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

Filing 728

MEMORANDUM in Support re 727 MOTION for Leave to File Under Seal Documents Containing Defendants' Trade Secrets Submitted in Connection with the Pendleton and Galvin Declarations filed by F. Hoffmann-LaRoche LTD, Roche Diagnostics GmbH, Hoffmann LaRoche Inc.. (Attachments: # 1 Exhibit A# 2 Exhibit B# 3 Exhibit C)(Toms, Keith)

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Amgen Inc. v. F. Hoffmann-LaRoche LTD et al Doc. 728 Att. 3 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 1 of 10 UNITED STATES DISTRICT COURT DISTRICT OF MASSACHUSETTS Ai\JGEN INC., )) v. ) Plaintiff, ) ) I Exhibit C ) ) C. A. No.: 05-CY-12237-WGY F. HOFFMANN-LA ROCHE LTD., ) a Swiss Company, ROCHE ) ) ) DIAGNOSTICS GmbH, a German ) Company and HOFFANN-LA ROCHE ) INC., a New Jersey Corporation, ) Defendants. ) ) PUBLIC VERSION Exhibit 48 Declaration of Robert M. Galvin in Support of Amgen Inc.' s Reply in Support of its Motion for Summary Judgment of Infringement of '422 Claim 1, '933 Claim 3, and '698 Claim 6 Dockets.Justia.com Case 1:05-cv-12237-WGY "I Document 728-4 Filed 07/16/2007 Page 2 of 10 209 ELUCIDATION OF STRUCTURE SUMMARY: EVIDENCE OF STRUCTURE In the current study, the ROO503821 registration batches G004.05E to G008.05E as well as the batches G011.05E, G013.05E and G014.05E (produced with EPO from building 354, denoted as EPO/354) were analyzed using a set of different analytical methods to investigate the structure of the R00503821 molecule. The methods were chosen to charactenze the R00503821 molecule with respect to the molecular mass, the positional isomer distribution, the glycostrctures and the higher order strcture. The purpose of the study was to demonstrate the consistency of the R00503821 manufactunng process and to compare R00503821 drug substance produced with the 3F process (registration batches and the three qualifcation batches for EPO/354) to drug substance produced previously according to the preliminary process, the 1 F process and the 2F process. For details regarding the developmental history of the process refer to 3.2.S.2.6 "Developmental History. Matnx ▀ssisted .baser Desorption ionization Mass žpectometry (MALDI-MS) was used to determine the molecular mass of the different R00503821 batches. The signals of the mass spectra obtained from the R00503821 registration batches and the R00503821 EPO/354 batches correspond to the signals that are also present in the spectra obtained from the measurements of the previous and current RS. The molecular masses of the main component are in accrdance to the molecular mass expected from R00503821. In addition, it was found that the molecular masses of the ROO503821 molecule of the registration batches as well as of the R00503821 batches (EPO/354) are highly consistent and comparable to the molecular masses of the previously used RS (R204421) as well as to the current RS (782 386 00). The sites as well as the degree of pegylation of the R00503821 batches were analyzed by means of Lyse-peptide mapping. The amino terminus, Lys 45 and Lys 52 constitute the major pegylation sites of the R00503821 molecule exhibiting a degree of pegylation of approximately 40 %, 15 % and 25 %, respectively. Lys 20, Lys 116 and Lys 154 are also signifcantly pegylated (degree of pegylation between approximately 5 % and 10 %). The remaining Lys 97, Lys 140 and Lys 152 are either not affected by pegylation or exhibit a degree of pegylation close to the detection limit (DL) of the method (detection limits calculated for different positional isomers range from 1 to 5 %). With the analysis of the registration batches it was demonstrated that with respect to the positional isomer distnbution, the 3F process is performed with a high degree of consistency. The positional isomer distributions determined are within the range defined by ROO503821 batches produced previously using the preliminary, 1 F and 2F production processes. In