Kidney International, Vol. 67 (2005), pp. 2346–2353
`
`The increased incidence of pure red cell aplasia with an Eprex
`formulation in uncoated rubber stopper syringes
`
`KATIA BOVEN, SCOTT STRYKER, JOHN KNIGHT, ADRIAN THOMAS, MARC VAN REGENMORTEL,
`DAVID M. KEMENY, DAVID POWER, JEROME ROSSERT, and NICOLE CASADEVALL
`
`Johnson and Johnson, Pharmaceutical Research and Development, L.L.C, Raritan, New Jersey; Centre National de la Recherche
`Scientifique, Ecole Sup´erieure de Biotechnologie de Strasbourg, France; Department of Microbiology, National University of
`Singapore, Singapore; Kidney Laboratory, Austin Research Institute, Austin, Australia; Service de N´ephrologie, H ˆopital Tenon,
`Paris, France; and Service d’H´ematologie Biologique, H ˆopital H ˆotel-Dieu, Paris, France
`
`The increased incidence of pure red cell aplasia with an Eprex
`formulation in uncoated rubber stopper syringes.
`Background. The incidence of pure red cell aplasia (PRCA)
`in chronic kidney disease patients treated with epoetins in-
`creased substantially in 1998, was shown to be antibody me-
`diated, and was associated predominantly with subcutaneous
`administration of Eprex(cid:1). Atechnical investigation identified
`organic compounds leached from uncoated rubber stoppers in
`prefilled syringes containing polysorbate 80 as the most proba-
`ble cause of the increased immunogenicity.
`Methods. This study investigated whether the incidence of
`PRCA was higher for exposure to the product form contain-
`ing leachates than for leachate-free product forms. Antibody-
`mediated PRCA cases were classified according to indication,
`product form, and route of administration. Exposure estimates
`were obtained by country, indication, route of administration,
`and product form.
`Results. For 2001 to 2003, the PRCA incidence rate for pa-
`tients with subcutaneous exposure to Eprex in prefilled syringes
`with polysorbate 80 and uncoated rubber stoppers (leachates
`present) was 4.61/10,000 patient years (95% CI 3.88–5.43) ver-
`sus 0.26/10,000 patient years (95% CI 0.007–1.44) for syringes
`with coated stoppers (leachates absent). The rate difference was
`4.35/10,000 patient years (95% CI 3.44–5.26; P < 0.0001); the
`rate ratio was 17 (95% CI 3.14–707). A substantial rate differ-
`ence remained in sensitivity analyses that adjusted for exposure
`to multiple product forms.
`Conclusion. The epidemiologic data, together with the
`chemical and immunologic data, support the hypothesis that
`leachates from uncoated rubber syringe stoppers caused the in-
`creased incidence of PRCA associated with Eprex. Currently,
`all Eprex prefilled syringes contain fluoro-resin coated stop-
`pers, which has contributed to decreased incidence of PRCA
`with continued surveillance.
`
`Key words: pure red cell aplasia, erythropoietin, antibodies, epoetin
`alfa.
`
`Received for publication October 22, 2004
`and in revised form December 21, 2004
`Accepted for publication January 11, 2005
`
`C(cid:1) 2005 by the International Society of Nephrology
`
`Pure red cell aplasia (PRCA) is a rare disorder that
`manifests itself as a severe, isolated anemia of sudden
`onset, characterized by an almost complete absence of
`red cell precursors in the bone marrow and a reticulo-
`cyte count below 10 × 109/L [1]. Many potential causes
`for PRCA have been reported, but most concern only
`isolated case reports, and about 50% of cases have no
`known cause [2]. Over the decade following its introduc-
`tion in 1989, three cases of PRCA were associated with re-
`combinant human erythropoietin (epoetin) treatment for
`anemia in patients with chronic kidney disease (CKD) [3–
`5]. From 1998 onward, however, an increasing number of
`cases were reported [2]. These patients developed PRCA
`due to neutralizing antibodies to epoetin that cross-react
`with endogenous erythropoietin, and therefore, the con-
`dition is termed antibody-mediated PRCA.
