10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`EP 03 748 438
`M/45063-EP
`
`TEST REPORT
`
`Additional Experimental Results
`for EP 03 748 438
`
`Abbott Biotechnology Ltd
`
`Example A: Potential Of The Formulation To Maintain Adalimumab Stability Over
`Broad Adalimumab Concentration Range
`
`The present formulations of the invention have improved properties as compared
`to other formulations used in the field. Particularly, the formulations of the invention
`were shown to maintain physical and chemical stability of Adalimumab (D2E7) over a
`broad protein concentration range. It is generally accepted by persons skilled in the art
`that with increasing protein concentration stabilization of liquid protein formulation
`presents a challenge, especially over longer periods of time (e. g., more tha n a year).
`Surprisingly, the formulations of the invention guarantee protein stability over at
`least 24 months in a liquid state with a Adalimumab concentration in the range of about
`1mg/ml to 158 mg/ml.
`
`Test Series 1: Adalimumab concentration in the range of 12,5 mg/ml to 158 mg/ml
`
`Table 1 lists formulations containing a broad concentration range of Adalimumab
`that were placed on long term stability for up to 24 months, during which time the
`stabilizing effects of the formulations were monitored
`
`Table l: Adalimumab Formulations Placed On Long-Term Stability Testing At 2-
`8°C
`
`
` Adalimumab
`Concentration
`
`
`
`
`
`
`12.5 1 ml 10mg/0.8ml)
`25
`1 ml 20 ; 0.8m]
`50m ml 40 ,; 0.8ml
`8O
`; ml (160 ; 2ml
`100
`; m1 80
`1 0.8m]
`
`158 mg/ml
`
`
`
`023010vo
`88-003HK _
`080100AW
`980200A0 _
`18228 HK
`
`
`15206HK
`
`
`
`Real Time Stability
`Data Available
`
`(Months)
`
`
`Table. 2 provides a detailed overview over the compositions of the protein
`formulations listed in Table 1.
`
`Table 2: Composition Of Adalimumab Formulations Placed On Long-Term
`Stability Monitoring
`
`AMGEN INC.
`
`Exhibit 1054
`
`Ex. 1054 - Page 1 of 26
`
`Ex. 1054 - Page 1 of 26
`
`AMGEN INC.
`Exhibit 1054
`
`

