrid piece containing (or marrying, if you will) the synthetic DNA primer and DNA copied from the target sequence.

Fusion PCR is an adaptation of conventional PCR methodology. As in regular PCR, the purpose of fusion PCR is to join two separately amplified PCR fragments together by means of an overlap between nucleotides they have in common on the ends of the fragments.

An example will help to explain fusion PCR where two separate DNA fragments A and B are involved. Following PCR, the amplified, double-stranded DNA fragments are mixed, denatured by heating to yield single-strands, and then cooled to allow base pairs to reform (“renature”). Different combinations of renatured DNA strands can result. Complementary strands can hybridize over their entire length to recreate separate molecules of fragment A or B. Another possible combination is that one strand from fragment A can cross-hybridize with a strand from fragment B by base-pairing over their complementary, overlapping ends. In the presence of DNA polymerase, enzymatic DNA synthesis will take place in a 5' to 3' direction to create a fusion of fragment A to fragment B. Once the initial fusion product has been formed, conventional PCR using the original flanking primer can be used to generate large quantities of the fused DNA fragment.

d. BM plasmids

1. Plasmid pBT95, encoding precursor t-PA

To make plasmid pBT95, BM isolated cDNA encoding precursor t-PA; that is, the DNA sequence for both (i) the mature t-PA coding region (i.e., the part of the gene coding for t-PA’s five domains), and (ii) the leader sequence located upstream of the 5' end of the mature t-PA coding region.

The precursor t-PA had been made using the enzyme reverse transcriptase to copy t-PA messenger RNA (“mRNA”) into cDNA. Plasmid vector DNA was combined with the cDNA by “cutting and pasting” the vector DNA and cDNA resulting in the recombinant plasmid designated pBT95.

pBT95 contained, in addition to the coding region for precursor t-PA, certain non-coding DNA sequences which affected the production of the t-PA protein, but are located outside the gene for t-PA. These included a promoter known as the tac promoter, a ribosome binding site (or Shine-Delgarno sequence), and an ATG start signal for translation, all located upstream of the 5' end. pB95 also included DNA known as the 3' untranslated region, located downstream of the 3' end.

2. Plasmid pePA98.1, encoding mature t-PA

In 1985, BM embarked on a research project to see if mammalian proteins could be expressed in E. Coli. It constructed a plasmid encoding mature t-PA by joining an organically synthesized linker designed to maximize expression to a cDNA fragment encoding t-PA and inserted that hybrid into a plasmid, designated pePA98.1. That hybrid was inserted into E. Coli for the purpose of expressing the cDNA encoding mature t-PA. This project was a failure because it produced improperly folded, biologically inactive protein. The improper folding problem was particularly acute with t-PA because the native protein possesses a very complex structure.

The t-PA leader amino acid sequence is not recognized, and thus, is not cleaved by E. Coli enzymes. It was therefore necessary, if .active (i.e., mature) t-PA were to be expressed in E. Coli, to make plasmid pePA98.1 in which the DNA coding for the leader sequence was removed from the precursor t-PA gene.

Plasmid pePA98.1 was made by cutting pBT95 at a restriction site close to the junction between the DNA coding for the leader sequence and the DNA coding for mature t-PA. A nuclease enzyme was used to “chew back” the sticky end left by the restriction enzyme, but this removed two nucleotides of the first codon coding for mature t-PA, which codes for the amino acid serine. Furthermore, in addition to the DNA coding for the leader sequence (35 codons) and the first two nucleotides of the codon for serine that were “chewed back,” the cutting and “chewing back” operations also removed the start codon (ATG). These operations resulted in a mature t-PA coding sequence that was two nucleotides too short. BM replaced these two nucleotides. It organically synthesized partially complementary oligonucleo-tides to make a double stranded “linker” that provided, in addition to the first two nucleotides for serine, the obligatory start codon (ATG) for translation (which by necessity had been removed along with the leader sequence), additional nucleotides to create a sticky end to facilitate ligation to the plasmid, and additional nucleotides to optimize the distance between the ribo-some binding site and the start codon.

