STABLE COMPOSITIONS OF FC MULTIMERS

Abstract

The disclosure relates inter alia to an Fc multimer composition, an Fc multimer composition comprising the Fc multimer composition of the invention in lyophilized form, as well as an Fc multimer composition according to the invention for use in the treatment of an autoimmune disease or an inflammatory disease.

Claims

1. An Fc multimer composition comprising a) an Fc multimer at a concentration between 60 mg/ml and 180 mg/ml, b) a pH value between 4.8 and 6.0, and c) a stabilizer at a concentration between 200 mM and 450 mM.

2. The Fc multimer composition of claim 1, comprising an Fc multimer at a concentration between 70 mg/ml and 110 mg/ml.

3. The Fc multimer composition of claim 1, comprising an Fc multimer at a concentration between 110 mg/ml and 180 mg/ml.

4. The Fc multimer composition of claim 1, wherein the composition comprises a buffer.

5. The Fc multimer composition of claim 4, wherein the pH value is between 5.0 and 5.5.

6. The Fc multimer composition of claim 4, wherein the buffer is an acetate buffer, a histidine buffer, or a citrate buffer.

7. The Fc multimer composition of claim 1, wherein the stabilizer is a polyol.

8. The Fc multimer composition of claim 1, wherein the molar ratio of Fc multimer to stabilizer is between 1:300 and 1:650.

9. The Fc multimer composition of claim 1, further comprising an antioxidant.

10. The Fc multimer composition of claim 1, further comprising a surfactant.

11. The Fc multimer composition of claim 1 in lyophilized form.

12. The Fc multimer composition of claim 1, wherein the Fc multimer is composed of 3 to 10 Fc portions.

13. The Fc multimer composition of claim 12, wherein the Fc multimer is composed of 3 Fc portions.

14. The Fc multimer composition of claim 13, wherein each Fc portion comprises 2 Fc polypeptides, which each comprise a hinge region, a CH2 constant domain, and a CH3 constant domain.

15. The Fc multimer composition of claim 14, wherein the Fc multimer comprises two polypeptides having the amino acid sequence of SEQ ID NO: 23 and two polypeptides having the amino acid sequence of SEQ ID NO: 24.

16. (canceled)

17. A method of treating an autoimmune disease or an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the Fc multimer composition of claim 1.

18. The Fc multimer composition of claim 7, wherein the polyol is a disaccharide.

19. The Fc multimer composition of claim 18, wherein the disaccharide is sucrose, trehalose, or sorbitol.

20. The Fc multimer composition of claim 9, wherein the antioxidant is methionine.

21. The Fc multimer composition of claim 10, wherein the surfactant is polysorbate 80.

Description

DESCRIPTION OF THE FIGURES

[0181] FIG. 1: Soluble (physical) aggregation (% HMW) over time at 35° C. for formulations 1-12

[0182] FIG. 2: Plot for pooled data for rates of aggregation at 25 and 35° C. as a function of measured pH

[0183] FIG. 3: Fragmentation (% LMW species) over time (NR-Caliper) at 35° C. for formulations 1-12

[0184] FIG. 4: Plot for pooled data for rates of fragmentation (±SE) at 35° C. as a function of measured pH

[0185] FIG. 5: Formation of acidic species over time (CEX) at 35° C. for all formulations

[0186] FIG. 6: Plot for pooled data for rates of formation of acidic species at 25 and 35° C. as a function of measured pH

[0187] FIG. 7: Subvisible particle analysis (a) Particle counts (particles/mL) for particles ≥10 microns (black bars) and for particles ≥25 microns (grey bars) after 3 months at 5° C. and 25° C. in F1, F2, F3, F4, F5, F9, F12, F17. (b) Overview of particle counts (particles/mL) for particles in all size ranges: 2-5 microns (black bars), 5-10 microns (grey bars), 10-25 microns (stripes across) and 10-25 microns (stripes diagonal) after 3 months at 5° C. and 25° C. in F1, F2, F3, F4, F5, F9, F12, F17.

[0188] FIG. 8: Initial (T0) results for % acidic and basic species as measured by cation exchange chromatography.

[0189] FIG. 9: Representative chromatogram from RP-HPLC to quantify oxidation of the Fc multimer molecule (CSL730) in a formulation

[0190] FIG. 10: Plot comparing initial (T0) % short chain oxidation species, long chain oxidation species and relative % total oxidation as measured by RP-HPLC for all formulations of the Fc multimer molecule (CSL730)

[0191] FIG. 11: Plot showing progress of % total oxidation, as measured by RP-HPLC, for all formulations of the Fc multimer molecule (CSL730) at 25° C. and 40° C. for up to 2 months of storage stability

[0192] FIG. 12: Illustration of an Fc construct containing three Fc domains formed from four polypeptides like CSL730. The first polypeptide (502) contains one Fc polypeptide containing different charged amino acids at the CH3-CH3 interface than the wt sequence (506) joined in a tandem series with a protuberance-containing Fc polypeptide (504). The second polypeptide (508) contains an Fc polypeptide containing different charged amino acids at the CH3-CH3 interface than the wt sequence (512) joined in a tandem series with another protuberance-containing Fc polypeptide (510). The third and fourth polypeptides (514 and 516, respectively) each contain a cavity-containing Fc polypeptide.

EXAMPLES

[0193] Part 1—pH-Buffer-Salt Screening Study

[0194] To study the effect of pH, buffer type and ionic strength on formulations comprising an Fc multimer molecule (CSL730) at high concentrations, CSL730 formulations with a protein concentration of 100 mg/mL were prepared and investigated for protein stability.

[0195] CSL730 is a trivalent Fc multimer, consisting of two long chains of SEQ ID NO: 24, and two short chains of SEQ ID NO: 23, produced essentially as described in WO 2015/168643 A2. This results in a trivalent Fc multimer as shown in FIG. 12.

Materials and Methods

[0196] Materials: The materials used for the examples, their catalog numbers and the suppliers are listed in Table 1.

TABLE-US-00001 TABLE 1 Materials used for the examples Catalog Material Number Supplier 15 ml 10K molecular weight cut 87731 Life off (MWCO) cassette Technologies Australia Pty Ltd 2 ml liquid vials with 13 mm neck BO2128 Schott L-Histidine 1.043521.1000 Merck Sucrose 1.00892.5000 Merck Sodium chloride 1.06400.5000 Merck Citric acid monohydrate 1.00244.500 Merck Acetic acid (glacial) 1.00056.2500 Merck Amicon Ultra-15 Centrifugal Filter UFC903024 Merck Devices (30 kDa) Millex-GP Syringe Filter Unit, SLGP033RS Merck 0.22 μm Hydrochloric acid, 6M 1.10164.1000 Merck Sodium hydroxide solution, 4N 1048 ACR Chemical Reagents

[0197] Preparation of formulations: The formulations were prepared with CSL730 (recombinant Fc multimer bulk purified protein (concentration: 146 mg/ml)) as starting material. The bulk purified protein was dialysed against each of the 12 buffers listed below. The pH of each buffer was measured after preparation. 30% overage (mg protein) was applied to account for losses. The formulations were dialysed against about 1 L of buffer for about 5 hours which was subsequently replaced with fresh buffer (about 1 L) and dialysed overnight. Dialysis occurred at 2-8° C. in 15 ml 10K MWCO cassettes. The following buffers were prepared for the dialysis:

[0198] 1. 20 mM glacial acetic acid, 50 mM Sucrose, pH 4.5

[0199] 2. 20 mM glacial acetic acid, 50 mM Sucrose, pH 5

[0200] 3. 20 mM glacial acetic acid, 50 mM Sucrose, pH 5.5

[0201] 4. 20 mM Histidine, 50 mM Sucrose at pH 5

[0202] 5. 20 mM Histidine, 50 mM Sucrose at pH 5.5

[0203] 6. 20 mM Histidine, 50 mM Sucrose at pH 6

[0204] 7. 20 mM histidine, 50 mM Sucrose at pH 6.5

[0205] 8. 20 mM Histidine, 50 mM Sucrose at pH 7

[0206] 9. 20 mM Histidine, 50 mM Sucrose, 100 mM NaCl at pH 6

[0207] 10. 20 mM citric acid monohydrate, 50 mM Sucrose, pH 5.5

[0208] 11. 20 mM citric acid monohydrate, 50 mM Sucrose, pH 6

[0209] 12. 50 mM Sucrose at pH 5.3 (buffer free)

[0210] After dialysis, the protein concentration of each sample was measured. When samples were found to be too dilute they were up-concentrated by centrifugation using Amicon tubes (30 kDa cut-off) with the aim of targeting 100±10 mg/mL. Protein concentration was confirmed afterwards.

[0211] Fill and finish was performed under a laminar flow hood. Each formulation was filtered through a 0.22 μm filter and filled into pre-labelled 2 ml glass vials with 1 ml of the formulation. Vials were then stoppered, and crimped.

[0212] A summary of the formulations prepared for this study are presented in Table 2. The target buffer concentration for all formulations was 20 mM. Acetate, histidine and citrate buffers were used at different respective pHs to achieve good buffer capacity within a pH range of 4.5-7. No surfactant was added to the formulations in this study. 50 mM Sucrose was added as a stabilizer to all formulations to provide a minimum level of stability. Note that sucrose does not impact ionic strength. One buffer-free formulation was prepared since proteins at high concentrations are known to be self-buffering. The pH of the buffer-free solution will depend on the protein itself.

TABLE-US-00002 TABLE 2 Summary of formulations prepared for the present study Buffer Sucrose Formulation Formulation Conc. Target NaCl Conc. No. Buffer pKa (mM) pH (mM) (mM)  1 acetate 4.76 20 4.5 — 50  2 20 5.0 — 50  3 20 5.5 — 50  4 histidine 6.04 20 5.0 — 50  5 20 5.5 — 50  6 20 6.0 — 50  7 20 6.5 — 50  8 20 7.0 — 50  9 20 6.0 100 50 10 citrate 3.13 (1), 20 5.5 — 50 11 4.76 (2), 20 6.0 — 50 6.40 (3) 12 buffer-free N/A — molecule — 50 dependent

[0213] After filling, the filled formulations were placed in stability chambers at 2-8° C., 25° C. or 35° C. Samples were analyzed immediately after fill/finish (for “t0”/t=0 analysis) and at predetermined subsequent time points for up to 4 weeks. In order to compare kinetics of degradation between the different formulations, rate constants of degradation by different pathways (with standard error) were measured using multiple linear regression spanning a minimum of 3 time points (t=0, t=2 weeks and t=4 weeks). Multiple linear regression was done using a general linear model (GLM) to plot and fit the assay data to the model (equation 1).

% P=P.sub.o+k.Math.t  [Equation 1]

Methods Used for Analyzing the Stability of the Formulations:

[0214] Stability indicating methods were used to analyze the formulations at the different time points, namely: Size exclusion chromatography (SEC), Cation exchange chromatography (CEX) and Capillary Gel Electrophoresis (CGE) (Caliper). In addition, pH and visual appearance (for color, turbidity and visible particles) of the formulations were monitored at the different time points.

[0215] Methods used for the study, purpose of each method and analyses used at each analysis time point are summarized in Table 3.