`These cases occurred mainly in patients receiving epo-
`etin alfa (EPREX(cid:1)/ERYPO(cid:1); Ortho Biotech, a divi-
`sion of Janssen-Cilag, Bridgewater, NJ, USA) outside the
`USA, although a limited number received other epo-
`etin products or a combination of products [6–10]. Ir-
`respective of the type of epoetin administered, virtually
`all of the cases occurred in CKD patients treated with
`subcutaneous epoetin; no cases have occurred in cancer
`patients [10]. To limit
`the increasing incidence of
`antibody-mediated PRCA attributed to Eprex, risk-
`mitigation initiatives were taken, including improved
`cold chain storage and handling of Eprex [11], and a
`switch to intravenous administration for CKD patients.
`Health authorities in Europe formally contraindicated
`subcutaneous administration of Eprex for CKD patients
`in December 2002.
`Technical and clinical investigations were initiated to
`identify the cause of the observed increase in the im-
`munogenicity of Eprex. Investigations into the manufac-
`turing process for bulk drug substance and characteri-
`zation of Eprex packaged into prefilled syringes did not
`uncover any irregularities in the manufacturing process
`
`2346
`
`Novartis Exhibit 2176.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`2347
`
`EPO
`
`EPREX syringe
`with coated stopper:
`no extra peaks
`
`EPREX syringe
`with rubber stopper:
`multiple extra peaks
`
`I
`
`mAU
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`70
`
`min
`
`Fig. 1. Reverse-phase high-performance liquid chromatography. The large peak at 47 minutes is epoetin (EPO). The small peaks at 54 and 57
`minutes are polysorbate 80. A series of extra peaks are present in the polysorbate 80 formulation of Eprex dispensed from prefilled syringes with
`uncoated rubber stoppers (red) that are not present in the same product dispensed from syringes with coated stoppers (blue). Reproduced with
`modification from Sharma et al with permission from the European Journal of Hospital Pharmacy [12].
`
`or finished product that could be linked to the increased
`incidence of PRCA [12]. During the course of this sys-
`tematic investigation, however, a series of peaks were
`observed on an experimental high-performance liquid
`chromatography (HPLC) elution profile of Eprex from
`prefilled syringes (Fig. 1) [12]. These peaks occurred
`after the large epoetin peak and were identified by
`mass spectrometry as organic compounds present in
`the rubber stoppers [12]. It was reported in 2002 that
`polysorbates and other nonionic detergents can leach
`compounds out of plastics and rubber materials [13].
`These leachates were only detected in the polysor-
`bate 80 formulation of Eprex from prefilled syringes
`with uncoated rubber stoppers. Eprex preparations con-
`taining polysorbate 80 with fluoro-resin coated stop-
`pers (FluroTec(cid:1), Daikyo Seiko, Tokyo, Japan) did not
`demonstrate these leachates, nor did those with uncoated
`stoppers containing human serum albumin (HSA) in-
`stead of polysorbate 80 as stabilizer. Leachates were
`not present in other epoetin products, all of which
`have coated stoppers. These data suggest
`that
`the
`polysorbate 80 in the formulation had extracted these
`compounds from the uncoated rubber stoppers [12].
`The concentration of leachates in the syringes was vari-
`able and increased with time and exposure to heat [12].
`Studies using the well-characterized antigen ovalbumin
`demonstrated that these leached compounds could act
`as adjuvants when administered subcutaneously in mice
`[12].
`The invivo findings for the adjuvant potential of
`leachates led to the hypothesis that leachates present in
`prefilled syringes of Eprex with uncoated rubber stop-
`
`pers increased the risk of PRCA when this product was
`administered subcutaneously in humans. To determine if
`this hypothesis was consistent with the clinical data, the
`incidence of PRCA by formulation, product form, and
`route of administration were determined in a retrospec-
`tive study using reported PRCA cases. This was possible
`because prefilled syringes with coated stoppers had al-
`ready been introduced in 2001 for some dosage strengths
`and, in 2003, all dosage strengths of prefilled syringes with
`the polysorbate 80 formulated Eprex were shipped with
`coated stoppers.
`
`METHODS
`Reverse-phase HPLC
`Reverse-phase HPLC analysis was carried out as previ-
`ously described [12]. Briefly, the contents of epoetin alfa
`syringes were injected into a Vydac C4 column. After a 5-
`minute hold at 5% mobile phase B (0.06% trifluoroacetic
`acid in acetonitrile), samples were eluted at a flow rate of
`1.0 mL/minute by a linear gradient of 5% to 90% of mo-
`bile phase B for 90 minutes. Monitoring was performed
`at 280 nm.