`

`
`
`[m mL]
`
`
`
`B
`[m_mL] m mL]
`25.00
`50.00
`12-00
`1200
`
`[m mL]
`
`0.71166
`
`1.04
`
`1.31
`
`1.36 1 82
`
`0.24
`0.75
`
`1.53
`
`1.53
`
`EP 03 748 438
`M/45063-EP
`
`Adalimumab
`
`Citricacid
`monoh drate
`
`Sodiumcitrate
`Disodium
`
`phosphate
`dih drate
`
`phosphate
`dih drate
`Sodiumchloride
`P01 sorbate 80
`
`
`
`
`
`
`
`
`
`
`
`
`
`E
`[mmL] m_mL]
`100.00
`12-00
`
`1.31
`
`0-31
`1.53
`
`1.53
`
`
`
`
`
`
`
`5.48-7.31
`
`.
`17
`_
`6.
`3.0
`ON
`H
`-0 “mu-mu
`adlmL
`
`
`
`Formulation A contained 12.50 mg/mL of Adalimumab and various concentration
`ranges of other excipients. Formulations C—F varied only in protein concentration.
`Criteria tested ranged from general quality parameters (e.g., protein content, pH),
`parameters indicating physical stability (e.g., clarity, color, particle contamination
`monitored by visual inspection and light obscuration particle counting, purity including
`fi'agmentation and aggregation monitoring by SEC), and parameters indicating chemical
`Adalimumab stability (e.g., IEC for deamidation, oxidation, general chemical stability;
`SEC).
`
`0 Clarity (analytical measurement)
`
`0 Color (visual inspection)
`
`0
`
`0
`
`0
`
`0
`
`0
`
`pH
`
`Particulate contamination: visible particles (visual inspection)
`
`Particulate contamination: subvisible particles (light obscuration particle count
`test)
`
`Purity: Size Exclusion I-IPLC (SEC)
`
`Purity: Cation Exchange HPLC (IEC)
`
`0 Protein content (for batch 080100AVV)
`
`The clarity and the color of the solution were determined by analysis of the
`undiluted solution. Clarity was measured by turbidimetry and reported as Nephelometric
`Turbidity Units (NTU). A value of 30 NTU translates to reference suspension IV as
`defined per European Pharmacopeia (E.P.). For determination of color the Adalimurnab
`formulation was visually compared with the reference solutions as described in the ER
`
`10
`
`15
`
`20
`
`25
`
`Ex. 1054 - Page 2 of 26
`
`Ex. 1054 - Page 2 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`Particulate contamination (e.g., visible particles) was determined by visual
`inspection as described in German Drug Codex (DAC, section Visual Testing for Visible
`Particles).
`
`Subvisible particles were monitored by the light blockage method according to
`United States Pharmacopeia (USP). In addition, the physicochemical stability of the
`formulations was assessed by SEC which allows detection of fragments and aggregates.
`
`To monitor chemical stability, size exclusion high pressure liquid chromatography
`(SE-HPLC) (detection of fragments and hydrolysis specimen) and CEX—HPLC (cation
`exchange I-IPLC) were applied. CEX—HPLC resolves different
`lysine isoforms and
`degradation products (e.g., deamidated and oxidized species) that may have formed
`during storage.
`-
`
`Surprisingly, all of the formulations listed in Table 2 did not show any significant
`instability phenomena when exposed to long—term (at least 24 months) real-time stability
`testing.
`
`The data are analyzed below.
`
`Clarity (reported as NTU values) did not change significantly during the long-
`term stability study, and even afier 24 months storage at a temperature typically
`recommended for storage of biologic products (i.e., 2-8°C), NTU values remained well
`below the quality specification (Table 3). Thus, the formulation of the invention is able
`to provide stability over a broad Adalimumab concentration range with regard to clarity.
`
`Table 3: Clarity 0f Formulations With Adalimumab Concentrations Ranging From
`12.5 mg/mL To 158 mg/mL After 24 Months Storage At 2—8°C
`
`10
`
`15
`
`20
`
`12. 5mg/ml
`
`50 mm]
`
`
`
`
`
`
`
`
`
`
`
`
`25.
`
`
`
`000 000000000000
`
`
`
`5.60
`
`7.6
`
`
`00 0000_—
`00 .0000 __
`25 rug/ml
`00 .0000 _—
`80mg/m21ml
`23.9
`SEW“ _—-0.8ml
`
`
`the Adalimumab
`contamination revealed that
`for particulate
`Testing
`formulations remained practically free from visible particles. Additionally, subvisible
`particle levels (indicating aggregation, precipitation and general physical
`instability
`
`Ex. 1054 - Page 3 of 26
`
`Ex. 1054 - Page 3 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`phenomena) did not change significantly throughout the 24 months stability testing. The
`drug product batches were virtually equivalent with regards to their physicochemical
`stability at all storage conditions tested. This is surprising, as it is well accepted that
`especially physical stability tends to decrease at higher protein concentrations. Thus, the