TABLE-US-00003 TABLE 3 Analytical methods used for stability studies of formulations Time point (weeks) and storage temperatures (° C.) t0 (initial 2 4 Method Purpose time point) weeks weeks Visual Visible particles, Initial 25° C., 2-8° C., inspection colour, opalescence, 35° C. 25° C., turbidity 35° C. pH measurement pH testing Initial 25° C., 2-8° C., 35° C. 25° C., 35° C. UV spectroscopy Protein content Initial 25° C., 2-8° C., (A280) 35° C. 25° C., 35° C. Size exclusion High molecular Initial 25° C., 2-8° C., chromatography weight species 35° C. 25° C., (SEC) (soluble aggregates) 35° C. Cation exchange Acidic, basic and Initial 25° C., 2-8° C., chromatography main species 35° C. 25° C., (CEX) 35° C. Capillary Gel Low molecular Initial 25° C., 2-8° C., Electrophoresis weight species 35° C. 25° C., (CGE) (Caliper) 35° C.

[0216] A description of the analytical methods is provided below:

[0217] Visual inspection: Visual inspection was conducted in an inspection station equipped with a white and black background and fluorescent light. Formulations in vials were gently swirled without producing bubbles then inspected for colour, clarity and the presence of visible particles. Inspections were conducted by two independent inspectors.

[0218] pH measurement: pH was measured using a Mettler Toledo SevenExcellence pH meter equipped with a InLab® Ultra Micro ISM electrode.

[0219] UV spectroscopy: Protein concentration was measured by using A280/UV determination on the formulations without dilution on an IMPLEN P360 Nanophotometer. Measurements were conducted 3 times per formulation and the mean values of the measurements calculated.

[0220] Size exclusion chromatography (SEC)-high performance liquid chromatography (HPLC): SEC-HPLC was used to determine the protein aggregation profile of the formulations. A Dionex system (Ultimate 3000) equipped with an Acquity BEH200 column (Waters, 4.6×150 mm) was used to analyse the samples. Samples were diluted to 10 g/L in appropriate buffer, 3.0 μL was injected and the separation was performed under isocratic conditions at a flow rate of 0.3 mL/min. Mobile phase consisted of BES buffer (pH 6.5) with a run time of 15 min. Intact protein was detected at 280 nm at a retention time of roughly 3.5 min. Monomer species, high molecular weight species (HMWS, aggregates) and low molecular weight species (LMWS, fragments) were reported as a relative area %. Internal and external references were used to validate the run.

[0221] Cation exchange chromatography (CEX): CEX-HPLC was used to determine the proportions of proteinaceous acidic, main and basics species. A Dionex system (Ultimate 3000) equipped with a Propack WCX-10 column (Thermo Fisher, 4×250 mm) was used to analyse the samples. Samples were diluted to 10 g/L in appropriate buffer, 3.0 μL injection volume was used and separation was conducted with a gradient method at 0.7 ml/min. Briefly, two aqueous buffers at pH 5 and pH 10 (Imidazole, Piperazine and TRIS based) were alternated over a period of 55 minutes. Species were detected at 280 nm, identified against a reference standard and reported as Relative Area percentage over the integrated area.

[0222] Capillary Gel Electrophoresis (CGE) “Caliper” method: The protein “banding pattern” was obtained by Capillary Gel Electrophoresis. Analysis was performed using a microfluidic LabChip GXII system (Perkin Elmer Australia Pty Ltd). The protein electrophoresis on the microfluidic chip was achieved by integration of the main features of one-dimensional SDS-PAGE: these include the separation, staining, de-staining, and detection. Denatured proteins were loaded onto the chip directly from a microtiter plate through a capillary sipper. The samples were then electrokinetically loaded and injected into the 14 mm long separation channel containing a low viscosity matrix of entangled polymer solution. The entire sample preparation procedures were done according to manufacturer protocol. For non-reducing samples, protein solution were diluted to 1 g/L with buffer and Milliq water. Reducing samples were diluted with 1M DTT. Denaturation occurred 40° C. for 20 min for non-reduced samples and at 80° C. for 15 min for reduced samples. Results were reported in relative area percentage for LMWS Intact and HMWS for non-reduced samples. For reduced samples, Long Chain and Short chain fraction were considered.

Example 1—Visual Inspection at t=0

[0223] Visual appearance of all formulations directly before filling was evaluated. Additionally, all filled vials of all formulations were assessed by visual appearance after fill/finish.

[0224] Due to high protein concentration, all formulations possessed a brownish yellow colour with slight opalescence. Opalescence was observed to increase with pH. CSL730 in formulations at pH 6.5 and 7 precipitated with more intense precipitate seen at pH 7. It is well known that higher pH can be used to precipitate Fc fragments. The precipitated fragments can be recovered later on by solubilization in low pH buffers. This has been confirmed with the precipitated portion of the recombinant Fc multimer (data not shown). However, the impact of this high pH exposure on storage stability is not well understood.

[0225] All formulations were filtered before filling to vials (including the precipitated formulations) and reinspected after stoppering and crimping. All formulations had a brownish-yellow colour, were slightly opalescent and were free of visible particles (including pH 6.5 and pH 7.0 formulations). No gelling was observed in any of the formulations prepared.

[0226] Overall, results showed that formulations at pH 6.5 led to protein precipitation. Formulations with lower pH values down to pH 4.5 were more suitable since lower pHs did not lead to protein precipitation.

Example 2—pH and Protein Concentration Measurements at t=0

[0227] pH and protein concentration measurements on all formulations 1-12 (see Table 2) were conducted at t=0. Differences from target values were also assessed. The results are summarized in Table 4

[0228] pH measurements showed that the measured pH values were within ±0.2 pH units for most formulations in comparison to the target pH-values, with the exception of the acetate formulation with the target pH 4.5 and the histidine formulation with the target pH 5.0, where the pH-values were within 0.4 and 0.3 units from the target pH, respectively. Overall, based on the measured pH values, the pH screening for this study covers a pH range of 4.88 to 6.92.

[0229] With regards to measured protein concentrations, most formulations were within the target concentration of 100+/−10 mg/mL. Exceptions were the acetate formulations with target pH 4.5 and 5.0. Those samples were over diluted by mistake, so this is considered a study deviation. On the other hand, histidine formulations at pH 6.5 and 7.0 had a lower concentration as a result of the precipitation that occurred.

[0230] The results show that the pH-value of the formulations was in most cases as expected. With the exception of the acetate formulations, a significant drop in protein concentration at pH 6.5 is associated with the significant precipitation of the protein under those conditions.

TABLE-US-00004 TABLE 4 Measured pH (and differences from target values) and protein concentration values for all formulations. Measured protein Formulation Formulation Target Measured concentration No. Buffer pH pH ΔpH (mg/mL)**  1 20 mM Acetate 4.5 4.9   0.4***   74 ± 1***  2 5.0 5.2 0.2   72 ± 1***  3 5.5 5.6 0.1 102 ± 1   4 20 mM Histidine 5.0 5.3   0.3*** 91 ± 1  5 5.5 5.7 0.2 107 ± 3   6 6.0 6.2 0.2 96 ± 5  7 6.5 6.6 0.1   82 ± 6****  8 7.0 6.9* N/A   19 ± 1****  9 20 mM Histidine/ 6.0 6.0 0.1 98 ± 5 150 mM NaCl 10 20 mM Citrate 5.5 5.5 0.1 106 ± 10 11 6.0 5.9 0.1 91 ± 8 12 Buffer free TBD 5.7 N/A 96 ± 4 *pH only measured after 4 weeks at cold storage, **Target 100 ± 10 mg/mL, ***sample overdilution (study deviation), ****protein concentration decreased due to sample precipitation

Example 3—High Molecular Weight (HMW) Profile at t=0 by Size Exclusion Chromatography

[0231] All formulations 1-12 (see Table 2) were analyzed by size exclusion chromatography for the quantitation of high molecular weight (soluble aggregates) species at t=0, as described above.

[0232] Soluble aggregates profile for all formulations showed relatively low % HMW in all formulations (overall <1.5%), with a rough trend showing that increasing pH is related to a higher % of HMW molecules (until the point of precipitation). Interestingly, the initial % HMW of the filtered solutions of the formulations after precipitation at pH 6.5 also fell below 1.5%. This indicates that while a pH 6.5 is not suitable for formulating the Fc multimer molecule at high protein concentrations, it could be suitable for formulating the recombinant Fc multimer molecule at lower protein concentrations.

Example 4—Low Molecular Weight (LMW) Profile at t=0 by Capillary Gel Electrophoresis

[0233] All formulations 1-12 (see Table 2) were analyzed by non-reducing capillary gel electrophoresis using Caliper (NR Caliper) for the quantitation of low molecular weight species (fragmentation) at t=0, as described above.

[0234] All formulations, with the exception of formulation 8 (precipitated at pH ˜7), had a relatively low % LMW (1.5-2.0%). Overall, the data shows no proportional relation between the pH and % LMWs. However, the increase in % LMWs at pH 6.9 indicates that there may be a threshold pH above which more measurable increases in % LMWs occurs.

Example 5—Acidic and Basic Species Profile at t=0 by Cation Exchange Chromatography (CEX)

[0235] All formulations were analyzed by cation exchange chromatography (CEX) for the quantitation of acidic and basic species at t=0, as described above.

[0236] With the exception of formulations 7 and 8, all formulations showed comparable levels of acidic species. The percentage of acidic species was lower in formulation 7 (52%) and lowest in formulation 8 (45%).

[0237] The data showed no relation between pH and initial % acidic species. Drop in initial % acidic species in histidine buffer at pH≥6.5 indicates there may be a threshold pH above which a significant decrease of acidic species is observed.

[0238] With the exception of formulations 7 and 8, all formulations showed comparable levels of basic species. In comparison with all formulations, the percentage of basic species was lower in formulations 7 and 8 (4%).

[0239] The data showed no relation between pH and initial % basic species. Drop in initial % basic species in histidine buffer at pH≥6.5 indicates there may be a threshold pH above which a decrease of basic species is observed.

[0240] However, the results of formulations 7 and 8 may be influenced by protein precipitation for both acidic and basic species, as described above.

Summary of Examples 1 to 5

[0241] Based on analyses performed during processing and immediately after fill/finish (T=0), it can be concluded that formulations with pH≥6.5 may not be suitable for the formulation of Fc multimer molecules at high concentrations (≥100 mg/mL).

[0242] Nevertheless, all formulations were placed on stability to cover the kinetics of degradation over the range of pH values in all formulations.

Example 6—Visual Inspection Over Time

[0243] Formulations 1 to 12 (see Table 2) were visually inspected after 2 and 4 weeks. The results are summarized in Table 5.

[0244] As described above, two inspectors (Insp 1 and Insp 2) examined visual appearance at each time point and temperature. There was a general alignment between examiners in most cases.

[0245] No changes were observed with regards to color and clarity for most formulations at all storage temperatures. Color of all formulations remained slight brown yellow while clarity remained slightly opalescent. Formulations at higher storage temperatures started showing visible particles, but the most significant formation of particles occurred at pH≥6.5. Interestingly, the formulation with salt showed no visible particles compared to the same formulation at pH 6.

[0246] The results indicate that particle formation at each time point of visual inspection was increased at a pH≥6.5 in comparison to samples having a lower pH.