The organically synthesized DNA fragment had the sequence:

5' AATTCTTATG TC 3'

3' GAATACAG 5'

As already noted, it was designed specifically to create the optimal distance between the ATG translation start signal (underlined above) and the ribosome binding site, and to facilitate insertion of the cDNA coding for mature t-PA into the expression vector. As shown, the last two nucleotides of the organically synthesized DNA fragment constituted two nucleotides removed by the chewing process, i.e., the TC of the TCT serine codon. The second T of the TCT serine codon was present on the cDNA.

In sum, plasmid pePA98.1 differed from pBT95 in that the DNA coding for the leader sequence of t-PA was removed, and the translation start signal ATG was moved from its previous location at the beginning of the leader to a new position next to the first codon (serine) of the DNA encoding mature t-PA.

3. Plasmid pePAl26.1, encoding mature t-PA

Plasmid pePA126.1 was constructed in order to delete the 3' untranslated region of the gene for t-PA. The 3' untranslated region is the DNA that is immediately adjacent to, but downstream of, the 3' end of the DNA coding region (ie., it is not in the coding region and therefore not translated).

The 3' untranslated region was removed by cutting pePA98.1 and pasting the cut DNA into a vector. The resulting plasmid was designated pePA126.1. All of the cutting and pasting occurred outside of the region encoding t-PA. Plasmid pePA126.1 contained the same t-PA coding sequence as pePA98.1 (ie., all of the DNA coding for mature t-PA).

4. Plasmid pePAl33, encoding mature t-PA

In 1985, BM undertook a limited project using a plasmid designated pePA133. This plasmid contained the same coding sequence as pePA98.1 but was a “low copy” plasmid. This project was successful and active t-PA was obtained by in vitro folding of t-PA isolated from inclusion bodies. A patent was originally filed in Germany in October 1985 and was issued in the U.S. in September ■ 1995 as United States Patent No. 5,453,363 to Rudolph et al., assigned to BM.

Plasmid pePA133 was made in order to change the number of plasmid copies inside the bacterial host cell. Somewhat oversimplified, each plasmid contains a DNA sequence controlling the level of its replication. Plasmid pePA126.1 contained a DNA sequence which resulted in a high plasmid copy number inside the bacterial cell.

Plasmid pePA133 was produced by excising the gene for mature t-PA, including its promoter, from plasmid pePA126.1, and combining this fragment, using cutting and pasting techniques, with a DNA fragment from another plasmid vector containing a DNA sequence yielding a low level of copies. It was thought that this change to low copy would help increase the amount of mature t-PA protein produced. Plasmid pePA133 contained the same t-PA coding sequence as pePA98.1 (ie., all of the DNA coding for mature t-PA). In other words, pePA133 does not have any modification in the nucleotide sequence encoding the semi-synthetic gene for t-PA. Only sequences outside the coding region were manipulated.

5. Plasmid pePAl26fd, encoding mature t-PA

To construct another of its plasmids, plasmid pePA126fd, BM added a strong fd terminator of transcription to pePA126.1. This was done to increase the stability of the t-PA mRNA and hence increase the yield of t-PA protein in the host cells. The fd terminator was cut from the fd phage (a virus that infects bacteria) and ligated to the 3' end (but outside) of the t-PA coding sequence carried in plasmid pePA126.1. Thus, as with plasmid pePA133, the coding sequence of the resulting plasmid, pePA126fd, remained the coding sequence for mature t-PA; nothing was added to or subtracted from that coding sequence.

6. Plasmid pREM7685, encoding FK2 P'

Using the DNA from plasmid pePA133 which codes for mature t-PA as a template, BM applied the Morinaga SDM technique to obtain a plasmid designated pREM7685 encoding only the F, K2, and P domains of t-PA. In this application of SDM, a cDNA fragment from the middle of the t-PA coding region is eliminated and a synthetic fragment which bonds to the coding information flanking the deleted region is incorporated into the plasmid. The sequences that encode the amino terminus end of the polypeptide are still synthetic DNA sequences that are provided from plasmid pePA98.1.