`
`Pharmacovigilance
`An expanded review was undertaken of all sponta-
`neous adverse events reported to Johnson & Johnson
`relating to any epoetin product and any indication
`for use, gathered from physicians, pharmacists,
`the
`literature, medical representatives, and the patients
`themselves. The Medical Dictionary for Regulatory
`
`Novartis Exhibit 2176.002
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`2348
`
`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`Activities’ code terms was used to ensure that all pos-
`sible cases of antibody-mediated PRCA were captured.
`Loss of effect was defined as an absent or decreased re-
`sponse to erythropoietin treatment in a patient who pre-
`viously responded to such therapy, and characterized by
`an increase in erythropoietin dosage and/or a sustained
`decrease in hemoglobin. Antibody-mediated PRCA was
`defined as either suspected or confirmed PRCA with a
`positive anti-erythropoietin antibody test with any testing
`method. Suspected PRCA was defined as loss of efficacy
`with a decrease in hemoglobin of ≥2 g/dL in 30 days, need
`for transfusions, reticulocytes <20,000 per mm3, platelet
`count and white blood cell count normal, and bone mar-
`row unavailable or unknown. Confirmed PRCA was de-
`fined as suspected PRCA plus bone marrow with isolated
`erythroblastopenia.
`Exposure was estimated for patients receiving Eprex
`(exclusively or in combination with another epoetin prod-
`uct) for anemia in five disease areas: chronic kidney
`disease receiving dialysis, chronic kidney disease not re-
`ceiving dialysis, oncology, infectious disease (infection
`with human immunodeficiency virus), and remaining use
`(typically surgery). Estimates of the actual patient years
`of exposure were obtained for each country by year (and
`monthly from July 2002) for each indication, route of
`administration, and formulation using drug-monitoring
`data. The PRCA incidence rate (per 10,000 patient years)
`was calculated by dividing the number of cases by the cor-
`responding exposure (in 10,000 patient years). Reports
`received by June 30, 2004 were included. When adjust-
`ing for market exposure for the cumulative time period,
`the incidence by date of onset is calculated with expo-
`sure through the end of April 2004, not to the end of
`June 2004. This accommodates an assumed minimum in-
`terval of two month’s delay from the occurrence of LOE
`to the determination of PRCA diagnosis and report to
`the manufacturer; however, the lag time, in practice, may
`be longer.
`
`Case series analysis
`Antibody-mediated PRCA cases in patients with
`CKD were identified in the Johnson & Johnson clinical
`database. The cases were attributed to Eprex if exposure
`was more than one month with an adequate response and,
`if there was a recent switch from another epoetin, there
`was an ongoing response to that epoetin at the time of
`the switch. Using information from treating physicians,
`Counsel for International Organizations of Medical Sci-
`ences forms, site assessments, and the company clinical
`database, each case was evaluated and assigned to a cate-
`gory based on exposure to a certain product presentation
`(vials or prefilled syringes containing HSA or polysorbate
`80 as stabilizer, coated or uncoated rubber syringe stop-
`pers) and route of administration (intravenous or subcu-
`
`taneous) of Eprex. The evaluation also took into account
`the first date of availability for the different formulations
`and product forms per country.
`The following criteria were predetermined for the
`purpose of retrospectively attributing the occurrence of
`PRCA to a particular product exposure or route of ad-
`ministration. The time frame of suspected exposure was
`defined as one to 12 months prior to the onset of loss of
`effect of Eprex. As there have been no cases of PRCA
`with only intravenous exposure to Eprex, the nine cases
`with mixed intravenous and subcutaneous exposure prior
`to loss of effect were classified together with cases with
`only subcutaneous exposure. Two cases of patients not on
`dialysis or on peritoneal dialysis whose route of admin-
`istration was unknown were attributed to subcutaneous
`exposure, a reasonable assumption, as most of these pa-
`tients do not routinely have intravenous access. Based on
`the information of product availability over time in the
`different countries, a decision tree was designed to allow
`classification of each case into a specific category. If both
`the product form and the dose were unknown, the case
`was classified as unknown.