addition, the positional isomer distnbutions of the R00503821 batches (EPO/354) were found to be comparable to the 'Pharma Biotechnology Ha~nn Anja Referenc: cmcO7081 Version: 1.0 b-Ro.05381_APl-3.2.S.3.1.STC.evs.sum 1/3 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 3 of 10 210 registration batches as well as to the database denved from previously produced batches. Besides the analysis of the positional isomers, the carbohydrate part of the R00503821 molecule was investigated and compared to the carbohydrate structures of EPO used for pegylation. The assessment was made with respect to the asialo N-Iinked oligosacchandes, the a-linked oligosacchandes and the total sialic acid content. The purpose of the analysis was to investigate the influence of the R00503821 manufactunng process on the carbohydrate structure of the EPO protein used for pegylation as well as to demonstrte the consistency of the R00503821 manufactunng process. The N-Iinked glycans were enzymatically released from the protein by means of N-glycosidase F treatment, the sialic acids were cleaved off simultaneously by neuraminidase treatment. The released asialo N-Iinked oligosacchandes were analyzed using high performance anion-exchange chromatography with pulsed amperometrc detection (HPAEe-PAD). With the analyses performed it was found that for the registration batches as well as for the batches produced with EPO/354, the relative amount of tetraantennary and lactosamine structures is slightly decreased in R00503821 as compared to EPO used for pegylation, whereas the amount of biantennary and tnantennary strctures is slightly increased. The differences in N-Iinked glycosylation observed between R00503821 and the respectve EPO used for pegylation are within the range defined by R00503821 batches produced previously with the preliminary process, the 1 F process and the 2F process. This also holds true for the R00503821 registration batches and the batches produced with EPO/354. The O-linked oligosaccandes of the R00503821 registration batches and the R00503821 batches produced with EPO/354 as well as of the respective EPO samples used for pegylation were investigated by means of Lyse peptide mapping with online mass detecton, the O-linked glycosylation of the samples was assessed using specific ion currnt (Sie) chromatograms of the Lyse peptide K6 carring the O-linked oligosacchandes. The data demonstrated that for the registration batches as well as for the batches produced with EPO/354, the relative ion count of the peptide K6_NANA2 is slightly decreased in R00503821 as compared to EPO used for pegylation, whereas the relative ion count of the peptides K6_NANAO and K6_NANA1 is slightly increased. The differences in O-Iinked glycosylation observed between R00503821 and the respective EPO used for pegylation are within the range defined by R00503821 batches produced previously with the preliminary process, the 1 F process and the 2F process. This also holds tre for the ROO503821 registration batches and the batches produced with EPO/354. The sialic acids were enzymatically released from the protein and analyzed by means of high performance anion-exchange chromatography with pulsed amperometric detection (HPAEe-PAD). It was found that for the registration batches as well as the batches produced with EPO/354, a slight difference in the total sialic acid content was observed as compared to EPO used for pegylation. 