TABLE-US-00005 TABLE 5 Results of visual appearance testing by the two inspectors in formulations stored for 2 or 4 weeks as 2-8° C., 25° C. and 35° C. 2 weeks 4 weeks Target 25° C. 35° C. 2-8° C. 25° C. 35° C. Buffer pH Insp 1 Insp 2 Insp 1 Insp 2 Insp 1 Insp 2 Insp 1 Insp 2 Insp 1 Insp 2 20 mM 4.5 0 0 <5 <5 <5 <5 0 0 0 0 Acetate 5.0 0 0 0 0 0 <5 0 0 0 <5 5.5 0 <5  0 0 0 0 <5 <5 0 <5 20 mM 5.0 0 0 0 <5 0 <5 <5 <5 <5 0 Histidine 5.5 0 0 0 <5 0 0 0 0 0 <5 6.0 0 0 <5 <5 0 <5 0 0 0 0 6.5 0 <5  <5 <5 Countless Countless <5 <5 0 <5 7.0 ND ND 0 <5 <5 Countless <5 <5 0 <5 His/NaCl 6.0 0 0 0 0 0 0 0 0 0 0 20 mM 5.5 0 5-10 <5 0 0 0 0 0 <5 <5 Citrate 6.0 0 0 <5 <5 0 0 <5 0 <5 <5 Buffer free TBD 0 <5  0 0 0 <5 0 <5 0 <5

Example 7—pH and Protein Concentrations Over Time

[0247] The pH and protein concentrations were monitored in formulations 1 to 12 (see Table 2) at 2-8, 25 and 35° C. for up to 4 weeks.

[0248] Overall, within experimental error, results showed minimum shifts in pH and protein conc. at 2-8, 25 and 35° C. for all formulations for up to 4 weeks of storage.

Example 8—Relationship Between the Formation of High Molecular Weight (HMW) and pH/Buffer/Salt Over Time (Size Exclusion Chromatography)

[0249] HMW species were monitored in formulations 1 to 12 (see Table 2) at 2-8, 25 and 35° C. for up to 4 weeks. Representative plots for changes in % HMW over time for formulations stored at 35° C. are shown in FIG. 1. FIG. 1 shows an initial high increase in % HMW species followed by a slower increase of species with time. The same trend was observed for the formulations stored at 25° C.

[0250] % changes in aggregate species (% HMW) after 4 weeks of storage at 2-8° C. are presented in Table 6. The aggregation rate constants, along with the corresponding standard error (SE), were determined for all formulations at 25 and 35° C. (from 3 time points) by multiple linear regression using Graphpad Prism software. The results are summarized in Table 6.

[0251] Results in Table 6 show that the rate of aggregation increased with temperature and with pH. Higher aggregation rates occurred at pH 6.0. The trend was more prominently observed at 35° C. Note that the formulation in histidine buffer with pH of 6.9 (formulation 8) appeared to be most stable. However, this is an artifact of the very low protein concentration in this formulation after protein precipitation (18 mg/mL), in comparison with much higher protein concentration in all other formulations (see Table 4).

[0252] The buffer type had a small impact on aggregation rate. At equivalent pH, different buffers performed similarly. The self-buffered formulation (formulation 12) was slightly less stable than the formulation with equivalent pH containing a buffer (formulation 5). The addition of salt (leading to a higher ionic strength) showed no significant impact on improvement in aggregation behaviour.

[0253] The trend for aggregation is summarized in FIG. 2 showing pooled data (aggregation rates at 25 and 35° C.) as a function of measured pH (without pH 7 formulation which is not representative of aggregation behavior). FIG. 2 shows overall a non-linear increase in the rate of aggregation as measured pH increased.

TABLE-US-00006 TABLE 6 Summary of SEC data for aggregation (% HMW) comparing changes in % HMW after 4 weeks of storage at 2-8° C. (5.sup.th column), aggregation rate constants k (±SE) at 25° C. (6.sup.th column) and at 35° C. (7.sup.th column). Δ% HMW after k at 25° C. k at 35° C. Formulation Target Measured T-4 weeks (±SE) (±SE) No. Buffer pH pH (2-8° C.) (%/week) (%/week)  1 20 mM 4.5 4.9 0.1 0.2 ± 0.0 0.3 ± 0.0  2 Acetate 5.0 5.2 0.1 0.1 ± 0.0 0.3 ± 0.0  3 5.5 5.6 0.7 0.4 ± 0.1 0.7 ± 0.1  4 20 mM 5.0 5.3 0.4 0.3 ± 0.0 0.4 ± 0.1  5 Histidine 5.5 5.7 0.7 0.4 ± 0.1 0.6 ± 0.1  6 6.0 6.2 1.0 0.5 ± 0.1 0.8 ± 0.2  7 6.5 6.6 1.8 0.8 ± 0.3 1.1 ± 0.3  8 7.0 6.9 ~0 0.06* 0.2 ± 0.0  9 20 mM 6.0 6.0 0.8 0.5 ± 0.1 0.7 ± 0.1 Histidine/NaCl 10 20 mM 5.5 5.5 0.5 0.4 ± 0.1 0.7 ± 0.1 11 Citrate 6.0 5.9 0.8 0.5 ± 0.1 0.8 ± 0.1 12 Buffer free TBD 5.7 1.0 0.5 ± 0.1 0.7 ± 0.1 *Rate estimate from 2 points.

Example 9—Relationship Between the Formation of Fragments (LMW) and pH/Buffer/Salt Over Time (Non Reducing Caliper)

[0254] Fragmentation of formulations 1 to 12 (see Table 2) was monitored at 2-8, 25 and 35° C. for up to 4 weeks. Representative plots for changes in % fragments over time for formulations stored at 35° C. are shown in FIG. 3. FIG. 3 shows an initial high increase in % LMW species (fragments) followed by a slower increase of species with time. The same trend was observed for the formulations stored at 25° C.

[0255] % changes in fragments after 4 weeks of storage at 2-8° C. are presented in Table 7. Rates constants of fragmentation, along with the corresponding standard error (SE), were determined for the formulations at 25 and 35° C. (from 3 time points) by multiple linear regression using Graphpad Prism software. Multiple linear regression was done using a general linear model (GLM) to plot and fit the assay data to the model (equation 1). The results are summarized in Table 7.

[0256] The data revealed that generally low fragmentation occurs at 2-8° C. However, the rate of fragmentation increased with storage temperature and with pH (pH≥6.0). More significant changes in % LMW were observed at 2-8° C. with the formulation at ˜pH 7. At 25° C. and 35° C. the rate of fragmentation was also highest in formulation 8 at ˜pH 7 (despite lowest protein concentration at 18 mg/mL) followed by pH 6.5. At the same pH, the buffer type only had a small impact on aggregation rate. No clear benefits were found for the addition of NaCl (higher ionic strength).

[0257] The trend for fragmentation is summarized in FIG. 4 showing pooled data (fragmentation rates at 35° C. with standard errors) as a function of measured pH. As was observed with aggregation, FIG. 4 shows overall that the rate of fragmentation increased as measured pH increased pH (≥6.0).

TABLE-US-00007 TABLE 7 Summary of NR-Caliper data for fragmentation (% LMW) comparing changes in fragments after 4 weeks of storage at 2-8° C. (5.sup.th column), fragmentation rate constants k (±SE) at 25° C. (6.sup.th column) and at 35° C. (7.sup.th column). Δ% fragments k at 25° C. k at 35° C. Sample Target Measured after T-4 (±SE)* (±SE)* No. Buffer pH pH weeks (2-8° C.) (%/wk) (%/wk)  1 20 mM 4.5 4.9 0.1 0.2 ± 0.1 0.5 ± 0.1  2 Acetate 5.0 5.2 0.1 0.1 ± 0.1 0.4 ± 0.1  3 5.5 5.6 0.1 0.1 ± 0.1 0.4 ± 0.1  4 20 mM 5.0 5.3 0.1 0.1 ± 0.1 0.4 ± 0.1  5 Histidine 5.5 5.7 0.1 0.1 ± 0.1 0.3 ± 0.1  6 6.0 6.2 ~0 0.2 ± 0.1 0.5 ± 0.1  7 6.5 6.6 0.1 0.3 ± 0.1 0.8 ± 0.1  8 7.0 6.9 0.3 0.5* 1.3 ± 0.1  9 20 mM 6.0 6.0 ~0 0.1 ± 0.1 0.4 ± 0.1 Histidine/NaCl 10 20 mM 5.5 5.5 0.1 0.2 ± 0.1 0.8 ± 0.2 11 Citrate 6.0 5.9 0.1 0.1 ± 0.1 0.4 ± 0.1 12 Buffer free TBD 5.7 0.1 0.1 ± 0.1 0.4 ± 0.1 *Rate estimate from 2 points.

Example 10—Relationship Between the Formation of Acidic Species and pH/Buffer/Salt Over Time (Cation Exchange Chromatography)

[0258] The formation of acidic species was monitored in formulations 1 to 12 (see Table 2) at 2-8, 25 and 35° C. for up to 4 weeks. Representative plots for changes in % acidic species over time for formulations stored at 35° C. are shown in FIG. 5. FIG. 5 shows (except for the histidine formulation at ˜pH 7) a more linear increase in % acidic species over time, as compared to plots for % HMW (FIG. 1) and % LMW (FIG. 3). The same trend was also observed for the formulations stored at 25° C.

[0259] % changes in acidic species after 4 weeks of storage at 2-8° C. are presented in Table 8. Rate constants of acidic species formation, along with the corresponding standard error (SE), were determined for the formulations at 25 and 35° C. (from 3 time points) by multiple linear regression using Graphpad Prism software. Multiple linear regression was done using a general linear model (GLM) to plot and fit the assay data to the model (equation 1). The results are also summarized in Table 8.

[0260] The data revealed that generally low fragmentation occurred at 2-8° C. However, the rate of formation of acidic species increased with storage temperature and with pH (pH≥6.0). The highest levels of acidic species formed after 4 weeks at 2-8° C. in the formulation at ˜pH 7. At 25° C. and 35° C. the rate of formation of acidic species was also highest in formulation 8 at ˜pH 7 (despite lowest protein concentration at 18 mg/mL) followed by pH 6.5.

[0261] At the same pH, the buffer type was also found to play a role in the rate of formation of acidic species, with formulations in histidine buffer showing more storage stability, as compared to the formulations in the same pH in acetate or citrate buffers.

[0262] The addition of NaCl (higher ionic strength) showed better storage stability, with regards to rate of formation of acidic species, than the formulation at the same pH without salt.

[0263] The trend for formation of acidic species with pH is summarized in FIG. 6 showing pooled data (rates of formation of acidic species at 25 and 35° C.) as a function of measured pH. As was observed with aggregation and fragmentation, FIG. 6 shows overall that the rate of formation of acidic species increased as measured pH increased (>pH 6.0).