`
`Statistical methods
`Rate ratios, rate differences, and P value calculations
`for the case series analyses were performed with STATA
`version 8.0 (Stata Corporation, College Station, TX,
`USA). Confidence intervals for the incidence rates were
`based on the Poisson distribution for rare events.
`
`RESULTS
`Incidence
`Based on reports received by Johnson & Johnson from
`January 1, 1989 to June 30, 2004, 217 cases of antibody-
`mediated PRCA were observed among patients with
`chronic kidney disease. Of these, 206 cases were at-
`tributed to epoetin alfa marketed under the EPREX/
`ERYPO trade name, 23 of which had exposure to both
`Eprex and another epoetin. Of the 206 cases, 192 had
`only subcutaneous exposure, nine had both intravenous
`and subcutaneous exposure, and for five, the route was
`unknown. Assigning those with mixed route exposure to
`the subcutaneous route and excluding those cases with
`unknown route, the overall rate of antibody-mediated
`PRCA was 1.61 (95% CI 1.40–1.85) per 10,000 patient
`years of subcutaneous exposure (201 cases/1,244,970 pa-
`tient years) versus 0 (95% CI 0–0.04) per 10,000 patient
`years of intravenous exposure (0 cases/871,098 patient
`years) for the cumulative time period.
`The occurrence of PRCA began to increase in 1998
`and peaked from 2001 to 2002 (analyzed by date of on-
`set rather than date of report). Subcutaneous exposure
`to the polysorbate 80 formulation with uncoated syringe
`
`Novartis Exhibit 2176.003
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`2349
`
`PRCA case count, in period
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`160,000
`
`140,000
`
`120,000
`
`100,000
`
`80,000
`
`60,000
`
`40,000
`
`20,000
`
`0
`
`EPREX exposure, person-years
`
`2003
`
`2002
`
`2001
`
`2000
`
`1999
`
`1998
`
`1997
`
`1996
`
`1995
`
`1994
`
`1993
`
`1992
`
`1991
`
`1990
`
`1989
`
`PRCA
`SC polysorbate 80 uncoated stopper
`
`IV all forms
`SC polysorbate 80 vials
`
`SC HSA
`SC polysorbate 80 coated stopper
`
`- D-
`
`Fig. 2. Onset of antibody-mediated pure red cell aplasia (PRCA) and worldwide Eprex exposure for nephrology by calendar time. The bar graph
`shows the number of antibody-mediated PRCA cases by year in which the loss of effect occurred. Four cases are not shown due to unknown year
`of loss of effect. Two additional cases occurred in the period January-April 2004. Eprex exposure is shown linearly for intravenous exposure to
`all product forms and subcutaneous exposure to the human serum albumin formulation, the polysorbate 80 formulation in syringes with uncoated
`stoppers, polysorbate 80 in vials, and polysorbate 80 in syringes with coated stoppers. The polysorbate 80 formulation of Eprex was introduced in
`1998.
`
`stoppers peaked with 158,650 patient years in 2002, while
`onset of PRCA peaked in 2003 with 71 cases (see Fig. 2).
`In the period January through April 2004, two new cases
`were reported.
`
`Case series analysis
`Classification of PRCA cases by formulation of Eprex
`within 1 to 12 months of onset is shown in Table 1. In all,
`182 cases had subcutaneous exposure to prefilled syringes
`containing a formulation with polysorbate 80 stabilizer
`and uncoated rubber stoppers. In 46 of the 182 cases, an
`assumption was made that the product form was a pre-
`filled syringe and not a single-use vial based on product
`availability in the country. Ten cases had exposure to only
`HSA-containing product (vials or prefilled syringes), and
`one had exposure to only prefilled syringes with polysor-
`bate 80 and coated stoppers. Insufficient information was
`available to classify 12 cases.