The following steps were performed in the construction of pREM7685:

(i) BM started with two identical pePA133 plasmids, each encoding mature t-PA (domains FEKjKy3). Plasmid 1(a) was cut with a restriction enzyme to make a single cut in the circle. Plasmid 1(b) was cut in two places with restriction enzymes outside the region to be deleted. In this case, plasmid 1(b) was cut just upstream of the F domain and also in the P domain.

(ii) One strand from plasmid 1(a) was then hybridized with one strand from plasmid 1(b) by heat denaturing each plasmid and allowing the strands to cross-hybridize producing a hybrid intermediate commonly referred to as a “gapped duplex.”

(iii) A single-stranded oligonucleotide primer was synthesized having a sequence complementary to a short sequence on each side of the DNA to be deleted. In this case, one-half of the primer consisted of a short sequence 0GCCTGTCAAA) complementary to the end of the F domain and the other half consisted of a short sequence (GGAAA-CAGTGA) complementary to DNA at the beginning of the K2 domain. In other words, the SDM elimination step was done using a synthetic oligonucleo-tide with a sequence of codons bonding to amino acid codons 46-49 and 176-179 of full length t-PA — coding already provided by the single stranded cDNA template — in order to effect a deletion of the cDNA sequences encoding the regions for amino acids 50-175.

(iv) The primer was then hybridized to the template DNA strand in the “gap” resulting in the E and Kx domains forming a “loop.”

(v) Next, the remaining gaps on either side of the primer were extended enzymatically by adding DNA polymerase and nucleotides, and the ends were then sealed with DNA ligase. The synthetic strand consisted of 21 nucleotides. The gap filled in enzymatically between the synthetic strand and the cDNA encoding portions of the P region constituted over 500 nucleotides. The resulting heteroduplex DNA was comprised of one DNA strand corresponding to the original parental sequence (FEKjK^P) and the other strand comprised of DNA corresponding to the mutant progeny sequence (FK2P).

(vi) This heteroduplex was then introduced into the bacterium, E. Coli. Once in E. Coli, each strand of the heterodu-plex was copied independently, resulting in two plasmid populations — one a copy of the original parental plasmid (which coded for FEKjKgP) and the other a plasmid carrying a deletion mutant of the t-PA gene (coding for FK2P), the nucleotides coding for the E and Kj domains having been “deleted” by use of the Morinaga method.

The synthetically synthesized primer used in SDM was not incorporated at the N-terminus of the gene (ie., the 5' end). On the contrary, it was incorporated into the middle part of the coding region for the mutant t-PA gene.

7. Plasmid pA27.3 encoding Reteplase (KZP)

Using the DNA from pREM7685 (encoding FK2P) as a template, BM again used the Morinaga SDM technique to remove the DNA region encoding the F domain of t-PA. The resulting plasmid, encoding only K2P, was designated pA27.3. Plasmid pA27.3 was the first plasmid made which contained DNA encoding the protein now known as Reteplase. Genentech contends that the production of this plasmid violates its patent. BM hotly disputes this contention. Understanding the production of this plasmid is key to understanding the infringement claim. See Attachment B,

The following steps were performed in the construction of pA27.3:

(i) BM started with two identical pREM7685 plasmids each encoding domains F, K2, and P of t-PA. Plasmid 1(a) was cut with a restriction enzyme to make a single cut in the circle. Plasmid 1(b) was cut in two places with restriction enzymes outside the region to be deleted. In this case, plasmid 1(b) was cut just upstream of the F domain and in the P domain.

(ii) One strand from one plasmid 1(a) was then hybridized with one strand from the other plasmid by heat denaturing each plasmid and allowing the strands to cross-hybridize to produce a hybrid “gapped duplex” intermediate.