'Pharma Biotechnology Haus~r: Anja Reference: cmcO7081 Version: 1.0 b-Ro.053821_APL3.2.S.3.1.STC.evs.sum 2/3 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 4 of 10 211 The differences observed are wihin the range defined by ROO503821 batches produced previously with the preliminary proŠss, the 1 F process and the 2F process. Taken together, it is concluded that with respect to N-Iinked glycosylation, O-Iinked glycosylation and the total sialic acid content of R00503821 as compared to EPO used for pegylation, R00503821 is manufactured with a high degree of consistency. For the registration batches as well as for the batches produced with EPO/354, the slight quantitative differences in glycosylation observed between R00503821 and EPO are well within the range defined by previous batches. The higher order structure of the R00503821 registation batches and the R00503821 batches (EPO/354) was investigated by circular dichroism (eD) spectroscopy. The spectra of the batches were compared to the specta of the reference standard 782 386 00 acquired concurrently. The eD spectra of the R00503821 solutions were acquired in the far-UV (185 nm - 260 nm) as well as in the near-UV (250 nm - 350 nm) spectral regions. The far-UV eD of proteins is due to chi rally asymmetnc relative onentations of neighboring peptide bonds that are different for the different classes of secondary strctures (e.g. a-helix, ▀-sheet, ▀-tums, random). Therefore, the far-UV CD spectrum reflects the composite secondary structure of the protein. The intensities as well as the locations of the near-UV eD bands are dependent on the structural and electric environment of the side chains of the individual aromatic amino acids immobilzed within the tertiary strcture of the folded protein. As a consequence, the near-UV eD spectm constitutes a fingerprint of the tertiary strcture. With the analyses performed it was found that the far UV and near UV eD spectra of the registration batches as wen as of the R00503821 (EPO/354) batches are comparable to the spectra obtained for the reference standard. The slight diferences observed are within the limits of precision of the methodology. It is concluded that with regard to the higher order structure, the R00503821 registration batches and the batches (EPO/354) were produced with a high degree of consistency. As a summary, it can be concluded that with respect to the molecular mass, the positional isomer distnbution, the glycostructures and the higher order structure the R00503821 registration batches as well as the batches (EPO/354) were produced in a highly consistent manner. 'Pharma Biotechnology Haus~nn Anja Reference: crcO7081 Version: 1.0 b-Ro.05381_APl-3.2.S.3.1.STC.evs.sum 3/3 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 5 of 10 212 ELUCIDATION OF STRUCTURE ANALYSIS OF THE PEGYLTION SITES 1. SUMMARY The R00503821 registration batches, R00503821 batches produced with Epoetin beta (EPO) from building 354 (denoted as EPO/354) as well as the former reference standard (RS) R204421 (derived from batch G001.03E produced accrding to the 1 F process) and the current RS 782 386 00 (derived from batch G002.04E produced with the 2F process) were analyzed by means of Lyse Peptide mapping of R00503821 in order to determine the positional isomer distnbutions. It was demonstrated that with respect to the positional isomer distribution, R00503821 drug substance is produced with a high degree of consistency. In addition, the positional isomer distributions obtained from the R00503821 registration batches G004.05E to G008.05E produced with the 3F process (60 g scale manufactunng process trnsferred to the commercial facility) and the batches G011.05E, G013.05E and G014.05E produced with EPO/354 were found to be comparable to the data obtained from R00503821 batches produced previously using the 1 F and 2F prOduction processes. 2. OBJECTIVE The producton of R00503821 includes a pegylation step of EPO with the MSBA30K PEG reagent. This pegylation is the result of the reacton of the succinimidyl ester group of the MSBA30K PEG reagent with a free amino group of EPO forming an amide bond. As a consequence, the ROO503821 molecule can theoretically be pegylated at the N-terminus or at the e-amino group of the different lysine residues. The R00503821 vanants where pegylation occurred at different amino groups are denoted as positional isomers. The name of a positional isomer indicates the amino group to which the PEG chain is attached to the EPO molecule. The main