`Exposure to Eprex over the cumulative period 1989
`to April 2004, and for the period 2001 to 2003, when
`
`exposure estimation and case ascertainment were most
`complete (due to publicity), and when both uncoated
`and coated stopper formulations were on the market, is
`shown in Tables 2 and 3. For the cumulative period, the
`incidence rate of PRCA in patients who received Eprex
`from syringes with polysorbate 80 and uncoated rubber
`stoppers was 3.43/10,000 patient years (95% CI 2.95–3.96)
`versus 0.23/10,000 patient years (95% CI 0.006–1.28) for
`products with polysorbate 80 and coated stoppers. The
`rate difference between these products was 3.20/10,000
`patient years (95% CI 2.53–3.87; P < 0.0001), and the
`rate ratio was 15 (95% CI 2.7–594). For the period 2001
`to 2003, the incidence rate of PRCA in patients who re-
`ceived Eprex from syringes with uncoated rubber stop-
`pers and polysorbate 80 was 4.61/10,000 patient years
`(95% CI 3.88–5.43) versus 0.26/10,000 patient years (95%
`CI 0.007–1.44) for products with coated stoppers and
`polysorbate 80. The rate difference between these prod-
`ucts was 4.35/10,000 patient years (95% CI 3.44–5.26; P <
`0.0001), and the rate ratio was 17 (95% CI 3.14–707). Sim-
`ilar results were obtained comparing the incidence rate of
`
`Novartis Exhibit 2176.004
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`2350
`
`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`Table 1. Cases of antibody-mediated pure red cell aplasia in chronic
`kidney disease patients reported between January 1, 1989 and June 30,
`2004 were classified by form of Eprex exposure
`
`Eprex form by subcutaneous
`route within 1 to 12 months
`of onset of loss of effect
`
`Uncoated rubber stopper, polysorbate
`80 formulation, prefilled syringe
`Coated stopper, polysorbate 80
`formulation, prefilled syringe
`Human serum albumin formulation
`Polysorbate 80 formulation, vials
`Mixed:
`Uncoated rubber stopper, polysorbate
`80 formulation, prefilled syringe,
`and coated stopper, polysorbate 80
`formulation, prefilled syringe
`Mixed:
`Uncoated rubber stopper,
`polysorbate 80 formulation,
`prefilled syringe, and human serum
`albumin formulation
`Unknown
`Total
`
`1989–April 2004
`(total)
`
`2001–2003
`(peak)
`
`151
`
`1
`
`10
`1
`
`27
`
`4
`
`12
`206
`
`116
`
`1
`
`2
`1
`
`25
`
`1
`
`9
`155
`
`PRCA in patients who received Eprex from syringes with
`uncoated rubber stoppers and polysorbate 80 against that
`for the subtotal of all other subcutaneous forms combined
`(Tables 2 and 3).
`To avoid bias, the analysis was adjusted to account for
`mixed exposure in both the cases and the reference popu-
`lation. Cases with mixed exposure accounted for less than
`20% of the total with known exposure. As shown in the
`footnotes to Tables 2 and 3, estimates for rate differences
`and ratios were decreased by less than 20% when cases
`with mixed exposure were excluded. For 2001 to 2003, ap-
`proximately 25% of subcutaneous exposure to prefilled
`syringes with polysorbate 80 and coated stoppers was at-
`tributed to replacement of syringes with uncoated stop-
`pers; lower mixed usage was anticipated for other forms.
`In sensitivity analyses, exposure estimates for product
`forms other than prefilled syringes with uncoated rub-
`ber stoppers and polysorbate 80 were decreased by 25%
`based on observed temporal trends in usage patterns. The
`sum of exposure subtracted from other forms was added
`to exposure to prefilled syringes with uncoated rubber
`stoppers and polysorbate 80. The rate difference between
`uncoated syringe stoppers and coated syringe stoppers
`for 2001 to 2003 was 3.89/10,000 patient-years (95% CI
`2.92–4.86; P = 0.0001), and the rate ratio was 12 (95%
`CI 2.2–448) (see Table 4). Results were similar for the
`cumulative period 1989 to April 2004.