(iii) A single-stranded oligonucleotide primer was synthesized having a sequence complementary to a short sequence on each side of the DNA to be deleted from the template strand. In this case, one-half of the primer consisted of a short sequence (TGTCTTAC-CAA) complementary to the codons for amino acids 1, 2 and 3 of mature t-PA and the other half consisted of a short sequence (GGAAACAGTGA) complementary to the beginning of the K2 domain.

(iv) The primer was then hybridized to the “template” strand such that it “straddled” the region between the DNA sequence that codes for amino acids 1-3 of mature t-PA and the beginning of the K2 domains causing the F domain to “loop out.”

(v) Next, the remaining gaps on either side of the primer were filled in enzymatically by DNA polymerase and the ends then sealed with DNA ligase, resulting in the formation of a heterodu-plex wherein the one strand comprised DNA corresponding to the pREM7685 parental plasmid (FK2P) and the other strand comprised DNA corresponding to the mutant progeny sequence (K2P) The gap filled in enzymatically for portions of the K2 and P domains consisted of over 500 nucleotides.

(vi) This heteroduplex was then introduced into the bacterium, E. Coli. Once in E. Coli, each strand of the heterodu-plex was copied independently, resulting in two populations' — one a copy of the original parental plasmid, which coded for FK2P, and the other a plasmid carrying a deletion mutant gene for t-PA, which coded for K2P (ie., Reteplase), the nucleotides coding for the F domain having been “deleted” by means of the Morinaga method.

Although plasmid pA27.3 expressed the Reteplase protein, the yield was very low and most of the host cells showed impaired viability. Thus, plasmid pA27.3 could not be used for commercial expression of Rete-plase.

Significantly, synthetic primer used in the SDM occurs at the amino acid sequences at the N-terminus. During SDM, synthetic DNA was used to create the deletion of a specific sequence which BM wanted to eliminate. The synthetic primers (of 22 nucleotides) for the SDM annealed with the cDNA template at amino acids 1-3 and 176-179, and were incorporated in the plasmid to create the deletion of the F domain. The codons for amino acids 50 to 175 had been deleted in the predecessor plasmid pREM7685.

8. pA27fd encoding Reteplase

To increase the yield of Reteplase, the fd terminator was added downstream of the 3' end of the region coding for the protein. As noted above, in connection with BM’s construction of the plasmid pePA126fd, the fd terminator is a naturally occurring DNA sequence found in a bacterial virus, known as the fd phage. The fd terminator does not code for any protein. It is a DNA sequence which regulates transcription. The expression of mammalian proteins in E. Coli. is significantly enhanced by adding the fd terminator downstream of the DNA coding for a protein.

This insertion of the fd terminator downstream of the K2P coding region to create plasmid pA27td was done by cutting and pasting DNA fragments from plasmids pA27.3 and pePA126fd (a plasmid containing DNA encoding mature t-PA and an fd terminator). These changes occur outside of the coding sequence for Reteplase.

9. The Final Production Plasmid for Reteplase pA27-T2L

To make the Reteplase production plasmid, pA27-T2L, the promoter in front of the DNA encoding Reteplase in plasmid pA27fd was switched. A promoter designated T2L was substituted for the existing “tac” promoter.

Plasmid pA27-T2L was obtained by combination of three fragments derived from plasmid pA27fd and another plasmid already containing the T2L promoter. One of the three fragments carrying the T2L promoter was made using a combination of conventional PCR and fusion PCR. This fusion PCR generated fragment contained the T2L promoter and part of the Reteplase coding region. The other two fragments used to construct pA27-T2L were isolated by cutting the DNA in plasmid pA27fd with restriction enzymes. These two fragments contained the other part of the Reteplase coding region, the fd terminator, and the remainder of the pA27-T2L plasmid vector outside the coding region. All three fragments were pasted together to make pA27-T2L.

None of the primers was incorporated into the DNA region encoding the N-terminus amino acid sequence of Reteplase.