TABLE-US-00008 TABLE 8 Summary of CEX data (% acidic species) comparing changes in % acidic species after 4 weeks of storage at 2-8° C. (5.sup.th column) and rate constants of acidic species formation k (±SE) at 25° C. (6.sup.th column) and at 35° C. (7.sup.th column). Δ% acidic species k at 25° C. k at 35° C. Formulation Target Measured after T-4 wks (±SE)* (±SE)* No. Buffer pH pH (2-8° C.) (%/wk) (%/wk)  1 20 mM 4.5 4.9 0.2 0.5 ± 0.1 2.5 ± 0.1  2 Acetate 5.0 5.2 0.2 0.6 ± 0.1 2.8 ± 0.1  3 5.5 5.6 0.3 0.7 ± 0.0 2.7 ± 0.1  4 20 mM 5.0 5.3 0.1 0.4 ± 0.1 2.0 ± 0.2  5 Histidine 5.5 5.7 ~0 0.4 ± 0.1 1.9 ± 0.2  6 6.0 6.2 0.3 0.6 ± 0.1 2.3 ± 0.0  7 6.5 6.6 ~0 1.2 ± 0.2 3.5 ± 0.2  8 7.0 6.9 1.7 2.6* 6.2 ± 0.5  9 20 mM 6.0 6.0 0.4 0.3 ± 0.1 1.5 ± 0.0 Histidine/NaCl 10 20 mM 5.5 5.5 0.1 0.7 ± 0.1 3.1 ± 0.1 11 Citrate 6.0 5.9 0.2 0.6 ± 0.1 2.6 ± 0.1 12 Buffer free TBD 5.7 0.5 0.5 ± 0.1 2.0 ± 0.1 *Rate estimate from 2 points

Conclusion of Examples 1 to 10

[0264] The results indicate that formulations of Fc multimer molecules (CSL730) at high concentrations (100 mg/mL) require specific conditions to prevent protein precipitation. It was shown that a pH≥6.5 may not be suitable for formulations of Fc multimer molecules (CSL730) at high concentrations (100 mg/mL) due to precipitation. Formulations of Fc multimer molecules (CSL730) at high protein concentrations (100 mg/mL) were more stable to aggregation, fragmentation and acidic species formation at pH lower than 6. Furthermore formulations of Fc multimer molecules (CSL730) at high protein concentrations (100 mg/mL) were more stable to acidic species formation with high salt concentration, as well as with histidine buffer.

Part 2—Stabilizer Screening Study

[0265] Excipient screening studies were performed to investigate the effect of stabilizer type (sugars/polyols and amino acids), stabilizer levels, surfactant levels, antioxidant type and antioxidant levels on liquid formulations comprising an Fc multimer molecule (CSL730) at protein concentrations of 10-100 mg/ml. A parallel short term (3 month) study was conducted to investigate storage stability as a function of protein concentration. Overall, CSL730 formulations with protein concentrations ranging from 10-100 mg/mL were prepared and investigated for protein stability.

Materials and Methods

[0266] Materials: The materials used for the examples were the same as in Part 1; additional materials used in this part, their catalogue numbers and the suppliers are listed in Table 9.

TABLE-US-00009 TABLE 9 Materials used for the examples, the corresponding catalog number from the supplier and the supplier. Catalog Material Number Supplier Slide-A-Lyzer ® G2 Dialysis 87732 Thermofisher Cassettes 10K MWCO, 30 ml Scientific Slide-A-Lyzer ® G2 Dialysis 87733 Thermofisher Cassettes 10K MWCO, 70 ml Scientific 2 ml Fiolax clear vials with 13 BO2128 Schott mm neck 13 mm Injection Stopper 19700004 West FluroTec Pharmaceutical Services 13 mm Flip Off Vial Seals F013DBL-1K West Pharmaceutical Services L-Proline 1.07430.1000 Merck L-arginine monohydrochloride 1.01544.1000 Merck D (+) Trehalose dihydrate T9531-100G Sigma D-Sorbitol S1876 Sigma Polysorbate 80 7815 NOF L-glutathione reduced G4251 Sigma L-Methionine M8439 Sigma

[0267] Preparation of formulations: The formulations were prepared from CSL730 recombinant Fc multimer bulk purified protein at 120 mg/mL formulated in 20 mM Histidine, 40 mM NaCl and 200 mM Proline at pH 6.0.

[0268] Histidine was the buffer of choice in all formulations at a target buffer concentration of 20 mM. pH was fixed in all but one formulation to 5.25±0.1. Polysorbate 80 (PS80) was the surfactant of choice for all formulations. PS80 concentration was fixed in all but one formulation to 0.02 w/v. Different stabilizers were used (proline, arginine, sucrose, trehalose and sorbitol) spanning a target concentration range of 200-300 mM. Four formulations contained varying levels (1-20 mM) of antioxidants (either methionine or reduced glutathione GSH). Study (and hence formulation differences) was designed to primarily change one factor at a time (OFAT). A summary of the formulations prepared for this study is provided in Table 10.

[0269] The following buffers were prepared for the dialysis: [0270] 1. 20 mM histidine, 250 mM sucrose, pH 5.25 [0271] 2. 20 mM histidine, 250 mM trehalose, pH 5.25 [0272] 3. 20 mM histidine, 250 mM sorbitol, pH 5.25 [0273] 4. 20 mM histidine, 250 mM arginine, pH 5.25 [0274] 5. 20 mM histidine, 167 mM arginine, 83 mM sucrose, pH 5.25 [0275] 6. 20 mM histidine, 225 mM sucrose, pH 5.25

[0276] To prepare the formulations (2-6, 8-16, 18), approximately 30-50 mL portions of the bulk purified protein were dialysed against each of the 6 buffers listed above. The pH of each buffer was measured after preparation. 30% overage (mg protein) was applied to account for losses. The formulations were dialysed against about 1 L of buffer for about 5 hours which was subsequently replaced with fresh buffer (about 1 L) and dialysed overnight. Dialysis occurred at 2-8° C. in 30 or 70 ml 10K MWCO cassettes.

[0277] After dialysis, the protein concentration of each dialysate was measured. When samples were found to be too dilute, they were concentrated up by centrifugation using Amicon tubes with the aim of targeting 120±5 mg/mL. Protein concentration was confirmed afterwards.

[0278] The following stock solutions were prepared and used as spiking solutions to aid in the preparation of the final formulations: [0279] 1. 20 mM histidine, 5.75% w/v (500 mM) proline, 0.02% w/v PS80, pH 5.25 [0280] 2. 20 mM histidine, 17.04% w/v sucrose, 0.02% w/v PS80, pH 5.25 [0281] 3. 10% w/v PS80 [0282] 4. 20 mM histidine, 200 mM proline, 40 mM sodium chloride, 0.02% w/v PS80, pH 5.8 (labelled “CSL730 Diluent”)

[0283] Drug substance (DS) was composed of 118 mg/mL recombinant Fc multimer CSL730, 20 mM Histidine, 40 mM NaCl, 200 mM Proline and 0.02% w/v PS80 at pH 6.0. The DS was diluted by the addition of appropriate volumes of stock solution 1 or of stock solution 2 to achieve protein concentrations of 100±5 mg/mL followed by adjusting pH to 5.25±0.1 using 0.1M HCl to prepare formulation 1 and formulation 7, respectively. Formulation 17 was prepared by direct dilution of the DS to 100±5 mg/mL using stock solution 4 while maintaining the pH at 5.8±0.1.

[0284] After an up-concentration step and confirmation of protein concentration (A280 measurements), formulations 2-6, 8-16 and 18 were appropriately diluted with buffers 1-6, as needed, and final pH was adjusted to 5.25±0.1 using 0.1 M HCl. Protein concentration was confirmed after pH adjustment step.

[0285] Fill and finish was performed under a laminar flow hood. Each formulation and its corresponding placebo was filtered through a 0.22 μm PES sterile filter and filled into pre-labelled 2 ml glass vials with 0.8 ml of the formulation. Vials were then stoppered and crimped. In total, 17 liquid formulations and 13 placebos were prepared.

[0286] After fill/finish, the vials of the filled liquid formulations were placed in stability chambers at 2-8° C., 25° C. or 40° C. For “T0”/T=0 analysis samples were analyzed immediately after fill/finish followed by analysis at predetermined subsequent time points for up to 6 months. In order to compare kinetics of degradation between the different formulations, rate constants of degradation by different pathways (with standard error) were measured using multiple linear regression using JMP and Excel software. Multiple linear regression was calculated using a general linear model (GLM) to plot and fit the assay data to the model (equation 1).

% P=Po+k.Math.t  [Equation 1]

TABLE-US-00010 TABLE 10 Summary of formulations prepared for the excipient screening study Target Target Stabilizer Antioxidant Formulation CSL730 conc. PS80 conc. Target Stabilizer Conc. type and conc. Molar ratio No. (mg/mL) (% w/v) pH type (mM) (mM) (protein:stabilizer) F1* 100 ± 5 0.02 5.25 ± 0.1 Proline 250 1:417 F2 100 ± 5 0.02 5.25 ± 0.1 Sucrose 250 1:417 F3 100 ± 5 0.02 5.25 ± 0.1 Trehalose 250 1:417 F4 100 ± 5 0.02 5.25 ± 0.1 Sorbitol 250 1:417 F5 100 ± 5 0.02 5.25 ± 0.1 Arginine 250 1:417 F6 100 ± 5 0.02 5.25 ± 0.1 Sucrose/Arginine 83/167 1:417 F7* 100 ± 5 0.02 5.25 ± 0.1 Sucrose/Proline 83/167 1:417 F8 100 ± 5 0.02 5.25 ± 0.1 Sucrose 225 1:375 F9 100 ± 5 0.02 5.25 ± 0.1 Sucrose 300 1:500 F10 70 0.02 5.25 ± 0.1 Sucrose 250 1:625 F11 10 0.02 5.25 ± 0.1 Sucrose 250  1:4167 F12 100 ± 5 0.02 5.25 ± 0.1 Sucrose 250 10 mM 1:417 Methionine F13 100 ± 5 0.02 5.25 ± 0.1 Sucrose 250 20 mM 1:417 Methionine F14 100 ± 5 0.02 5.25 ± 0.1 Sucrose 250 1 mM 1:417 reduced GSH F15 100 ± 5 0.02 5.25 ± 0.1 Sucrose 250 10 mM 1:417 reduced GSH F17* 100 ± 5 0.02  525 ± 0.1 Proline 200 1:333 F18 100 ± 5 0.04 5.25 ± 0.1 Sucrose 250 1:417 *F1, F7 and F17 were the only formulation not dialysed. Thus, the formulations had low levels of NaCl (~30-40 mM).

Methods Used for Analyzing the Stability of the Formulations:

[0287] Stability indicating methods were used to analyze the formulations at the different time points namely: Size exclusion chromatography (SEC), Cation exchange chromatography (CEX), Capillary Gel Electrophoresis (CGE) (Caliper) and reverse phase HPLC (RP-HPLC) for oxidation. In addition, pH, visual appearance (for color, turbidity and visible particles) and subvisible particles (SVP) of the formulations were monitored at the different time points.

[0288] Methods used for the study and purpose of each method are summarized in Table 11. Storage conditions and analysis time points are summarized in Table 12.