`formulation of the invention is able to provide stability over a broad Adalimumab
`concentration range with regard to particulate contamination.
`Tables 4 and 5 provide detailed data of particulate formation (visible and
`subvisible particles) of formulations with various Adalimumab concentrations up to 24
`months:
`
`10
`
`Table 4: Visible Particle Data Of Formulations With Adalimumab Concentrations
`
`Ranging From 12.5 mg/mL To 158 mg/mL After 24 Months Storage At 2-8"C
`
`concentration
`
`
`
`10mg/0m81ml
`25Omg/ml
`'0. 8m]
`50Otng/ml
`
`(4O _.0 8m]
`
`
`
`100 mg/m]
`80m_ 0ml8m]
`Maximum Allowable NMT < 4.5
`
`E
`
`Particulate Contamination (Visual Score)
`
`
`
`
`
`
`
`15
`
`Table 5: Subvisible Particle Data (Determined Via Light Obscuration) Of
`
`Formulations With Adalimumab Concentrations Ranging From 12.5 mg/mL To
`
`158 mg/mL After 24 Months Storage At 2-8°C
`
` Subvisible Particles
`
`W011)
`
`Z lOum
`
`0
`
`11111111101)10811 _—O
`
`81 118111 01181181)
`
`
`
`0
`
`
`
`
`
`
`
`
`
`Ex. 1054 - Page 4 of 26
`
`Ex. 1054 - Page 4 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`
`
`
`W...
`
`
`Maximum Allowable :
`
`2 10pm NMT < 6000 partJunit
`
`2 25pm NMT < 600 part/unit
`
`
`
`
`
`
`
`
`
`23
`
`30
`
`0
`
`
`
`
`
`Table 6 provides the results of SE—HPLC testing afier 24 months of storage of
`Adalimumab formulations, showing the levels of monomer over time. Table 6 clearly
`demonstrates that the formulations are able to guarantee virtually identical monomer
`levels with Adalimumab concentrations ranging from 12.5 mg/mL up to 158 mg/mL,
`even after 24 months storage. All formulations reveal monomer levels clearly above the
`95% monomer quality level (indicative for very good stability, even over 24 months real-
`time testing. The formulations actually maintain Adalimumab long—term stability to an
`extent that even an extremely strict quality specification for real-time stability testing of
`98% would be met. Thus, the formulations of the invention are able to provide physical
`stability over a very broad Adalimumab concentration range and over a long time with
`regard to monomer level as well.
`This is very surprising, since it is well accepted in the scientific community that
`aggregation (leading to a decrease of monomer) strongly correlates with increased protein
`concentration.
`
`Table 6: Adalimumab Monomer Levels Of Various Formulations After 24
`
`Months Storage At 2—8°C Difl'ering Significantly In Adalimumab Concentration
`
`(i.e., Ranging From 12.5 mglmL To 158 mg/mL)
`
`10
`
`15
`
`20
`
`
`
`Puri HPLC %
`
`
`
`
`
`
`
`50 mg/ml (40mg/O.8ml)
`
`Monomer
`
`somg/rnl (160mg/2ml) - Monomer
`
`
`Ex. 1054 - Page 5 of 26
`
`Ex. 1054 - Page 5 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`100 ngml (80mg/O.8ml)
`
`989
`
`Quality Target NLT 95%, with greater than 98 providing excellent stability
`
`CEX-HPLC is a very powerful tool for monitoring the chemical stability of
`antibodies. Table 7 shows that
`the formulation of invention is able to maintain
`Adalimumab chemical stability (expressed as the sum of lysine variants, deamidation,
`and oxidation) for up to 24 months and over a broad Adalimumab concentration range.
`No correlation between protein concentration and decreasing chemical stability was
`observed. The ability to maintain chemical stability over a long period of time and over a
`broad concentration range is an important quality criterium of pharmaceutical
`formulations. Table 7 clearly demonstrates that the formulation is able to guarantee
`virtually identical
`chemical
`stability levels
`in
`formulations with Adalimumab
`concentrations ranging from 12.5 mg/mL up to 158 mg/rnL, even after 24 months
`storage. All formulations revealed a sum of lysine levels clearly above the quality
`specification of 75% sum of lysine determining acceptable Adalimumab stability.
`'
`
`Table 7: Adalimumab Lysine Variant Levels Of Various Formulations After 24
`Months Storage At 2-8°C Differing Significantly In Adalimumab Concentration
`(i.e., Ranging From 12.5 mg/mL To 158 mg/mL)
`
`5
`
`10
`
`15
`
`
`
`
`Sum of Lysine Variants
`
`
`
`
`
`"I;-
`
`"E—
`WWW-w
`
`
`
`
`
`Quality target: Not less than 70%, with greater than 75% providing exceptional
`
`
`
`
`
`20
`
`stability
`
`These quprising stability study results continued the findings that the invention
`comprising a protein formulation with protein concentrations ranging fiom 12.5 mg/ml
`
`
`
`Ex. 1054 - Page 6 of 26
`
`Ex. 1054 - Page 6 of 26
`
`