`
`DISCUSSION
`Before 1998, Eprex was formulated with HSA as stabi-
`lizer. It was replaced with polysorbate 80 for most coun-
`tries outside the USA to preclude hypothetical risk of
`
`transmission of infectious diseases by HSA [10]. It should
`be noted that multidose vials containing HSA continue
`to be available in Canada, and prefilled syringes contain-
`ing HSA are available in Turkey. Overall, the case series
`shows a clear temporal association between the increased
`incidence of antibody-mediated PRCA and the replace-
`ment of HSA with polysorbate 80 in countries outside
`the USA (Fig. 2). Detailed analysis of the case series re-
`vealed a substantial difference in incidence rates between
`patients with subcutaneous exposure to prefilled syringes
`with polysorbate 80 and uncoated rubber stoppers and
`all other product forms, suggesting that the introduction
`of the stabilizer polysorbate 80 in combination with the
`uncoated rubber stoppers had a major impact on the im-
`munogenicity of Eprex given subcutaneously.
`Subcutaneous administration of Eprex in CKD pa-
`tients has decreased markedly since its contraindication
`in Europe; however, a low level of subcutaneous use con-
`tinues. For January through April 2004, subcutaneous
`exposure to forms containing polysorbate 80 was 5000
`patient years, and for forms containing human serum al-
`bumin, 4600 patient years. This population was included
`in the case series reported here. The analysis shows that
`the immunogenicity of Eprex in prefilled syringes with
`coated stoppers is substantially lower than that of the
`product with uncoated rubber stoppers. These findings
`correlate with the results of the technical investigation,
`which demonstrated that the combination of polysor-
`bate 80 and uncoated rubber syringe stoppers introduced
`leachates from the stoppers into the product [12].
`Although rubber stoppers were in use from the first
`introduction of Eprex prefilled syringes for subcuta-
`neous administration in 1994, the replacement of HSA
`with polysorbate 80 in 1998 appears to have effected a
`change in the leaching of potentially immunogenic com-
`pounds from the rubber stoppers of epoetin presenta-
`tions most commonly used in patients with CKD. Epoetin
`beta and darbepoetin syringes for subcutaneous injection
`were introduced after those of Eprex and have coated
`stoppers; no leachates were detected in these products,
`despite the presence of polysorbate stabilizers [12]. The
`enhanced immune response in mice in the presence of
`rubber leachates supports the hypothesis that these com-
`pounds could act as adjuvants to increase the immuno-
`genicity of Eprex in humans [12].
`The question remains why relatively few cases of
`antibody-mediated PRCA have occurred, and why some
`(a very few) cases have occurred in patients treated only
`with products other than Eprex. Multiple factors are re-
`quired to trigger a T-cell–mediated immune response and
`loss of tolerance. These include the presence of a suffi-
`cient number of erythropoietin-recognizing T cells and B
`cells in the patient, as well as erythropoietin and an adju-
`vant. The relatively low frequency of cases is most likely
`due to the relatively low and variable concentration of
`
`Novartis Exhibit 2176.005
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`2351
`
`Table 2. Case count, Eprex exposure, and incidence rate of antibody-mediated pure red cell aplasia in chronic kidney disease patients received
`from January 1, 1989 to June 30, 2004
`
`Eprex form by subcutaneous route only
`
`Case
`count
`
`Exposure
`(patient-years)b
`
`Incidence rate
`(per 10,000 patient-years)
`
`95% confidence interval
`(Poisson exact)
`
`182a
`
`1
`
`10
`1
`194
`12
`
`530,991
`
`43,659
`
`598,690
`71,630
`1,244,970
`713,979
`
`Uncoated rubber stopper, polysorbate 80
`formulation, prefilled syringe
`Coated stopper, polysorbate 80 formulation,
`prefilled syringe
`Human serum albumin formulation
`Polysorbate 80 formulation, vials
`Total (case count where onset and form are known)
`Subtotal: all subcutaneous forms except uncoated
`stopper polysorbate 80 prefilled syringe
`Comparison of uncoated stopper versus coated stopper polysorbate 80 prefilled syringe with mixed-exposure cases included (N = 31) and without adjustment of
`exposure denominator for mixed exposure: rate difference = 3.20/10,000 patient-years (95% CI 2.53–3.87); P < 0.0001, rate ratio = 15 (95% CI 2.7–594). Comparison of
`uncoated stopper versus coated stopper polysorbate 80 prefilled syringe with mixed-exposure cases excluded (N = 31) and without adjustment of exposure denominator
`for mixed exposure: rate difference = 2.62/10,000 patient-years (95% CI 1.98–3.25); P < 0.0001, rate ratio = 12 (95% CI 2.2–494).