TABLE-US-00011 TABLE 11 Analytical methods used for stability studies of formulations and their purpose Method Purpose Visual inspection Visible particles, colour, opalescence, turbidity Dynamic Imaging Particle Analysis Sub-visible particle count (DIPA) pH measurement pH testing Osmolality Osmolality measurement Polysorbate 80 (PS80) testing Quantitation of PS80 UV spectroscopy Protein content (A280) Size exclusion chromatography (SEC) High molecular weight species (soluble aggregates) Cation exchange chromatography Acidic, basic and main species (CEX) Capillary Gel Electrophoresis Low molecular weight species (CGE) (Caliper) Reverse phase high performance Quantitation of Oxidation liquid chromatography (RP-HPLC)

TABLE-US-00012 TABLE 12 A summary of storage conditions and analysis time points Storage condition Time points 2-8° C.  0, 3, 6 months 25° C. 0, 1, 2, 3, 6 months 40° C. 0, 1, 2, 3 months

[0289] A description of the analytical methods is provided below:

[0290] Visual inspection, pH measurement, UV spectroscopy, size exclusion chromatography, cation exchange chromatography, and capillary gel electrophoresis were carried out as described in Part 1 above.

[0291] Osmolality: Osmolality of the formulations was measured at the initial time point (T0) by freezing point depression using a Gonotec Osmomat 3000 Osmometer. Sample volumes were 300 μL. Measurements were conducted 3 times per formulation and the mean values of the measurements calculated.

[0292] Subvisible particle count testing: Limited analysis of subvisible particles using a FlowCam Biologics instrument (a Dynamic/flow Imaging Particle Analysis—DIPA—technique) was performed on formulations of interest at 2-8° C. and 25° C. only after 3 months storage time. A minimum sample volume of 0.5 mL was used. Measurements were conducted 3 times per formulation and the mean values of the measurements calculated.

[0293] Analysis of oxidation by reverse phase high performance liquid chromatography (RP-HPLC): A RP-HPLC method was used to determine the total amount of oxidized species as a percentage of the total area, and the relative amount of oxidation of both short and long chain. Sample preparation involved sample dilution to 10 g/L in appropriate buffer followed by the use of guanidine as denaturing reagent and of DL-Dithiothreitol (DTT) for the reduction of disulphide bonds. This step generates two identical long chains and two identical short chains. To prevent oxidation of the fragments, a subsequent dilution step with a solution containing high concentration of L-methionine was performed followed by sample analysis at a sample concentration of 2 μg/μl. A Thermo Ultimate 3000 (or equivalent) equipped with an AdvanceBio RP-mAb Diphenyl 3.5 μm, 2.1×50 mm column was used to analyse the samples. 3 μL injection volume was used and separation was conducted with a gradient method at 0.35 ml/min. Column temperature was set to 65° C. Briefly, two buffers (0.1% trifluoroacetic acid in water and 0.08% TFA in acetonitrile) were alternated over a period of 20 minutes. Species were detected at 280 nm, identified against a reference standard and reported as Relative Area percentage over the integrated area.

[0294] Analysis of polysorbate 80 (PS80): RP-HPLC was used to quantify the amount of PS80 at the initial time point (T0) in the different formulations. In short, PS80 standard and the samples were treated with ethanol followed by 0.1M KOH at 40° C. followed by sample analysis of oleic acid resulting from hydrolysis by a reverse phase HPLC method. A Dionex (Ultimate 3000) System (or equivalent) equipped with a Nova-Pak® C18 3.9×150 mm, 4 μm reverse phase column (Waters) was used to analyze the samples. Injection volume was 15 μL and separation was conducted using an isocratic method at 2.0 ml/min. Mobile phase was 80% acetonitrile with 20% potassium dihydrogen phosphate buffer at pH 2.8. Column temperature was set to 40° C. Species were detected at 250 nm, and quantified using a standard calibration curve generated by the PS80 standard solutions. Data is reported as (w/v) of PS80.

Example 1—Visual Inspection at T0 and Over Time

[0295] All filled vials were examined by visual appearance after fill/finish (100% visual inspection) by two inspectors (Insp 1 and Insp 2, as described above) and at each time point and temperature. No significant discrepancies were found in the visual description between examiners.

[0296] Except for the 10 mg/ml formulation, all other formulations possessed a slight brownish yellow (BY) (70 mg/mL formulation) to BY (100 mg/mL formulations) colour with slight opalescence due to high protein concentration. 0-2 visible particles were observed randomly in some formulations and placebos. A close examination, with knowledge about the protein, revealed that the visible particles were exogenous in nature (probably introduced during the fill/finish process). Formulations at high protein concentrations did not undergo gelling.

[0297] Formulations 1 to 18 (see Table 10) were visually inspected for up to 6 months at different temperatures. The color of most formulations remained unchanged at all storage temperatures from T0 (colorless for 10 mg/ml formulation, slight BY for 70 mg/ml formulation and BY for 100 mg/ml formulations). Formulations with reduced GSH had a slightly more BY color, as compared to formulations at the same protein concentration. Clarity remained unchanged (clear in 10 mg/ml formulation and slightly opalescent in all others). Particle counts remained unchanged from T0 (0-2), regardless of temperature and time. None of the formulations gelled during the storage time period at any temperature.

[0298] With time, GSH-containing placebos exhibited discoloration to a slight BY or BY colour. The colour intensified with storage time, storage temperature and with increasing GSH concentration. Placebos for other formulations remained clear and colourless regardless of temperature and time. Overall, particle counts remained unchanged from T0.

Example 2—Osmolality, pH, Protein Concentration and Polysorbate 80 Measurements at T0 and Over Time

[0299] Osmolality, pH, protein concentration and polysorbate 80 (PS80) concentration measurements on all formulations 1-18 (see Table 10) were conducted at the initial time point (T0). The results are summarized in Table 13.

[0300] The results in Table 13 show that protein concentration was within 100±5 mg/mL for the high concentration formulations. pH measurements showed that the measured pH values were within ±0.1 pH units for all formulations in comparison to the target pH-values. Osmolality measurements were generally within 300-390 mOsm/kg, with the exception of arginine-containing formulations (formulations 5, 6) where osmolality values were above 400 mOsm/kg. PS80 concentrations in all formulations were within a maximum of ±0.003 w/v of the PS80 target concentrations.

[0301] The pH and protein concentrations were monitored in formulations 1 to 18 (see Table 10) at 2-8, 25 and 40° C. for up to 6 months.

[0302] Table 14 summarizes results of pH measurements at different storage temperatures and times. Within experimental error, results showed minimum shifts (within a maximum of ±0.1 units) in pH at all temperatures for all formulations within the time frame measured.

[0303] Table 15 summarizes results of protein concentration measurements at different storage temperatures and times. Within experimental error, results showed small shifts in protein concentration with time. Three formulations showed relatively larger variations in protein concentration with time (F3 at 40° C. and F15 at 25° C. and 40° C.), but no protein precipitation appeared in any of these formulations. Average variation in protein concentration was 1.0 mg/mL at 2-8° C., 1.7 mg/mL at 25° C. and 1.6 mg/mL at 40° C. within the period measured. This is presumed to be a result of relatively assay variability of the high throughput technique used.

TABLE-US-00013 Table 13: Measured protein concentration (±standard deviation), pH, osmolality (±standard deviation) and polysorbate 80 (PS80) concentration for all formulations at initial time point (T0) Target Measured protein protein Measured PS80 Form. conc. conc. Target Measured Osmo. conc. Code (mg/mL) (mg/mL) pH pH (mOsm/kg) (% w/v) F1 100 102.8 ± 2.2  5.25 5.25 343 ± 2 0.019 F2 100 95.9 ± 1.1 5.25 5.20 298 ± 2 0.020 F3 100 100.7 ± 0.6  5.25 5.13 308 ± 3 0.019 F4 100 97.5 ± 0.0 5.25 5.18 297 ± 4 0.020 F5 100 99.2 ± 2.8 5.25 5.18 473 ± 2 0.021 F6 100 103.8 ± 1.3  5.25 5.12 424 ± 5 0.020 F7 100 100.5 ± 1.9  5.25 5.20 389 ± 4 0.017 F8 100 102.3 ± 0.5  5.25 5.18 285 ± 1 0.019 F9 100 95.2 ± 0.6 5.25 5.20 364 ± 3 0.020 F10 70 67.8 ± 1.5 5.25 5.20 302 ± 3 0.019 F11 10  9.9 ± 0.3 5.25 5.15 311 ± 1 0.020 F12 100 92.6 ± 0.8 5.25 5.16 320 ± 4 0.021 F13 100 97.4 ± 3.3 5.25 5.22 324 ± 2 0.022 F14 100 96.6 ± 0.7 5.25 5.20 301 ± 2 0.021 F15 100 95.4 ± 0.9 5.25 5.15 302 ± 3 0.021 F17 100 101.2 ± 2.3  5.25 5.13 291 ± 4 0.018 F18 100 100.3 ± 1.2  5.25 5.16 296 ± 1 0.043

Example 3—Limited Analysis of Subvisible Particles at 6 Month Time Point

[0304] Low subvisible particle counts were measured for most of the formulations tested after storage for 3 months at both 5 and 25° C. in all particle-size ranges tested (see FIG. 7).

[0305] High sub visible particle counts were observed for F1 (at both 5° C. and 25° C.). This could be related to the process to generate the formulation. The high level of particles could be related to the pH adjustment, by spiking and not by dialysis.

Example 4—Study of Aggregation Behavior (High Molecular Weight Species) by Size Exclusion Chromatography at T0 and Over Time

[0306] All formulations 1-18 (see Table 10) were analyzed by size exclusion chromatography for the quantitation of high molecular weight (soluble aggregates) species at T0, as described above.

[0307] Initial (T0) % HMW for all formulations are presented in Table 17. The aggregation rate constants, along with the corresponding standard error (SE), were determined for all formulations at 2-8, 25 and 40° C. by multiple linear regression as described before. The results are also summarized in Table 16.

[0308] Initial (T0) soluble aggregates profile for all formulations showed a variation in % HMW. This is attributed to differences in process when preparing different formulations. For example, some dialysates had to undergo up-concentration post dialysis while others did not. Overall, a relatively low % HMW was measured in all formulations at T0 (overall <1.5%). Interestingly, a systematic increase in aggregate (% HMW) levels was observed in formulations with increasing protein conc. (F2, F10, F11). Note that these formulations were prepared by simple dilution using the same buffer from the same stock dialysate solution.

[0309] Analysis of all formulations was discontinued after 3 months of storage at 40° C. since aggregation in all formulations reached levels sufficient to distinguish between performance of the formulations in terms of aggregation stability (best to worst performing). Analysis of F10, F11, F14 and F15 was also discontinued after 3 months at 2-8° C. and 25° C. Aggregation data collected to 3 months at 2-8° C. and 25° C. for F10 and F11 were sufficient to determine the impact of protein concentration on physical stability (by aggregation). F14 and F15, on the other hand, showed poor stability by this method and by other methods (to be discussed) sufficient to warrant their elimination. In general, % main species (monomer) was observed to decline with time along with a concurrent increase in aggregation (% HMW) and fragments (% LMW). Fragmentation however is not reported by this method but by a more reliable method for LMWs (CGE), as will be discussed later.

[0310] As expected, results in Table 16 show that the rate of aggregation increased with storage temperature. For every 10° C. increase in temperature, aggregation rate increased by 1.75 times on average up to 40° C. where more significant changes were observed, indicating possibly a different mechanism of aggregation of the molecule at this higher storage temperature as compared to lower temperature storage conditions.