`

`EP 03 748 438
`M/45063—EP
`up to 158 mg/ml maintains Adalimumab stability in liquid solutions for at least 24
`months.
`
`Test Series 2: Adalimumab at protein concentrations from 2 to 150 mg/ml
`
`In order to further verify the surprising stabilization potential of the formulation
`of the invention over a broad protein concentration range, Adalimumab was formulated at
`protein concentrations from 2 up to 150 mg/ml and subjected to repeated freeze thaw
`processing (freezing performed at —80°C, thawing performed at 25°C). It is well known
`that freeze—thaw processing can result in substantial protein denaturation and aggregation,
`resulting in soluble and insoluble aggregate formation. Subvisible particle testing is
`known to be a very sensitive tool for detecting physical instability at very early stages of
`a potential instability pathway.
`.
`
`Adalimumab was formulated in a citrate—phosphate buffer system within a 0.2—
`150 mg/mL Adalimumab concentration range at pH 7 and subjected to repeated
`freeze/thaw processing (-80°C/25°C). Whereas at low protein concentration (0.2 mg/mL)
`the citrate—phosphate buffer system did not maintain stability over repeated freeze/thaw
`cycles, within a 2—150 mg/mL concentration range the citrate—phosphate buffer system
`maintained stability very well, and subvisible particle levels remained well below the
`6,000 particles sized 10 micrometer and above per container set for small volume
`parenteral products by regulatory pharmaceutical compendia (Figure 1). Similarly, the
`citrate-phosphate buffer system maintained Adalimumab stability Within a 2-150 mg/mL
`concentration range to an extent that even after 4 freeze/thaw cycles monomer levels
`exceeded 90% in all formulations tested, and were even well above 95% within a 15-150
`mgmL concentration range (Figure 2).
`
`Similar data have been generated for pH 4 conditions (Figure 3), and the citrate—
`phosphate buffer system was shown to provide Adalimumab stability over a broad
`concentration range (02-150 mg/mL) at pH 4, as demonstrated by virtually identical
`aggregate levels as monitored by SEC. Subvisible particle data was within USP and EU
`specification requirements for low volume parenterals (data not shown; available upon
`request) .
`
`10
`
`15
`
`20
`
`25
`
`Ex. 1054 - Page 7 of 26
`
`Ex. 1054 - Page 7 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`140
`
`Subvisble particle measurement Freezd'lhaw Experinent
`
`Particles>=10um
`
`aCI0O
`
`5D
`
`120
`
`100
`
`NO
`
`0.2
`
`2
`
`50
`15
`Protein Concentration [mglml]
`
`100
`
`150
`
`BTO IT1 IT2 01113 .T4
`
`Figure 1: Subvisible Particle Levels ( 2 10 um/mL) In Adalimumab Solution During
`Up To Four Freeze/Thaw Cycles In Citrate-Phosphate Buffer System At pH 7
`(Quality Target: Not More Than 6000 Particles 210 NIicrometer per mL)
`
`SEC HPLC: Freeze/Thaw wlth protein solufion-monomets
`
`110
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`40
`
`30
`
`20
`
`10
`
`
`
`monomer(”/o)
`
`
`
`
`protein concentration [mg/ml]
`
`ElTO IT1 ETZ INTI! IT4
`
`Figure 2: Monomer Levels (SEC Analysis) In Adalimumab Solution During Up To
`Four Freeze/Thaw Cycles In Citrate—Phosphate Buffer System At pH 7
`-
`
`Ex. 1054 - Page 8 of 26
`
`Ex. 1054 - Page 8 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`Mneahrhilfl)
`
`10
`
`Figure 3: SEC Analysis Showing Aggregate Levels Of Adalimumab Formulations
`During Up To Four Freeze/Thaw Cycles In Citrate-Phosphate Buffer System At pH
`
`4 T
`
`est Series 3: Adalimumab at concentrations ranging from 0.2 — 100 mg/mL
`
`15
`
`20
`
`25
`
`Based on these surprising results, Adalimumab solutions were prepared with
`various concentrations (ranging fi'om 0.2 — 100 mg/mL at pH 4, and ranging fi'om 2-100
`mg/mL at pH 6) in a citrate-phosphate buffer system. The final formulations were then
`subjected to long—term stability studies (at least 12 months) at 2—8°C. SEC analysis was
`conducted to monitor physical and chemical stability (fragmentation, aggregation), and
`CEX-HPLC was used to monitor chemical stability (reported as sum of lysine variants).
`Quality (stability) targets for these experiments were set to 95% monomer (physical
`stability monitored by SEC) afier 12 months storage at 2-8°C, and about 70% or higher
`for % sum of lysine variants (chemical stability monitored by IEC data).
`
`Data shown by Table 8 clearly indicate that a citrate-phosphate bufi‘er system
`provides stability for Adalimumab for at least 12 months over a broad Adalimumab
`concentration range and over a broad pH range. In all samples tested, monomer levels
`surprisingly exceeded 95%, and lysine variant data in all samples tested exceeded 70%