pnnclple of this test method is based on the fact that a pegylated lysine prohibits cleavage at this site by the Lyse protease. It was the objective of the current study to determine the positional isomer distributions of the R00503821 registration batches and batches produced with EPO/354, and the RS (former RS R204421 derived from batch G001.03E, 1 F process and current RS 782 386 00 denved from G002.04E, 2F process) in order to demonstrate consistency of the production process as well as comparabilty of the registration batches and the batches produced with EPO/354 to the 1 F and 2F processes. 'Pharm Biotecnology Haussm~i: Anja Reference: cm7082 Version: 1.0 b-Ro.053821_APl-3.2.S.3.1.STC.evs.pe 116 Case 1:05-cv-12237-WGY i Document 728-4 Filed 07/16/2007 Page 6 of 10 213 3. MATERIALS AND METHODS The data given in this report were obtained with the registration batches G004.05E to G008.05E and the R00503821 batches (EPO/354) G011.05E, G013.05E and G014.05E. In addition, the former reference standard RS R204421 (denved from G001.03E, iF) or the current RS 782 386 00 (denved from G002.04E, 2F) were analyzed concurrently with the R00503821 batches. For calculation of the positional isomer distnbutions, the analysis of the EPO RS G030.06 was also included in each senes of measurements. Testing was performed according to the procedure descnbed in report 3.2.S.4.2 "Peptide Mapping (Lys-e) and Polyethylene Glycol (PEG) Positional Isomer Distnbution of ROO503821 Drug Substance", the characteristics of the method are also descnbed in the respective validation report 3.2.S.4.3. 4. RESUL TS Four senes of measurements were performed in order to analyze the pegylation of the R00503821 registation batches and the R00503821 (EPO/354). The positional isomer distnbutions obtained for these batches are compiled in Table 1. As can be derived from Table 1, the R00503821 registration batches and the batches produced with EPO/354 are produced with a high degree of consistency. the positional isomer distnbutions determined are comparable to the previous reference standard RS R204421 and the current RS 782 386 00 measured concurrently with the registration batches. Table 1 Pegylation Analysis of R00503821 Batches Site of Pegylation NH2 ILys 20 ILvs 45 ILvs 52 ILys 97 ILys 116 lLys 140lLys 1521Lys 154 Serles Batch 1 1 RS R204211) GOO4.05E RS 782 386 00 ~J 2 2 2 3 3 3 G005.05E G006.05E GOO7.05E G008.05E RS 782 386 00 2) RS 782 386 00 ┐) IRedacted I 4 4 4 4 1) G011.05E G013.05E G014.05E 4 RS 782 386 00 2) Reference Standard denved from batc GOO1.03E prduced accrding to 1he 1 F process 2) Reference Standard denved frm batc GOO2.04E produce accrding to 1he 2F prcess To facilitate companson of the positional isomer distnbution of batches from the 3F process to the positional isomer distnbutions obtained previously with the preliminary process, the 1 F and the 2F processes, the data obtained from the 'Pharma Biotecnology Hauss~i:n Anja Reference: cmcO7082 Version: 1.0 b-Ro.05381_APl-3.2.S.3.1.STC.evs.peg 216 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 7 of 10 214 registration batches and the R00503821 batches (EPO/354) were compared to the ranges observed with previous batches. Table 2 summanzes the statistics of positional isomer distributions of R00503821 batches produced previously. The statistics are also ilustrated in Rgure 1 for the registration batches and in Figure 2 for the R00503821 batches produced with EPO/354. The data demonstrate that the positonal isomer distnbutions of R00503821 batches produced accrding to the 3F process are within the ranges observed previously with batches derived from the preliminary process as well as from the 1 F and 2F processes. Therefore it is concluded that with respect to the positional isomer distrbution of the R00503821 drug substance, the 3F process is comparable to the previously used production processes of R00503821. 