[0311] To evaluate the effect of stabilizer type, the storage stability of F1-F7 was compared. Stabilizers used in the formulations were sugars/polyols, amino acids or mixtures of amino acids and sugars. Total stabilizer levels were fixed at 250 mM for these formulations. The results in Table 16 show that the Fc multimer molecule (CSL730) in the sucrose and trehalose based formulations (F2 and F3, respectively) had better storage stability as compared to all other formulations at all temperatures, as evidenced by slightly lower aggregation rates at 2-8° C. and 25° C. and significantly lower aggregation rates at 40° C. The sorbitol-based formulation showed superior stability to the amino acid based formulations (regardless of the presence or absence of sucrose with the amino acids) at 40° C., but comparable stability at lower temperatures for up to 6 months. Similarly, the proline-based formulations showed superior stability to the arginine-based formulations (regardless of the presence or absence of sucrose) at 40° C., but comparable stability at lower temperatures for up to 6 months.

[0312] To evaluate the effect of protein concentration at fixed stabilizer level, the storage stability of F2, F10 and F11 was compared for sucrose as a sugar type stabilizer. Protein concentration range was from 10-100 mg/mL. As expected, results in Table 16 show that the Fc multimer molecule (CSL730) exhibited superior stability at very low concentration (10 mg/mL) in F11 followed by 70 mg/mL (F10) followed by 100 mg/mL (F2). A plot of the aggregation rate constant as a function of protein concentration for all three formulations at all temperatures demonstrate a non linear increase in aggregation rate with protein concentration (results not shown). Note that at fixed stabilizer levels, the molar ratio of stabilizer to protein increases as protein concentration decreases (Table 10)—a factor that may contribute to the significant improvement in storage stability.

[0313] To evaluate the effect of stabilizer levels at fixed (high) protein concentration (100 mg/mL), the storage stability of F2, F8 and F9 was compared for sucrose as a sugar type stabilizer. Stabilizer levels ranged from 200-300 mM. Results in Table 16 show that sucrose at 225 mM (F8) was the least stable sucrose formulation as compared to 250 mM (F2) and 300 mM (F9) formulations at all storage temperatures. F9 showed incrementally better storage stability than F2 at all temperatures. Similarly, the Fc multimer molecule (CSL730) in proline at 200 mM (F17) was significantly less stable than in proline at 250 mM at all storage temperatures. Interestingly, the Fc multimer molecule (CSL730) was more stable in all sucrose formulations as compared to the proline formulations at 40° C. storage temperature.

[0314] To evaluate the effect of antioxidant (type and level) at a fixed stabilizer level, the storage stability of F2, F12, F13, F14 and F15 was compared. Methionine (at 10 and 20 mM) and reduced glutathione GSH (at 1 and 10 mM) were used as antioxidants. Results in Table 16 show that with methionine as an antioxidant, the Fc multimer molecule (CSL730) showed incrementally (small) improvements in the storage stability (physical) with increase in methionine levels at 40° C. However, there were no significant differences between the physical stability of the formulations with and without methionine. Formulations containing reduced GSH showed significantly greater instability at 40° C., as compared to the formulation without antioxidant (F2) and with methionine as an antioxidant (F12, F13). Smaller differences in storage stability were observed between the GSH-containing formulations, as compared to F2, F12 and F13 at 25° C., but with the same outcome (less stability with reduced GSH formulations). Nevertheless, reduced GSH formulations (F14, F15) were eliminated after 3 months of storage stability since stability data by other methods showed significant instability of F14 and F15 to warrant their elimination.

[0315] To evaluate the effect of surfactant (PS80) level at a fixed stabilizer level, the storage stability of F2 and F18 was compared. PS80 levels were evaluated at 0.02% w/v and 0.04% w/v. No significant differences were observed between the storage stability (aggregation) of both formulations at 2-8° C. and at 25° C. for up to 6 months. However, the Fc multimer molecule (CSL730) was more stable at a lower PS80 concentration (F2) at 40° C.

Example 4—Study of Fragmentation Behavior (Low Molecular Weight Species) by Non Reducing Capillary Gel Electrophoresis (NR-cGE) at T0 and Over Time

[0316] All formulations (see Table 10) were analyzed by non-reducing cGE using Caliper (NR Caliper) for the quantitation of low molecular weight (LMW) species (i.e., fragmentation) at T0, as described above. Placebo samples were also run and no interference from placebo ingredients (i.e., formulation inactive ingredients) with protein peaks was observed in cGE chromatograms (data not shown).

[0317] Initial (T0) % LMW total species for all formulations were measured. F15 had the highest T0 level of fragments, an observation also confirmed using SEC (data not shown). The initial (total) % LMW species measured in all formulations, except for F15, was relatively low ranging between 2.1-2.5%.

[0318] Except for F10 and F11, fragmentation behavior of the formulations was monitored using the NR-cGE “Caliper” method over time at different temperatures for select formulations since it is a relatively higher throughput method. The fragmentation rate constants, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described before. The results are also summarized in Table 17.

[0319] Analysis of all formulations was discontinued after 3 months of storage at 40° C. since fragmentation in all formulations reached levels sufficient to distinguish between performance of the formulations in terms of fragmentation stability (best to worst performing). Analysis of F14 and F15 was also discontinued after 3 months at 2-8° C. and 25° C. since they showed poor stability by this method and by other methods (aggregation, oxidation) sufficient to warrant their elimination. In general, as was observed with SEC analysis, % main species was observed to decline with time along with a concurrent increase in aggregation (% HMW) and fragments (% LMW). Aggregation, however, is not reported by this method since it has been reported by SEC, a more reliable method for soluble aggregates.

[0320] Small fluctuations in fragmentation (within assay error) occurred in all formulations studied at 2-8° C. with time, as evidenced by very small changes in % LMW after 3 and after 6 months.

[0321] As expected, results in Table 17 show that the rate of fragmentation increased with storage temperature.

[0322] To evaluate the effect of stabilizer type, the storage stability of F1-F7 was compared. Table 17 results show that the Fc multimer molecule (CSL730) exhibited more significant fragmentation potential in the presence of arginine (regardless of the presence or absence of sucrose: F5 and F6) at both 25° C. and 40° C. Fragmentation was comparable for all other formulations at 25° C. At 40° C., less fragmentation was observed with sucrose and sorbitol (F2 and F4, respectively) formulations as compared to proline and trehalose formulations (F1 and F3, respectively), evidenced by lower fragmentation rates at 40° C.

[0323] To evaluate the effect of stabilizer levels at fixed protein concentration (100 mg/mL), the storage stability of F2, F8 and F9 was compared for sucrose as a sugar type stabilizer. Stabilizer levels ranged from 200-300 mM. Results in Table 17 show no major differences in fragmentation stability at 25° C. as a function of sucrose level. Surprisingly, the formulation with sucrose at 250 mM (F2) showed better fragmentation stability than the corresponding formulations at 225 mM and 300 mM sucrose at 40° C. With proline as a stabilizer, the Fc multimer molecule (CSL730) in proline at 200 mM (F17) showed slightly better stability with regards to fragmentation as compared to the formulation with 250 mM proline at 25° C. and at 40° C.

[0324] To evaluate the effect of antioxidant (type and level) at a fixed stabilizer level, the storage stability of F2, F12, F13, F14 and F15 was compared. Results in Table 17 show that there were small differences in fragmentation storage stability of the Fc multimer molecule (CSL730) in the presence versus absence of methionine as an antioxidant at 40° C. Stability with regards to fragmentation was slightly better in the absence of methionine at 25° C. for reasons we cannot explain. More significant instability with respect to fragmentation was observed only after 3 months of storage at both 25° C. and 40° C. with formulations containing reduced GSH. Thus, reduced GSH formulations (F14, F15) were eliminated after 3 months of storage since stability data by this method and other methods showed sufficient instability of F14 and F15 to warrant their elimination.

[0325] To evaluate the effect of surfactant (PS80) level at a fixed stabilizer level, the storage stability of F2 (0.02% w/v PS80) and F18 (0.04% w/v PS80) was compared. No differences were observed between the storage stability (fragmentation) of both formulations at 25° C. for up to 6 months. However, the Fc multimer molecule (CSL730) was more stable at a lower PS80 concentration (F2) at 40° C.

Example 5—Study of Acidic and Basic Species by Cation Exchange Chromatography (CEX) at T0 and Over Time

[0326] All formulations (see Table 10) were analyzed by CEX for the quantitation of % acidic species and basic species at T0, as described above. Placebo samples were also run by CEX and no interference from placebo ingredients with protein peaks was observed in CEX chromatograms (data not shown).

[0327] FIG. 8 compares the initial (T0) results for % acidic and basic species for all formulations. % acidic species at T0 for most formulations were comparable. Starting levels of % acidic species in F15 (with high reduced GSH levels) and F17 (with lower proline levels) was ˜2.5-3.5% lower as compared to the other formulations (also see Table 9). Similarly, % basic species at T0 for most formulations were comparable (˜4-5%). However, F15 had the highest starting levels of % basic species (6.1%) as compared to the other formulations.

[0328] % acidic species for all formulations was monitored using the CEX method over time at different temperatures. The rate constants for formation of acidic species, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described before. The results are also summarized in Table 18.

[0329] Analysis of all formulations was discontinued after 2 months of storage at 40° C. since the levels of acidic species in all formulations were different and high enough to distinguish between performance of the formulations in terms of stability of acidic species (best to worst performing). Analysis of F10, F11, F14 and F15 was also discontinued after 3 months at 2-8° C. and 25° C. F14 and F15 showed poor stability by this method and by other methods sufficient to warrant their elimination. In general, % main species was observed to decline with time along with a concurrent increase in % basic species and a greater increase in acidic species.

[0330] F15 showed a significant rise in % acidic species after only 3 months of storage at 2-8° C. followed by F17. Small fluctuations in % acidic species (within assay error) were observed with the other formulations at the same temperature. For the formulations continued after the 3 month time point at 2-8° C., small fluctuations in % acidic species were observed in the same formulations. Among these formulations, F17 still showed higher levels of % acidic species after 6 months of storage at 2-8° C. (Table 18).

[0331] As expected, results in Table 18 show that the rate of formation of acidic species increased with storage temperature.

[0332] To evaluate the effect of stabilizer type, the storage stability of F1-F7 was compared. Table 18 results show that the Fc multimer molecule (CSL730) exhibited surprisingly better stability with regards to rate of formation of acidic species in the presence of arginine (F5 and F6) at both 25° C. and 40° C. (unlike fragmentation and aggregation stability trends).

[0333] To evaluate the effect of protein concentration at fixed stabilizer level, the storage stability of F2, F10 and F11 was compared for sucrose as a sugar type stabilizer. Results in Table 18 show that the Fc multimer molecule (CSL730) exhibited small differences in stability with regards to acidic species formation between the three formulations at different protein concentrations at both 25° C. and 40° C.