`(except for 64.8% ofthe pH 4, 100 mymL, 12 month stability data).
`
`30
`
`Table 8: SEC And IEC Data 01' Adalimumab Formulated In Citratel’hosphate
`Buffer System Over A Broader pH Range (pH 4 - pH 6) And Over a Broader
`Concentration Range (12 Months, 2-8°C)
`
`Ex. 1054 - Page 9 of 26
`
`Ex. 1054 - Page 9 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`
`
`SEC
`
`
`
`Aggregate [%]l
`
` Monomer [%]l
`
`Fragment [%l
`
`
`-_——
`
`
`
`
`
`_--——_-—
`
`_——
`u--_
`
`
`
`_-_-
`____
`
`SEC Quality Target: Not less than 95% monomer
`
`
`
`
`
`IEC Quality Target: Not less than about 65% sum of lysine variants)
`
`The data presented in Table 8 are very surprising, since aggregation is
`an acknowledged problem with protein pharmaceuticals (refer to Manning et a1. (1989)
`Pharm Res. 62903-918), and protein aggregation is expected to correlate and to accelerate
`with increased protein concentration (refer to Lumry and Eyering (1954) J. Phys. Chem.
`58:110-120).
`
`the stability study results
`to conclude that
`These surprising data led us
`demonstrate
`that
`the
`invention
`comprising
`a
`formulation with Adalimumab
`concentrations ranging within several orders of magnitude and within a broader pH range
`provide excellent Adalimumab stability in liquid solutions over at least 12 months.
`
`Summary
`
`In summary, these data demonstrate that a citrate-phosphate buffer system is able
`to maintain Adalimumab stability over broad pH and broad concentration range. These
`findings were very surprising, since it is well known for persons skilled in the art that an
`increase in protein concentration almost
`inevitably is associated with increased
`instability, and accelerated aggregation of proteins at high concentrations has been
`reported repeatedly, e.g., for IL—1beta in the range of 0.1-0.5 mg/mL (refer to Gu et a1.
`
`10
`
`Ex. 1054 - Page 10 of 26
`
`
`
`
`
`
`5
`
`10
`
`15
`
`Ex. 1054 - Page 10 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`,
`(1991) Pharm. Res. 8:485-490), for low molecular weight urokinase in the range of 0.2—
`0.9 mg/mL (refer
`to Vrkljan et al.
`(1994) Pharm Res. 11:1004-1008), or
`for
`apomyoglobin in the range of 4-12 mg/mL in the presence of urea (De Young et a1.
`(1993) Biochemistry 32:3877-3886).
`
`It is further surprising that the citrate-phosphate buffer system provided stability
`for Adalimumab over a broad pH range spanning several pH units, since it is generally
`accepted that proteins usually are stable only in a narrow pH range, such as pH 6.5—7.0
`for recombinant factor VIII (Fatouros et al., 1997), pH 6-7 for low molecular weight
`urokinase (Vrkljan et al. (1994) Pharrn Res. 11:1004—1008), or pH 4.5-5.0 for relaxin
`(Nguyen and Shire (1996) Stability and characterization of recombinant human relaxin,
`In: Pearlman, R., Wang, Y.J. (eds), Formulation, characterization, and stability of protein
`drugs, Plenum Press, New Zork, pp. 247-271).
`
`10
`
`11
`
`Ex. 1054 - Page 11 of 26
`
`Ex. 1054 - Page 11 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`Example B: Variation of Buffer System and Antibody
`
`Test Series 1: Different Buffers
`
`Adalimumab formulated in the formulations of invention did not show significant
`changes in conformational stability as assessed by CD spectroscopy in a pH range of 4 to
`7 when formulated in a citrate—phosphate bufi‘er system (refer to Figure 4).
`
`Figure 4: Overlay Of CD Spectra For pH Range 1 To 7 Clearly Shows That
`Adalimumab Stability As Determined By CD Data Is Virtually Identical At pH 4
`And Above When Formulated In The Formulation Of Invention (Formulation
`Composition Of Buffer And Excipients As Listed In Table 2)
`300
`
`200
`
`.29;
`
`_ gm
`
`wave length mm
`
`
`
`Based upon the above CD data, bufi‘er systems that allowed for the control of pH
`between pH 3 and 8 were selected and screened for their potential to maintain the
`stability of Adalimumab in solution. Systems of citrate/phosphate/NaCl buffer (pH 3,4,5),
`acetate/phosphate/NaCl bufi‘er (pH 4,5) and phosphate bufl‘er/NaCl buffer (pH 6,7,8)
`were evaluated. These formulations were subjected to short term stability testing under
`accelerated conditions. Purity (using stability indicating CEX—HPLC and SE—HPLC),
`particulate matter
`(subvisible
`and visible particles),
`biological
`activity (TNF
`neutralization testing) and clarity of the solutions were selected as parameters to monitor
`Adalimumab stability. Table 10 shows the clarity data. Especially for physical stability of
`protein solutions, clarity is a highly differentiating parameter between the formulations
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`12
`
`
`
`Ex. 1054 - Page 12 of 26
`
`Ex. 1054 - Page 12 of 26
`
`