DISCUSSION

1. Summary Judgment Standard

“Summary judgment is appropriate when ‘the pleadings, depositions, answers to interrogatories, and admissions on file, together with the affidavits, if any, show that there is no genuine issue as to any material fact and that the moving party is entitled to judgment as a matter of law.’ ” Barbour v. Dynamics Research Corp., 63 F.3d 32, 36 (1st Cir.1995) (quoting Fed.R.Civ.P. 56(c)), cert. denied, 516 U.S. 1113, 116 S.Ct. 914, 133 L.Ed.2d 845 (1996). “To succeed [in a motion for summary judgment], the moving party must show that there is an absence of evidence to support the nonmoving party’s position.” Rogers v. Fair, 902 F.2d 140, 143 (1st Cir.1990); see also Celotex Corp. v. Catrett, 477 U.S. 317, 325, 106 S.Ct. 2548, 91 L.Ed.2d 265 (1986).

“Once the moving party has properly supported its motion for summary judgment, the burden shifts to the non-moving party, who ‘may not rest on mere allegations or denials of his pleading, but must set forth specific facts showing there is a genuine issue for trial.’ ” Barbour, 63 F.3d at 37 (quoting Anderson v. Liberty Lobby, Inc., 477 U.S. 242, 256, 106 S.Ct. 2505, 91 L.Ed.2d 202 (1986)). “There must be ‘sufficient evidence favoring the nonmoving party for a jury to return a verdict for that party. If the evidence is merely colorable or is not significantly probative, summary judgment may be granted.’ ” Rogers, 902 F.2d at 143 (quoting Anderson, 477 U.S. at 249-50, 106 S.Ct. 2505) (citations in Anderson omitted). The Court must “view the facts in the light most favorable to the non-moving party, drawing all reasonable inferences in that party’s favor.” Barbour, 63 F.3d at 36.

2. The ’832 Patent

Genentech asserts that the Rete-plase production plasmids infringe the ’832 patent. The determination of whether an accused process infringes a claim in a patent involves two steps. First, the court must construe the claim asserted to be infringed to determine its meaning and scope. Second, it must compare the properly construed claim to the accused process. See Tanabe Seiyaku Co. v. United States Int'l Trade Comm’n, 109 F.3d 726, 731 (Fed.Cir.1997) (“Tanabe ”). To prove infringement, Genentech must prove by a preponderance of the evidence that the accused process embodies each and every claim limitation either literally or by equivalence. See Id.; Conroy v. Reebok Int'l, Ltd., 14 F.3d 1570, 1573 (Fed.Cir.1994); Warner-Jenkinson Co., Inc. v. Hilton Davis Chemical Co., 520 U.S. 17, 21, 117 S.Ct. 1040, 137 L.Ed.2d 146 (1997). Whether a process infringes literally or under the doctrine of equivalents is a question of fact. Tanabe, 109 F.3d at 731.

a. The Claim

Entitled “Method of Constructing a Replicable Cloning Vehicle Having Quasi-Synthetic Genes,” the ’832 patent “provides a method of general applicability” for the production of useful proteins of known amino acid sequence, including antibodies and enzymes, and is “particularly suited to the expression of mammalian polypeptide hormones and other substances having medical applications.” (’832 Patent, Col. 8, 11. 3-11.) The inventors claim that their application represents “the first occasion upon which a medically significant human polypeptide was directly expressed micro-bially, rather than in conjunction with extraneous protein.” (’832 prosecution history, Markman Hearing, Pl.Ex. 2, at p. 103.)

To do this, the ’832 patent teaches a method of making a plasmid using two types of DNA, synthetic DNA, and cDNA derived from messenger RNA. {See ’832 patent, Col. 10, 11. 35 et seq.) Specifically, the ’832 patent describes “a method of using organically-synthesized DNA to create an optimal 5' end (encoding the amino terminus of a protein) for the desired protein. The organically-synthesized DNA is joined to the cDNA, to create a chimeric [or semi-synthetic] gene.” (Cepko Report at p. 1.)