5. CONCLUSION It is concluded that with regard to the positional isomer distnbution of the PEGchain, R00503821 is produced with high consistency. The data obtained for the registration batches G004.05E through G008.05E as well as for the R00503821 batches (EPO/354) are within the range defined by the batches produced with previously used producton processes. . 'Pharma Siotechnology HaussrTr:n Anja Reference: cmcO7082 Version: 1.0 b-Ro.053821_APl-3.2.S.3.1.STC.evs.peg 3/6 Case 1:05-cv-12237-WGY ~I Document 728-4 Filed 07/16/2007 Page 8 of 10 215 Table 2 Statistics of the Positional Isomer Distnbution of R00503821 Batches Produced with Different Production Processes Site of Pegylation Batch NHi 154 Statistics of RS 204421 Mean Standard Deviation LL 1) UL 2) Statistics of RS Mean 782 386 00 standard Deviation LL UL Statistics of Batches from the Preliminary Mean Standard Deviation LL 1) Process Statistics of UL 2) Mean íRedactedl Batches from the 1 FProcess 3) Stndard Deviation LL 1) UL 2) Statistics of Batches from the 2FProcess 4) Mean standard Deviation LL 1) UL 2) Statistics of Registrtion Batches Mean standard Deviation Minimum Maximum Mean Statistics of R00503821 produced with EPO/354 standard Deviation Minimum Maximum 2) Upper Confidence Limit calculated using 3 stndard deviations 3) The mean of RS R201440 denved frm batch GOO1.03E was included into the sttistcal analysis Lower Confidence Limit calculate using 3 stndard deviations 4) The mean of RS 78238600 denved from batc G002.04E was included into the statistcal analysis 'Pharma Biotechnology Hausm~nn Anja Reference: cmcO7082 Version: 1.0 b-Ro.05381_APl-3.2-S.3.1.STC.evs.peg 4/6 Case 1:05-cv-12237-WGY .1 Document 728-4 Filed 07/16/2007 Page 9 of 10 216 Figure 1 Degree of Pegylation of the Amino Terminus (NH2) and the Lysine Residues of ROO503821 Registration Batches as Compared to R00503821 Derived from Different Production Processes RS R204421 mean of all measurements performed Gnnn.02E mean of the R00503821 batches G008.02E through G013.02E from the preliminary process Gnnn.03E mean of the ROO503821 batches G001.03E through G005.03E from the 1 F process Gnnn.04E mean of the R00503821 batches G001.04E through G004.04E from the 2F process Registration batches G004.05E through G008.05E from the 3F process Error bars indicate the confidence limits calculated with 3 standard deviations ...eo 100 , . . RS R204421 90 . . 13 G˛n'rÝ.02E . Gnnn.03E :'~. ŕÍ. e. : :c.. 7:0 . . . , iÝ Gnnn.04E . IS GQ04.0$E: : :~ ~˛ il GOO::.QSE . ::~~30: (l 20 i: íRedacted I .., ., .. .., . . GQ06.05E. IS G007.05E . .. G˛OB.05E : :.:e: ',1.0.: :: i:' ::~ :0 : .:"-0 " .gO IRedacted I , Site˛f Pegylation 'Pharm Biotechnology Hausm~r:n Anja Reference: cmcO7082 Version: 1.0 b-Ro.053821_AP1_3.2.S.3.1.STC.evs.peg 5/6 Case 1:05-cv-12237-WGY Document 728-4 Filed 07/16/2007 Page 10 of 10 217 Figure 2 Degree of Pegylation of the Amino Terminus (NH2) and the Lysine Residues of R00503821 Batches Produced with EPO/354 as Compared to R00503821 Derived from Previous Production Processes RS 782 386 00 mean of all measurements performed Gnnn.02E mean of the R00503821 batches G008.02E through G013.02E from the preliminary process Gnnn.03E mean of the R00503821 batches G001.03E through G005.03E from the 1 F process Gnnn.04E mean of the R00503821 batches G001.04E through G004.04E from the 2F process Gnnn.05E Registration batches G004.05E to G008.05E from the 3F process G011.05E, G013.05E and G014.05E (EPO/354) from the 3F process Errr bars indicate the confidence limits calculated with 3 standard deviations '1:00. . , .. RS 782 386 00 ~ Ghnh.O:zE : . . : . . Ghnh.O:3E: ' :90. ,~ .~. . l,.' :80" . .70 ž:Úfr .:;:: :,g':4÷ .. Ghnn.04E. :. ~ Ghnn.O.5E. .. .. G01 1".O.5E' . .ns t\ -s - 511 .. G013.05E. íí G014.05E Q; . . 'ii.' 30 ,0 ci20 (. . ij .:0;' '10 IRedacted I íRedacted (j' ' Q.0 '-10 'Pharma Biotechnology Hausrn-in AnJa . Reference: cmcO7082 Version: 1,0 b-Ro.053821_APl-3.2S.3.1.STC.evs.peg 6/6 .__ _ _. . _""'''A''''I'

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