`

`EP 03 748 438
`Ml45063—EP
`tested and therefore formulations with the best clarity were selected (see shaded area in
`Table 10). The SE-HPLC and CEX-HPLC data were included for reference of chemical
`stability at the respective pH.
`Based upon the results of these extensive stress studies, citrate/phosphate/NaCl
`buffer at pH 5 and phosphate/NaCI buffer at pH 6 and at pH 7 were conSidered to be the
`most suitable buffer systems for maintaining Adalimumab stability, since in these
`systems clarity levels were lowest (indicating highest physical stability levels) and %
`lysine variants were highest (indicating highest chemical stability levels).
`
`10
`
`13
`
`Ex. 1054 - Page 13 of 26
`
`Ex. 1054 - Page 13 of 26
`
`

`

`
`
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`
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`
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`
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`
`Ex. 1054 - Page 14 of 26
`
`Ex. 1054 - Page 14 of 26
`
`
`
`
`
`
`

`

`EP 03 748 438
`M/45063—EP
`
`In order to verify the surprising results of a citrate—phosphate buffer system providing
`Adalimumab stability over a broad pH range, accelerated and long—term stability experiments
`were performed. Adalimumab was formulated in citrate-phosphate buffer at 2 mg/mL, and
`physical stability during storage was tested using SEC. Since it is well known fiom the
`literature that even small changes in formulation pH can have a tremendous impact on protein
`stability within a short period of time (see literature referenced in Example A), it was
`surprising to see that a citrate-phosphate buffer system maintained the stability of
`Adalimumab over 12 months at 2—8°C storage within a pH 4-7 range (see Figure 5a). Even at
`very high temperatures (e.g., 50°C), the citrate—phosphate formulated samples revealed more
`than 95% monomer within a pH 5—7 range afier 3 weeks, and more than 95% monomer within
`a pH 4-7 range after 3 weeks.
`At 2-8°C, the citrate-phosphate bufi‘er system provided stability within a pH 4-7 range
`to such a degree that all samples still revealed more than 98% monomer afier 12 months
`storage.
`
`100
`
`99
`
`
`
`98
`
`9, -
`
`92
`
`96
`
`94
`
`93
`
`
`
`monomer[%1
`
`10
`
`15
`
`20
`
`25
`
`3
`
`3.5
`
`4
`
`4.5
`
`5,5
`5
`pH values
`
`6
`
`6.5
`
`7
`
`7.5
`
`Figure 5a: SEC data, amount of monomer [%], Adalimumab (2mg/mL), formulated in
`citrate-phosphate buffer, 2-8°C over 21 days, Smonths, 9 months and 12 months storage.
`Addtionally, data of 40°C, 50°C over 21 days storage, pH 4, 5, 6 and 7 are provided.
`
`Surprisingly, when comparing the monomer loss of the individual formulations over
`12 months at 2-8°C, a citrate-phosphate buffer resulted in a monomer loss of not more than
`0.5% for all samples during 12 months storage when compare to T0 data, i.e., pH 4: 0.5%
`monomer decrease, pH 5: 0.3% monomer decrease, pH 6: 0.4% monomer decrease, and pH 7:
`0.2% monomer decrease.
`
`Given the background that it is well known to persons skilled in the art that even very
`small changes in buffer pH can have huge impact on protein stability (refer to literature
`references listed in Example A), it is very surprising that a citrate-phosphate buffer system
`
`15
`
`Ex. 1054 - Page 15 of 26
`
`Ex. 1054 - Page 15 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`maintains Adalimumab stability for at least 12 months within a pH range spanning at least 3
`pH units (pH 4-7).
`
`Given the exceptional stability a citrate-phosphate bufi‘er system was found to provide
`for Adalimumab, the stabilizing potential of this buffer system was challenged by extending
`the solution pH to a pH 3-8 range (Figure 5b). Since it is well accepted in the scientific
`community and regulatory agencies that accelerated short-term stress testing at elevated
`temperatures (e. g., 40°C storage temperature are typically applied within industry)
`is
`predictive for long—term storage events at the recommended storage temperature (ive., 2-8°C),
`temperatures of up to 50°C were applied in a 21 day accelerated stress test of Adalimumab
`formulated in citrate-phosphate bufi‘er system. Over the complete pH range evaluated at high
`temperatures (40°C) after 21 days more than 95% were monomer. Even when exposing the
`Adalimumab formulations to extreme temperature stress at 50°C, more than 95% monomer
`levels were obtained afier 21 days stress testing within a pH range covering pH 4 to pH 8.
`This is very surprising, since it is well known that high temperatures or changes in pH impair
`protein stability. However, the formulation of invention maintained Adalimumab stability
`even when those two stress parameters were applied simultaneously over time (i.e., extreme
`pH changes, extreme stress temperature).
`
`Manor
`
`[St]
`
`Figure 5b: SEC data, amount of monomer [%], Adalimumab (2mg/mL), formulated in
`citrate-phosphate buffer, 2-8°C over 21 days. Additionally, data of 40°C, 50°C over 21
`days storage, pH 4, 5, 6 and 7 are provided.
`
`16
`
`10
`
`15
`
`20
`
`25
`
`Ex. 1054 - Page 16 of 26
`
`Ex. 1054 - Page 16 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`Test Series 2: Different Monoclonal Antibodies
`
`In order to verify the surprising stabilization potential of the formulation of invention,
`these exceptional stability findings (i.e., stabilizing over broad pH range and ideally over
`longer time) were verified for proteins other than Adalimumab. Figure 5c shows data of an
`experiment where an anti-1L 13 antibody (IgGl, 150 kDa) was formulated at 2 mgmL over a
`broad pH range (pH 3—8)
`in a citrate-phosphate bufi‘er system and subjected to both
`accelerated and long-term storage testing. Surprisingly, not only the buffer system maintained
`stability of the anti—IL 13 antibody at higher storage temperatures over a broad pH 4-8 range
`(more than 90% monomer was observed after 21 days of stress testing), but even afier 1 year
`storage at 25°C within a pH 4 — 8 range the citrate—phosphate buffer system maintained
`stability (all samples revealed at least 90% monomer). When stored at temperatures typically
`recommended for biologics (i.e., 2-8°C), all samples revealed about 95% monomer and above,
`even after at least 18 months of storage. Additional analysis by anionic exchange data verified
`these surprising results.
`
`10
`
`15
`
`100.00
`
`_ 95.00
`é.
`
`
`
`\o
`
`90.00
`
`85.00
`
`80.00
`
`
`
`
`
`
`
`+T0
`——s>——3wks/40°C
`—0—12ml25°C
`+18m5,0
`
`L0 g
`
`g
`E
`
`20
`
`25
`
`30
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`solution pH
`
`Figure 5c: SEC data, amount of monomer [%], anti-IL 13 mAb (2mg/mL), formulated
`in citrate-phosphate buffer, stored at various temperatures up to 18 months. Stability
`monitored by SEC and expressed as % monomer remaining
`
`The stabilizing features of the citrate-phosphate buffer system were verified with yet
`another protein. Figure 5d shows data of an experiment where an anti-1L 12 antibody (IgGl,
`150 kDa) was formulated at 2 mg/mL over a broad pH range (pH 3-8) in a citrate-phosphate
`bufl‘er system and subjected to both accelerated and long-term storage testing. Again, not only
`the bufi‘er system maintained stability ofthe anti-IL 12 antibody at higher storage temperature
`over a broad pH 4-8 range (more than 90% monomer was observed afier 21 days of stress
`testing at 40°C), but even after 1 year storage at 25°C within a pH 5 — 8 range the citrate-
`phosphate buffer system maintained stability (all samples revealed at least 90% monomer).
`17
`
`Ex. 1054 - Page 17 of 26
`
`Ex. 1054 - Page 17 of 26
`
`