`aPRCA cases with mixed exposure (N = 31) to both uncoated stopper polysorbate 80 prefilled syringe and either coated stopper polysorbate 80 prefilled syringe (N
`= 27) or human serum albumin formulations (N = 4) were assigned to the uncoated stopper category. Eprex form was unknown for 12 cases.
`bWhen adjusting for market exposure, the actual exposure data used were calculated to the end of April 2004. This accommodated an assumed minimum interval of
`two months’ delay from identification of loss of effect to the actual report to the sponsor (June 2004), and helped address the latency from exposure to development of
`disease. Exposure was not adjusted for patients that switched forms or routes within a 12-month period.
`
`3.43
`
`0.23
`
`0.17
`0.14
`1.56
`0.17
`
`2.95–3.96
`
`0.006–1.28
`
`0.08–0.31
`0.004–0.78
`1.35–1.79
`0.09–0.29
`
`Table 3. Comparison by form 2001 to 2003: Case count, Eprex exposure, and incidence rate of antibody-mediated pure red cell aplasia in chronic
`kidney disease patients with onset of loss of effect during 2001 to 2003 and received by June 30, 2004
`
`Eprex form by subcutaneous route only
`
`Uncoated rubber stopper, polysorbate 80
`formulation, prefilled syringe
`Coated stopper, polysorbate 80 formulation,
`prefilled syringe
`Human serum albumin formulation
`Polysorbate 80 formulation, vials
`Total (case count where onset and form are known)
`Subtotal: all subcutaneous forms except uncoated
`stopper polysorbate 80 prefilled syringe
`
`Case
`count
`
`142a
`
`1
`
`2
`1
`146
`4
`
`Exposure
`(patient-years)b
`
`Incidence rate
`(per 10,000 patient-years)
`
`95% confidence interval
`(Poisson exact)
`
`308,232
`
`38,608
`
`42,305
`27,794
`416,939
`108,707
`
`4.61
`
`0.26
`
`0.47
`0.36
`3.50
`0.37
`
`3.88–5.43
`
`0.007–1.44
`
`0.057–1.71
`0.009–2.00
`2.96–4.12
`0.10–0.94
`
`Comparison of uncoated stopper versus coated stopper polysorbate 80 prefilled syringe without adjustment of exposure denominator for mixed exposure: rate
`difference = 4.35/10,000 patient-years (95% CI 3.44–5.26); P < 0.0001, rate ratio = 17 (95% CI 3.14–707). Comparison of uncoated stopper versus coated stopper
`polysorbate 80 prefilled syringe with mixed cases excluded without adjustment of exposure denominator for mixed exposure: rate difference = 3.50/10,000 patient-years
`(95% CI 2.65–4.36); P < 0.0001, rate ratio = 15 (95% CI 2.6–579).
`aPRCA cases with mixed exposure to both uncoated stopper polysorbate 80 prefilled syringe and either coated stopper polysorbate 80 prefilled syringe (N = 25) or
`human serum albumin formulations (N = 1) were assigned to the uncoated stopper category. Eprex form was unknown for nine cases.
`bWhen adjusting for market exposure, the actual exposure data used were calculated to the end of April 2004. This accommodated an assumed minimum interval of
`two months’ delay from identification of loss of effect to the actual report to the sponsor (June 2004), and helped address the latency from exposure to development of
`disease. Exposure was not adjusted for patients that switched forms or routes within a 12-month period.
`
`leached adjuvants in Eprex, and the low probability of a
`patient having a population of T cells and B cells that can
`recognize erythropoietin. Other as yet unidentified pre-
`disposing patient characteristics could also play a role.
`The cases with other products probably represent
`the very rare background incidence rate of antibody-
`mediated PRCA that occurs due to a combination of pres-
`ence of a sufficient number of erythropoietin-recognizing
`T cells and B cells (loss of erythropoietin tolerance), and
`perhaps the presence of some natural or bystander ad-
`juvant. The clinical experience with antibody-mediated
`PRCA—such as the prevalence in CKD patients and as-
`sociation with subcutaneous administration [2, 6], which
`would exacerbate any immunogenicity [14]—suggests
`that its causality is likely to be multifactorial. Thus, only
`
`in relatively few patients would all the necessary factors
`occur together. Possible reasons for the apparent protec-
`tion of cancer patients may include a shorter duration of
`epoetin treatment and nonspecific immunosuppression
`[2].