Claim 1 of the ’832 patent teaches the method of constructing the “replicable cloning vehicle” capable of expressing a gene for a particular polypeptide of known amino acid sequence by combining cDNA and synthetic DNA. {See ’832 patent, Col. 13, 11. 6-43.) Steps (a) and (b) involve “obtaining by reverse transcription from messenger RNA a first gene fragment” which encodes less than all of the amino 'acid sequence of the polypeptide. Critical to both step (a) and step (b) is that the whole reading sequence is not contained in the cDNA fragment. Step (c) involves providing by “organic synthesis one or more synthetic non-reverse transcript-gene fragments” for the “remainder” of the sequence. At least one of the synthetic fragments is described as coding for the amino terminus portion of the polypeptide. Step (d) involves joining the synthetic gene fragment(s) of Step (c) to the gene frag-mentes) described in Steps (a) and (b) and inserting these gene fragments into a plasmid in proper reading phase. According to Ravetch, “[i]n order for the synthetic and cDNA fragments to combine so that a particular polypeptide can be expressed, they must be correctly positioned relative to each other so that gene code will be read to result in the particular polypeptide.” (Ravetch Dir. Test. ¶ II.B.21.) See Attachment C.

Claim One of the ’832 patent reads:

In the method of constructing a replicable cloning vehicle capable, in a microbial organism, of expressing a particular polypeptide of known amino acid sequence wherein a gene coding for the polypeptide is inserted into a cloning vehicle and placed under the control of an expression promoter, the improvement which comprises:

(a) obtaining by reverse transcription from messenger RNA a first gene fragment for an expression product other than said polypeptide, which fragment comprises at least a portion of the coding sequence for said polypeptide;

(b) where the first fragment comprises protein-encoding codons for amino acid sequences other than those contained in said polypeptide, eliminating the same while retaining at least a substantial portion of said coding sequence, the resulting fragment nevertheless coding for an expression product other than said polypeptide;

the product of step (a) or, where required, step (b) being a fragment encoding less than all of the amino acid sequence of said polypeptide;

(c) providing by organic synthesis one or more synthetic non-reverse transcript-gene fragments encoding the remainder of the amino acid sequence of said polypeptide, at least one of said fragments coding for the amino-terminus portion of the polypeptide; and

(d) deploying the synthetic gene frag-mentes) of step (c) and that produced in step (a) or (b), as the case may be, in a replicable cloning vehicle in proper reading phase relative to one another and under the control of an expression promoter;

whereby a replicable cloning vehicle capable of expressing the amino acid sequence of said polypeptide is formed.

In the Markman hearing, the Court determined that the term “gene fragment” may refer to either single or double-stranded DNA, of any length, even two nucleotides. Genentech, 989 F.Supp. at 366. The Court construed the term “organically synthesized” to mean “the production of an oligonucleotide or fragment of a gene using organic chemistry without the use of an enzyme. It includes DNA made by replication of synthetic DNA.” Id. at 364.

b. “Material Change” Under Section 271(g)

Under this Court’s claim construction, BM concedes that the process for producing the intermediate plasmid pePA 98.1 which produces t-PA literally infringes the ’832 patent because it combines synthetic DNA which codes for two of the nucleotides for serine in the t-PA molecule with cDNA to direct expression of a known gene sequence in E. Coli.

However, BM argues that the Reteplase production plasmids do not infringe the patent. Stressing that these plasmids were produced outside the United States, in Germany, and have been materially changed from intermediate plasmid pePA98.1, BM contends that it is not liable for infringement under the Process Patent Amendments Act of 1988, Pub.L. No. 100-418, §§ 9001-9007, 102 Stat. 1107, 1563 (1988) (codified in various sections of 35 U.S.C.). This Act deems it an infringement to import, sell, offer to sell, or use in this country a product that was made abroad by a process protected by a United States patent. See 35 U.S.C. § 271(g). However, the Act does not apply if the product made by the patented process is “materially changed by subsequent processes” before it is imported. 35 U.S.C. § 271(g)(1).