`

`EP 03 748 438
`W45063—EP
`
`When stored at temperatures typically recommended for biologics (i.e., 2-8°C), all samples
`within a pH 4-8 range were more than 95% monomer. Additional analysis by anionic
`exchange data verified these surprising results.
`
`5
`
`E:
`'5
`E
`2
`g
`g
`g
`
`95.00
`
`90.00
`
`85.00
`
`80.00
`
`75.00
`
`
`
`
`
`
`
`
`
`—I—-—TO
`+12m, 25'0
`+12m.5"c
`x 3wks,40‘c
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`solution pH
`
`10
`
`Figure 5d: SEC data, amount of monomer [%], anti-IL 12 mAb (2mg/mL), formulated
`in citrate-phosphate buffer, stored at various temperatures up to 18 months. Stability
`monitored by SEC and expressed as % monomer remaining.
`
`18
`
`Ex. 1054 - Page 18 of 26
`
`Ex. 1054 - Page 18 of 26
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`

`

`EP 03 748 438
`M/45063-EP
`
`Example C: Excinient Potential To Maintain Adalimumab Stability Over A Long Period
`Of Time
`
`The present formulations of the invention have improved properties as compared to
`other formulations used in the field. Particularly, the formulations of the invention were
`shown to maintain physical and chemical stability of Adalimumab over a longer period of
`time, especially in liquid state. It is generally accepted by persons skilled in the art that
`successful development of stable liquid protein formulations provide a challenge, and no
`guarantee can be provided that excipients can be identified that maintain and/ or improve
`protein stability, especially over longer periods of time over a broader protein concentration
`range.
`
`Test Series 1: Mannitol
`
`a
`formulation, mannitol was used in
`isotonic
`an
`produce
`to
`In order
`citrate/phosphate/NaCl/Tween80 buffer system. Based on results of stress stability tests,
`citrate/phosphate/NaCl/mannitol was chosen as the formulation matrix due to its surprisingly
`superior stability profile.
`Mannitol added to the antibody solution (5, 10, 25 and 50 mg/ml) improves the
`solution clarity (see Table 11) and substantially contributed to protein stabilization.
`
`Table 11: Impact Of Mannitol Concentration In Adalimumab Formulations On Clarity
`Upon Stress Experiments
`
`Protein solution
`without
`mannitol
`
`Mannitol
`
`(Note That The Clarity Was Below The Quality Target Value Of 30 NTU For A Broad
`Mannitol Concentration Range.)
`
`Mannitol substantially contributed to protein stabilization, as both protein aggregation
`and fragmentation were impeded by mannitol. Eventually, a mannitol concentration of 12
`mg/mL was chosen for final Adalimumab Drug Product to best accommodate stability
`requirements and to comply with tonicity and local tolerability requirements, respectively.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`19
`
`Ex. 1054 - Page 19 of 26
`
`Ex. 1054 - Page 19 of 26
`
`