[0334] To evaluate the effect of stabilizer levels at fixed protein concentration (100 mg/mL), the storage stability of F2, F8 and F9 was compared for sucrose as a sugar type stabilizer. Stabilizer levels ranged from 200-300 mM. Results in Table 18 show that the Fc multimer molecule (CSL730) exhibited small differences in stability with regards to acidic species formation between the three formulations at different sucrose levels at both 25° C. and 40° C. In spite of starting higher levels of % acidic species with F17, the Fc multimer molecule (CSL730) in proline at 200 mM (F17) was comparable in stability with regards to acidic species formation in proline at 250 mM at both 25° C. and 40° C. Overall, the Fc multimer molecule (CSL730) stability with regards to acidic species formation in sucrose and proline formulations were comparable at both 25° C. and 40° C.

[0335] To evaluate the effect of antioxidant (type and level) at a fixed stabilizer level, the storage stability of F2, F12, F13, F14 and F15 was compared. Results in Table 18 show that there were small differences in storage stability with regards to acidic species formation of the Fc multimer molecule (CSL730) in the presence versus absence of methionine as an antioxidant at both 25° C. and 40° C. Similar to observations with aggregation and fragmentation stability, more instability with respect to acidic species formation was observed in formulations containing reduced GSH. A systematic increase in the rate constant for formation of acidic species with an increase in reduced GSH content up to 3 months at 25° C. and 2 months at 40° C. Thus, reduced GSH formulations (F14, F15) were eliminated after 3 months of storage stability since stability data by this method and other methods showed sufficient instability of F14 and F15 to warrant their elimination.

[0336] To evaluate the effect of surfactant (PS80) level at a fixed stabilizer level, the storage stability of F2 (0.02% w/v PS80) and F18 (0.04% w/v PS80) was compared. Small differences were observed between the storage stability (acidic species formation) of both formulations at 25° C. and at 40° C.

Example 6—Study of Oxidation Behavior by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) at T0 and Over Time

[0337] All formulations (see Table 10) were analyzed by RP-HPLC for the quantitation of total oxidation and % oxidation in long chain (LC) and short chain (SC) at T0, as described above. FIG. 9 shows representative chromatograms for the oxidation profile of the Fc multimer molecule (CSL730) in a sample formulation. FIG. 9 shows the elution of SC oxidized species followed by SC, then LC oxidized species followed by LC. The chromatogram profile also contains SC and LC fragments, as well as SC and LC N-tertiary pyruvate. Placebo samples were also run by the RP-HPLC method and no interference from placebo ingredients with protein peaks was observed in the chromatograms (data not shown). This was especially important to run for the formulations with reduced GSH.

[0338] FIG. 10 compares the initial (T0) results for % SC oxidized species, % LC oxidized species and relative area (%) for total oxidation for all formulations. % acidic species at T0 for most formulations were comparable. Starting levels of % SC oxidized species, % LC oxidized species and relative area (%) for total oxidation for most formulations were comparable. On the other hand, higher levels of oxidation were observed by all measures in formulations with reduced GSH (F14 and F15) (also see Table 19). % oxidized species (SC and LC) and total % oxidation increased at T0 as reduced GSH levels increased.

[0339] % SC oxidized species, % LC oxidized species and relative area (%) for total oxidation for all formulations were monitored using the RP-HPLC method over time at different temperatures. The rate constants for total oxidation, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described before. The results are also summarized in Table 19.

[0340] A few select formulations were analysed for up to 6 months at 2-8° C. and at 25° C. Oxidation analysis of some formulations was discontinued after only 2 months at 25° C. (based on their performance at 40° C.). Analysis of all formulations was discontinued after 2 months of storage at 40° C. since oxidation reached levels high enough to enable the identification and elimination of the worst performing formulations. In general, % unoxidized SC and unoxidized LC were observed to decline with time along with a concurrent increase in oxidized SC and % oxidized LC species. Overall, the total % oxidation in the different formulations increased at varying rates with time and temperature, as demonstrated in FIG. 11.

[0341] Table 19 shows that for formulations tested for oxidation after 6 months of storage at 2-8° C., they showed small increases in total % oxidation. However, the formulation containing arginine as the principle stabilizer (F5) showed the most oxidation after 6 months of storage—F14 and F15 not included at 2-8° C.

[0342] As expected, results in Table 19 show that the rate of oxidation increased with storage temperature. Peculiarly, the increase in the rate of oxidation at 25° C. and at 40° C. was very slight in presence of 10 mM reduced GSH (F15). This could possibly indicate that at high levels of GSH in the system studied (F15), oxidation becomes less temperature dependent and driven more by GSH concentration thus resulting in an alteration of oxidation kinetics.

[0343] To evaluate the effect of stabilizer type, the storage stability of F1-F7 was compared. Table 19 results show that the Fc multimer molecule (CSL730) exhibited better stability to oxidation in the presence of polyols/sugars at 25° C., as compared to amino acids. The outcome at 40° C. however, was not consistent with that at 25° C. Thus, no trend can be concluded from the results. Considering assay variability at the levels of oxidation measured, differences between the formulations would be considered small.

[0344] To evaluate the effect of protein concentration at fixed stabilizer level, the storage stability of F2, F10 and F11 was compared for sucrose as a sugar type stabilizer. Results in Table 19 show that the Fc multimer molecule (CSL730) exhibited more stability to oxidation as protein concentration increased at both 25° C. and 40° C. This appeared to be especially true at 40° C. when comparing the stability of the formulation at 10 mg/mL protein (F11) against the formulations at 100 mg/mL and 70 mg/mL protein (F2 and F10, respectively). F2 and F10 were 2.4-2.8 times more stable than F11 at 40° C.

[0345] To evaluate the effect of stabilizer levels at fixed protein concentration (100 mg/mL), the storage stability of F2, F8 and F9 was compared for sucrose as a sugar type stabilizer. Stabilizer levels ranged from 200-300 mM. Results in Table 19 show that the Fc multimer molecule (CSL730) exhibited slight differences in stability with regards to oxidation between the three formulations at different sucrose levels at both 25° C. and 40° C. The same outcome held true in the presence of proline as a stabilizer at a level of 200 mM and 250 mM at both 25° C. and 40° C.

[0346] To evaluate the effect of antioxidant (type and level) at a fixed stabilizer level, the storage stability of F2, F12, F13, F14 and F15 was compared. Results in Table 19 show small but systematic improvements in stability to oxidation with increasing levels of methionine at 25° C. (F12 and F13), as compared to its absence. A systematic but more significant improvement in stability to oxidation was observed at 40° C. with increasing levels of methionine. Formulations containing reduced GSH exhibited the poorest stability to oxidation, in contrast to formulations containing methionine (or even other formulations without antioxidants) (also see FIG. 11). A non-linear increase in the oxidation rate constant with an increase in reduced GSH content was observed for up to 2 months of storage at both 25° C. and 40° C. At both temperatures, just 1 mM GSH resulted in 4.7-9.3 times more oxidation that its counterpart formulations with methionine at both 25° C. and 40° C. Thus (as with other methods discussed previously), reduced GSH formulations (F14, F15) were eliminated based on only 2 months of oxidation storage stability.

[0347] To evaluate the effect of surfactant (PS80) level at a fixed stabilizer level, the storage stability of F2 (0.02% w/v PS80) and F18 (0.04% w/v PS80) was compared. No measurable differences were observed between the storage stability (oxidative) of both formulations at 40° C.

Conclusions of Part 2

[0348] Formulation of a stable liquid DP of CSL730 at high concentration (≥100 mg/mL) is feasible [0349] Stable to aggregation, acidic species formation, fragmentation and oxidation. [0350] Factors contributing to stabilization of Fc multimer molecules, especially at high protein concentrations [0351] Lower pH (5-5.8) [0352] Use of disaccharides/polyols as stabilizers, and to a lesser extent amino acids [0353] Optimum molar ratio for protein stability is a protein-to-sucrose ratio of 1:420. Stability also achieved with lower protein-to-sucrose ratios. Formulation robustness increases above that ratio. [0354] Antioxidants may be required to prevent yellowing and/or side oxidation reactions that may occur due to other excipients during storage, or to protect against specific manufacturing conditions (e.g. light exposure, exposure to vaporized hydrogen peroxide residual from sterilization of filling machines in isolators) or unusual handling conditions during clinical administration. [0355] Higher PS80 levels may not be needed for storage stability but may be needed to ensure stability during manufacturing and clinical handling/administration.
Part 3 Stabilizer Screening Study at 120-160 mg/ml Protein Conc.

[0356] A follow up study to the excipient screening studies was performed to investigate the effect of stabilizer type (stabilizers of interest based on Part 2 were: sucrose, trehalose and proline) in the presence of 20 mM Histidine, 0.02% w/v polysorbate 80 and 10 mM methionine on liquid formulations comprising an Fc multimer molecule (CSL730) at high protein concentrations. Protein concentration was varied but molar ratio of protein to stabilizer was fixed at approximately 1:420. pH of all formulations was fixed at 5.25±0.1. Overall, CSL730 formulations with protein concentrations ranging from 120-160 mg/mL were prepared and investigated for protein stability. Formulations prepared for this study are summarized in Table 20.

TABLE-US-00014 TABLE 20 Summary of formulations prepared for the follow-up study at higher protein conc. Target Target CSL730 PS80 Stabilizer Antioxidant Formulation conc. conc. Target Stabilizer Conc. type and Molar ratio No. (mg/mL) (% w/v) pH type (mM) conc. (mM) (protein:stabilizer) F1 120 ± 5 0.02 5.25 ± 0.1 Sucrose 305 10 mM 1:417 Methionine F2 160 ± 5 0.02 5.25 ± 0.1 Sucrose 380 10 mM 1:417 Methionine F3 120 ± 5 0.02 5.25 ± 0.1 Trehalose 305 10 mM 1:417 Methionine F4 160 ± 5 0.02 5.25 ± 0.1 Trehalose 380 10 mM 1:417 Methionine F5 120 ± 5 0.02 5.25 ± 0.1 Proline 305 10 mM 1:417 Methionine F6 160 ± 5 0.02 5.25 ± 0.1 Proline 305 10 mM 1:417 Methionine

Methods Used for Analyzing the Stability of the Formulations:

[0357] Stability indicating methods were used to analyze the formulations at the different time points namely: Size exclusion chromatography (SEC), Cation exchange chromatography (CEX), Capillary Gel Electrophoresis (CGE) (Caliper) and reverse phase HPLC (RP-HPLC) for oxidation. In addition, pH, visual appearance (for color, turbidity and visible particles) and subvisible particles (SVP) of the formulations were monitored at the different time points.

[0358] Methods used for the study and purpose of each method are summarized in Table 21. Storage conditions and analysis time points up to now are summarized in Table 22.

TABLE-US-00015 TABLE 21 Analytical methods used for stability studies of formulations and their purpose Method Purpose Visual inspection Visible particles, colour, opalescence, turbidity Dynamic Imaging Particle Analysis Sub-visible particle count (DIPA) pH measurement pH testing Osmolality Osmolality measurement Polysorbate 80 (PS80) testing Quantitation of PS80 UV spectroscopy Protein content (A280) Size exclusion chromatography (SEC) High molecular weight species (soluble aggregates) Cation exchange chromatography Acidic, basic and main species (CEX) Capillary Gel Electrophoresis Low molecular weight species (CGE) (Caliper)

TABLE-US-00016 TABLE 22 A summary of storage conditions and analysis time points Storage condition Time points 2-8° C.  0, 1, 3, 6, 9, 12 months 25° C. 0, 1, 2, 3, 6, 9, 12 months 35° C. 0, 1, 2, 3, 6 months

[0359] The analytical methods were carried out as described in Part 1 and Part 2 above.