Section 271(g) requires two separate inquiries. First, it requires that a patentee establish that an accused infringer imported a product made by a process falling within the scope of one or more of the claims of the patent. See Novo Nordisk of North America, Inc. v. Genentech, Inc., 77 F.3d 1364, 1367-68 (Fed.Cir.1996) (“Novo Nordisk ”). Second, once the pat-entee proves that the process falls within the literal scope of the patent, the court must determine whether the “materially changed” provision of Section 271(g) applies. “The ‘materially changed’ exception of Section 271(g) requires, at a minimum, that there by a real difference between the product imported, offered for sale, sold, or used in the United States and the products produced by the patented process.” BioTechnology General Corp. v. Genentech, Inc., 80 F.3d 1553, 1560 (Fed.Cir.1996) (“BTG ”). To determine whether the “materially changed” provision applies, the court must look to the substantiality of the change between the product of the patented process and the product that is being imported. See Eli Lilly & Co. v. American Cyanamid Co., 82 F.3d 1568, 1573 (Fed.Cir.1996).

C. Allocating the Burden of Proof under Section 271(g)

The statute is unclear as to whether the patentee or alleged infringer bears the burden of demonstrating “material change.” The issue has been addressed, in passing, by two federal courts. In Ajinomoto Co., Inc. v. Archer-Daniels-Midland Co., 1996 WL 621837 (D.Del. Oct.21, 1996), the court discussed whether, in the circumstances of the case, the burden of proof for showing a “material change” under Section 271(g) was on the alleged infringer. See 35 U.S.C. § 295 (1994). The court’s analysis hinged on Section 295’s burden-shifting mechanism. Under Section 295, the accused infringer’s product is presumed to have been made by the patented process if the trial court finds that (1) a substantial likelihood exists that the challenged product was made by the patented process, and (2) the patentee made a reasonable effort to determine the process actually used in the production of the product, but was unable to do so. “If the trial court makes these findings, the burden of establishing that the product was not made by the patented process is on the accused infringer.” Novo Nordisk, 77 F.3d at 1368 n. 6. Section 295 is inapplicable where the pat-entee is able to determine the process which the alleged infringer used. See Id. In Ajinomoto, the court concluded that, under section 295, if this “initial hurdle is surmounted, it is [the alleged infringer’s] burden to demonstrate that its products have been materially changed.” Ajinomoto, 1996 WL 621837, *12. However, because the initial burden was not met, the patentee was not afforded the benefit of the burden-shifting presumption. Id.

In Eli Lilly & Co. v. American Cyanamid Co., 896 F.Supp. 851 (S.D.Ind.1995), aff'd 82 F.3d 1568 (Fed.Cir.1996), involving a motion for a preliminary injunction, the Court held that, “when read as a whole, the two parts of section 271(g) require the plaintiff to demonstrate ... that the product made by the patented process is neither materially changed by subsequent processes nor a trivial and nonessential component of another product.” Id. at 855-56. The Federal Circuit did not directly address the issue on appeal.

This case law suggests that a patentee must bear the burden of proof on the issue of “material change” unless it satisfies the burden-shifting requirements of Section 295. Here, because there has been ample opportunity for discovery and Genentech has in fact determined the process that BM used, Section 295 is inapplicable. Novo Nordisk, 77 F.3d at 1368 n. 6. Accordingly, Genentech bears the burden on the issue of material change.

d. Analysis

As a preliminary matter, this Court must determine whether Reteplase is the “product” made by the patented process or whether the plasmid is the “product” for purposes of Section 271(g). BTG answers this question. Addressing a claim of the infringement of Genentech’s ’832 patent under Section 271(g) by a company that produced human growth hormone (hGH), the Federal Circuit determined that hGH was “a product which is made by a process patented in the United States,” even though claim 1 of the ’832 patent was literally directed to a method for producing a replicable cloning vehicle (e.g. a plasmid), not hGH. See BTG, 80 F.3d at 1560-61. Even though there was “little doubt that the plasmid product of the claimed process and hGH [were] entirely different materials, one being more than materially changed in relation to the other,” the Federal Circuit held:

The legislative history precisely anticipated this fact situation and indicated Congress’s intent that infringement of a process for making a plasmid is not to be avoided by using it to express its intended protein. Moreover, the ’832 patent itself explicitly contemplates that the patented process will be used as part of an overall process for producing hGH; indeed the patent discloses in detail how to make hGH by carrying out the claimed process and other necessary steps. Thus, it cannot be said as a matter of law that the production of hGH is too remote from the claimed process of making a replicable cloning vehicle. We therefore find no error in the court’s conclusion that hGH is a product that is “made by” the ’832 patented process.

Id. at 1561.

BTG is also helpful in resolving the present dispute because of the Federal Circuit’s rebanee on legislative history, specifically the “two-phased test” that the Senate Judiciary Committee recommended for determining whether a product was “materially changed” prior to its importation. Id. at 1576. This report outlined the test as follows:

In order to give the courts Congressional guidance in what may be a difficult determination, the Committee notes that the bill would establish the following two-phased test:

1. A product will be considered made by the patented process regardless of any subsequent changes if it would not be possible or commercially viable to make that product but for the use of the patented process. In judging commercial viability, the courts shall use a flexible standard which is appropriate to the competitive circumstances.

2. A product will be considered to have been made by a patented process if the additional processing steps which are not covered by the patent do not change the physical or chemical, properties of the product in a manner which changes the basic utility of the product by the patented process. However, a change in the physical or chemical properties of a product, even though minor, may be “material” if the change relates to a physical or chemical property which is an important feature of the product produced by the patented process. Usually, a change in the physical form of a product (e.g., the granules to powder, solid to liquid) or minor chemical conversion (e.g., conversion to a salt, base, acid, hydrate, ester, or addition or removal of a protection group) would not be a “material” change.

S.Rep. No. 83, 100th Cong., 1st Sess. 50 (1987). As the touchstone for determining material change, the Committee admonished that the courts must “exercise careful judgement (sic) in distinguishing those products that are too far removed from the patented process, and those that have been changed only in insignificant ways.” Id. at 49.

With respect to the first infringement inquiry under Section 271(g), Genentech argues that the process for constructing the BM production plasmids (pA27.3, pA27fd and pA27-T2L) violates claim one of the ’832 patents because organically synthesized fragments are combined with the reverse transcriptase produced sequence of the Reteplase gene.

BM makes four arguments to refute Genentech’s claim of literal infringement. Three are flawed; the last is not. First, BM argues that the first three steps of the method proscribe a process for constructing gene fragments for a “particular polypeptide of known amino acid sequence.” Because BM did not perform step (a)— obtaining a gene fragment of cDNA by reverse transcription from mRNA — at a time when it knew the amino acid sequence of Reteplase, it insists, it did not infringe the patent. However, this “conflation” theory — as Genentech dubs it — is not persuasive. As Dr. Ravetch said: “[Claim 1] provides the method allowing a researcher to work backwards, starting with the idea of a particular polypeptide sequence he desires to make and instructing him on how to create a DNA plasmid capable of expressing that polypeptide sequence.” (Ravetch Dir. Test. ¶ II.A.5.) At each of the stages of producing a plasmid, BM knew the amino acid sequence it desired.

Second, BM argues that in the construction of pA 27.3, there was no performance of step (a) of the ’832 patent — obtaining a gene fragment by reverse transcription— because the cDNA was obtained by removing the cDNA from one plasmid and transferring it to another. Therefore, according to BM, there could be no performance of step (b) of the patent — shortening the product of step (a).

The patent refers to cDNA as a “[gene] fragment derived by reverse transcription from mRNA.” (’832 patent, Col. 5, 11. 2-4.) Dr. Ravetch states: “cDNA is always obtained by reverse transcription by definition, whether it is immediately by reverse transcription or obtained by r