`

`EP 03 748 438
`M145063—EP
`
`Test Series 2: Polysorbate 80
`
`Polysorbate 80 was evaluated for its potential to stabilize Adalimumab in a variety of
`stress experiments.
`
`The formulations listed in Table 12 were subjected to long—term stability testing to
`evaluate the impact of polysorbate 80 on long-term stability (2—8°C).
`v
`
`Table 12: Adalimumab Formulations Put On Long-Term Stability At 2-8°C
`
`
`
`
`__n_
`— With Pol sorbate 80
`Without Pol sorbate so
`
`
`—‘__
`mam—“n
`—-I_——
`_——E_
`—_—E_
`
`
`
`
`
`
`10
`
`15
`
`Stability data provided in Table 13 reveal the stabilizing effect of Polysorbate 80 in
`0.1% concentration. Polysorbate 80 had a stabilizing effect during long-term storage, since
`both visual score data and subvisible particle levels of both 10 and 25 micrometer sized
`particles are lower in the Adalimumab formulation containing 0.1% polysorbate 80.
`This is very surprising, as polysorbate 80 has repeatedly been reported to increase
`protein stability upon air-liquid interface or ice-water interface stress, but not during quiescent
`storage (where polysorbate 80 is known to be very prone to autoxidation, eventually causing
`protein oxidation resulting in aggregation) :
`
`20
`
`Table 13: Impact Of Polysorbate 80 On Long-Term Stability Of Adalimumab (50
`mg/mL Protein, pH 5.2, 24 Month Testing At 2—8°C)
`
`
`
`
`
`
`Particles
`
`2 lOum
`
` Subvisible
`
`Visible Particles
`
` (Visual Score)
`
`
`[German Drug
`Codex, DAC] 2 25pm
`
`
`
`
`
`
`50 mg/ml (40mg/0.8ml)
`0.1% Tween 80 added
`
`50 mg/ml (40mg/0.8ml)
`Without Tween 80
`
`
`
`17
`0
`
`105
`11
`
`5.2
`
`Subvisible Particles: Quality Target: Not more than 6000 Particles 210 Micrometer per
`mL, and not more than 600 Particles 2 25 Micrometer per mL
`Visible Particles: Quality Target: NMT 4.5
`
`20
`
`Ex. 1054 - Page 20 of 26
`
`Ex. 1054 - Page 20 of 26
`
`

`

`EP 03 748 438
`M/45063-EP
`
`Additionally, Adalimumab was formulated in the citrate/phosphate/NaCl/ mannitol
`buffer system at 25 mg/mL as listed in detail in Table 14 and was tested for physico-chemical
`stability by using extended magnetic bar stirring and shaking. Table 15 shows that these
`conditions of shear stress can lead to an increase in subvisible and vi51ble particles. The
`inclusion of 0.1% polysorbate 80 eifectively stabilized the formulation,
`inhibiting the
`formation of particulate matter under stirred conditions and substantially reducing particulate
`matter formation during shaking.
`
`Table 14: Composition Of The Solution Subjected To Stir Stress And To Shaking Stress,
`Respectively
`
`Ingredient
`
`
`
`Formulation
`A
`
`m; mL
`
`m_—
`
`—_
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`10
`
`15
`
`20
`
`Table 15: Stabilizing Effect Of Polysorbate 80 Under Stir Stress And Shaking Stress
`Conditions
`
`Formulation A
`
`Condition
`
`
`
`
`
`
`Particles
`[German Drug
`[Counts/mL]
`Codex]
`—_ 2 10 m
`M—9.7
`without polysorbate so
`17 h shaken
`49724
`26191
`with 0.1 % polysorbate 80
`17 h shaken
`151 17
`30
`without polysorbate 80 —— 35073
`with 0.1 % polysorbate 80
`17 h stirred
`
`
`
`
`
`
`
`
`26923
`
`
`The protein concentration in the fonnulation listed in Table 16 was increased from 25
`mg/ml to 50 mg/ml in order to reach an administration dose of 40 mg in 0.8 mL of injection
`volume. Adalimumab was formulated in solutions as listed in Table 16 and the solutions were
`repeatedly subjected to stress via freezing (-80 °C, 12 hours) and thawing (5 °C, 12 hours) as
`well as to accelerated stability testing in liquid state at 40°C, and the effect of various
`polysorbate 80 co