`The reported mean time between first exposure to
`Eprex and the initial diagnosis of antibody-mediated
`PRCA is 9.1 months (range 0.3–82) [15]. With some delay
`in reporting, it is anticipated that a few new cases from
`exposure to uncoated stopper forms and leachates may
`still occur. Once a sufficient time period has elapsed since
`the replacement of the uncoated stopper form with the
`coated stopper form and the last exposure to leachates,
`the incidence rate for Eprex is expected to drop to the
`background incidence rate observed for all subcutaneous
`
`Novartis Exhibit 2176.006
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`2352
`
`Boven et al: Identifying the probable cause of Eprex immunogenicity
`
`Table 4. Sensitivity analysis for mixed usage of forms 2001 to 2003: Incidence rate of antibody-mediated pure red cell aplasia in chronic kidney
`disease patients with onset of loss of effect during 2001 to 2003 and received by June 30, 2004 with exposure adjusted for mixed usage of forms
`
`Eprex form by subcutaneous route only
`
`Uncoated stopper, polysorbate 80
`formulation, prefilled syringe
`Coated stopper, polysorbate 80
`formulation, prefilled syringe
`Human serum albumin formulation
`Polysorbate 80 formulation, vials
`Subtotal: all subcutaneous forms except
`uncoated stopper polysorbate 80
`prefilled syringe
`
`Unadjusted
`Case market exposure
`count
`(patient-years)
`
`Exposure adjusted 25% Incidence rate (per 10,000
`for mixed usage of
`patient-years) using adjusted
`forms over timea
`exposure for mixed use
`
`95% confidence
`interval
`(Poisson exact)
`
`142b
`
`1
`
`2
`1
`4
`
`308,232
`
`38,608
`
`42,305
`27,794
`108,707
`
`335,409
`
`28,956
`
`31,729
`20,846
`81,530
`
`4.23
`
`0.34
`
`0.63
`0.48
`0.49
`
`3.57–4.99
`
`0.009–1.92
`
`0.08–2.28
`0.01–2.67
`0.13–1.26
`
`Comparison of uncoated stopper versus coated stopper polysorbate 80 prefilled syringe with 25% adjustment of exposure denominator for mixed exposure: rate
`difference = 3.89 per 10,000 patient-years (95% CI 2.92–4.86); P = 0.0001, rate ratio = 12 (95% CI 2.2–448). Comparison of uncoated stopper versus all other
`subcutaneous forms with 25% adjustment of exposure denominator for mixed exposure: rate difference = 3.74 per 10,000 patient-years (95% CI 2.90–4.59); P < 0.0001,
`rate ratio = 8.6 (95% CI 3.3–32).
`aExposure for the uncoated stopper polysorbate 80 prefilled syringe is adjusted by adding the sum of exposure subtracted from the other forms.
`bPRCA cases with mixed exposure to both uncoated stopper polysorbate 80 prefilled syringe and either coated stopper polysorbate 80 prefilled syringe (N = 25) or
`human serum albumin formulations (N = 1) were assigned to the uncoated stopper category. Eprex form was unknown for nine cases.
`
`forms, excluding uncoated rubber syringe stoppers, of
`approximately 0.5/10,000 patient-years of subcutaneous
`exposure.
`The limitations of this study include the retrospec-
`tive nature of the analysis and reliance on postmarket-
`ing surveillance data, which can be incomplete. The re-
`porting of PRCA with Eprex could have been stimulated
`by the medical literature compared to the reporting of
`PRCA with other products. Additionally, different man-
`ufacturers may use different definitions or methods for
`calculating the incidence rate for their product and may
`not specify the peak period of reporting, subcutaneous
`versus all routes of administration, and the renal indica-
`tion versus all indications. Consequently, it is not valid
`to quantitatively compare rates between manufacturers.
`However, all of the cases analyzed in this manuscript were
`classified by the same definition. Since there is no reason
`to expect a bias in reporting by formulation, we believe
`the comparison across Eprex forms is valid. The incidence
`rates reported here are consistent with a recent report by
`Benn