Example 1—Visual Appearance at T0 and Over Time

[0360] All filled vials were examined by visual appearance after fill/finish (100% visual inspection) by two inspectors (Insp 1 and Insp 2, as described above) and at each time point and temperature. There was a general alignment between examiners in most cases.

[0361] All formulations possessed a brown yellow (BY) colour with slight opalescence due to high protein concentration. 0-1 visible particles were observed randomly in some formulations and placebos. A close examination, with knowledge about the protein, revealed that the visible particles were exogenous in nature (probably introduced during the fill/finish process). In spite of high viscosity, none of the formulations were found to be gelled.

[0362] Formulations 1 to 6 (see Table 20) were visually inspected for up to 12 months at different temperatures. The color of all formulations remained unchanged at all storage temperatures from T0 (BY). Clarity remained unchanged (slightly opalescent). Particle counts remained unchanged from T0 (0-1), regardless of temperature and time. None of the formulations gelled during the storage time period at any temperature.

Example 2—Osmolality, pH, Protein Concentration and Polysorbate 80 Measurements at T0 and Over Time

[0363] Osmolality, pH, protein concentration and polysorbate 80 (PS80) concentration measurements on all formulations 1-6 (see Table 20) were conducted at the initial time point (T0). The results are summarized in Table 23.

[0364] The results in Table 23 show that protein concentration was within 120±5 mg/mL for F1, F3 and F5 and that protein concentration was within 160±5 mg/mL for F2, F4 and F6. pH measurements showed that the measured pH values were within ±0.1 pH units for most formulations in comparison to the target pH-values. A slightly higher deviation in pH was observed with F6 (within 0.17 units). Osmolality was generally within 382-414 mOsm/kg for formulations at 120 mg/ml whereas osmolality was generally higher (529-551 mOsm/kg) for formulations at 160 mg/ml. PS80 concentrations in all formulations were within a maximum of ±0.003% w/v of the PS80 target concentrations.

[0365] The pH and protein concentrations were monitored in formulations 1 to 6 (see Table 20) at 2-8, 25 and 35° C. for up to 12 months. Within experimental error, results showed minimum shifts (within a maximum of ±0.1 units) in pH at all temperatures for all formulations within the time frame measured. Also within experimental error, results showed small shifts in protein concentration with time.

TABLE-US-00017 TABLE 23 Measured protein concentration, pH, osmolality and polysorbate 80 (PS80) concentration for all formulations at initial time point (T0) Target Measured protein protein Measured PS80 Form. conc. conc. Target Measured Osmo. conc. Code (mg/mL) (mg/mL) pH pH (mOsm/kg) (% w/v) F1 120 122 5.25 5.26 414 0.018 F2 160 160 5.25 5.30 551 0.017 F3 120 124 5.25 5.33 403 TBD F4 160 159 5.25 5.38 549 0.017 F5 120 121 5.25 5.36 382 0.021 F6 160 159 5.25 5.42 529 0.019

Example 3—Study of Aggregation Behavior (High Molecular Weight Species) by Size Exclusion Chromatography at T0 and Over Time

[0366] All formulations 1-6 (see Table 20) were analyzed by size exclusion chromatography for the quantitation of high molecular weight (soluble aggregates) species at T0, as described above.

[0367] Initial (T0) % HMW for all formulations are presented in Table 24. The aggregation rate constants, along with the corresponding standard error (SE), were determined for all formulations at 2-8, 25 and 35° C. by multiple linear regression as described before. The results are also summarized in Table 24.

[0368] As shown in Table 24, initial (T0) soluble aggregates levels (% HMW) were comparable and low (within 0.8-1% HMW) in spite of high protein concentrations.

[0369] Analysis of all formulations was conducted up to 12 months of storage at 2-8° C., 25° C. and up to 6 months of storage at 35° C. As expected, results in Table 24 show that the rate of aggregation increased with storage temperature. Within the temperature ranges for the stability study, for every 10° C. increase in temperature, aggregation rate increased by up to 2 times.

[0370] The stability of the Fc multimer molecule (CSL730) at higher protein concentration (160 mg/ml) was on average 1.3 times less stable at 2-8° C., 25° C. and 35° C. as opposed to the same formulation at lower protein concentration (120 mg/ml).

[0371] For up to 3 months at fixed molar ratio of approximately 1:420, all stabilizers appeared to perform similarly with respect to stabilizing the Fc multimer molecule (CSL730) at all temperatures studied. This was also confirmed to be true up to 12 months.

TABLE-US-00018 TABLE 24a Summary of SEC data for aggregation (% HMW) comparing initial (T0) % HMW and aggregation rate constants k (±SE) at 2-8° C., 25° C. and at 35° C. for 3 months Δ at 2-8° C. Form. after T = 3 Code T0 month k.sub.2-8° C. k.sub.25° C. k.sub.40° C. F1 0.8 0.7 0.06 ± 0.01 0.15 ± 0.03 0.30 ± 0.05 F2 0.9 0.8 0.06 ± 0.01 0.21 ± 0.04 0.40 ± 0.07 F3 0.9 0.7 0.05 ± 0.01 0.16 ± 0.03 0.30 ± 0.05 F4 1.0 0.9 0.06 ± 0.01 0.21 ± 0.04 0.40 ± 0.10 F5 0.8 0.7 0.06 ± 0.01 0.15 ± 0.03 0.27 ± 0.05 F6 0.9 0.9 0.07 ± 0.02 0.20 ± 0.04 0.36 ± 0.08

TABLE-US-00019 TABLE 24b Summary of SEC data for aggregation (% HMW) comparing initial (T0) % HMW and aggregation rate constants k (±SE) at 2-8° C. and 25° C. for 12 months and at 35° C. for up to 6 months Δ at 2-8° C. Form. after T = 12 Code T0 month k.sub.2-8° C. k.sub.25° C. K.sub.35° C. F1 0.8 1.86 0.15 ± 0.01 0.30 ± 0.04 0.98 ± 0.10 F2 0.9 2.07 0.17 ± 0.02 0.40 ± 0.05 1.59 ± 0.28 F3 0.9 1.79  0.14 ± 0.011 0.31 ± 0.04 0.97 ± 0.11 F4 1.0 2.37 0.19 ± 0.01 0.39 ± 0.06  (2.45 ± 0.14)* F5 0.8 1.79 0.15 ± 0.01 0.28 ± 0.04 0.94 ± 0.10 F6 0.9 2.40 0.19 ± 0.02 0.36 ± 0.05 1.14 ± 0.15 *Rate estimate not representative. Not enough sample available to perform time points >2 months

[0372] F1 to F6 all showed good stability for at least 12 months. At 4° C., all 6 formulations contained less than 3.5% HMW species after 12 months; at 25° C., all 6 formulations contained less than 6.5% HMW species.

Example 4—Study of Fragmentation Behavior (Low Molecular Weight Species) by Non Reducing Capillary Gel Electrophoresis (NR-cGE) at T0 and Over Time

[0373] All formulations (see Table 20) were analyzed by non-reducing cGE using Caliper (NR Caliper) for the quantitation of low molecular weight (LMW) species (i.e., fragmentation) at T0, as described above.

[0374] Initial (T0) % LMW species for all formulations are presented in Table 25. As shown in Table 25, % LMWs at T0 for all formulations were comparable. The same outcome was observed with LMWs as measured by the SEC method at T0 (measured % LMWs in all formulations were in the range of 2.6-2.7%).

[0375] % LMW species for all formulations was monitored using the NR-cGE “Caliper” method over time at different temperatures, initially for 3 months, but now continued for 12 months. The rate constants for fragmentation, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described before. The results are summarized in Table 25.

[0376] As expected, results in Table 25 show that the rate of fragmentation increased with storage temperature. Small fluctuations in fragmentation (within assay error) occurred in all formulations studied at 2-8° C. with time, as evidenced by small changes in % LMW for up to 12 months. However, for a 10° C. increase in storage temperature (from 25° C. to 35° C.), the rate of fragmentation increased on average by 3.6 to 4 times, regardless of protein concentration. A similar outcome was observed with rate of fragmentation as measured by the SEC method (results not shown). With the SEC method, the rate of fragmentation increased on average by 3.5 times for a 10° C. increase in storage temperature (from 25° C. to 35° C.).

[0377] At the same storage temperature, no differences were observed between the stability of the Fc multimer molecule (CSL730) at higher protein concentration (160 mg/ml) as opposed to the same formulation at lower protein concentration (120 mg/ml). That is, there were no significant differences between stability of the formulations at both 25° C. and 35° C. as a function of protein concentration. The same outcome was observed with LMWs as measured by the SEC method (results not shown).

[0378] For up to 12 months at a fixed molar ratio of approximately 1:420, all stabilizers appeared to perform similarly with respect to stabilizing the Fc multimer molecule (CSL730) against fragmentation at 2-8° C., 25° C. and at 35° C.

TABLE-US-00020 TABLE 25a Summary of data from NR-cGE from “Caliper” method for fragmentation (% LMW) comparing initial (T0) % LMW, changes in fragmentation at 2-8° C. after 3 months, and fragmentation rate constants k (±SE) at 25° C. and at 35° C. for 3 months of storage time. Δ at 2-8° C. Form. after T = 3 k.sub.25° C. K.sub.35° C. Code T0 months (T = 3 months) (T = 3 months) F1 2.8 0.3 0.12 ± 0.01 0.36 ± 0.04 F2 2.8 0.5 0.12 ± 0.01 0.40 ± 0.01 F3 2.8 0.3 0.12 ± 0.04 0.40 ± 0.02 F4 2.8 0.2 0.13 ± 0.04 0.40 ± 0.01 F5 2.8 0.5 0.11 ± 0.04 0.42 ± 0.03 F6 2.7 0.2 0.12 ± 0.04 0.41 ± 0.01

TABLE-US-00021 TABLE 25b Summary of NR-cGE data from “Caliper” method for fragmentation (% LMW) comparing initial (T0) % LMW and fragmentation rate constants k (±SE) at 2-8° C. and 25° C. for 12 months and at 3 5° C. for up to 6 months Δ at 2-8° C. Form. after T = 12 Code T0 month k.sub.2-8° C. k.sub.25° C. K.sub.35° C. F1 2.8 0.4 ≤0.05 0.48 ± 0.03 1.74 ± 0.12 F2 2.8 0.2 ≤0.05 0.45 ± 0.04 1.60 ± 0.03 F3 2.8 0.2 ≤0.05 0.43 ± 0.03 1.74 ± 0.06 F4 2.8 0.4 ≤0.05 0.41 ± 0.03  (2.40 ± 0.40)* F5 2.8 0.2 ≤0.05 0.44 ± 0.03 1.74 ± 0.05 F6 2.7 0.3 ≤0.05 0.46 ± 0.03 1.83 ± 0.06 *Rate estimate not representative. Not enough sample available to perform time points >2 month

Example 5—Study of Acidic and Basic Species by Cation Exchange Chromatography (CEX) at T0 and Over Time

[0379] All formulations (see Table 20) were analyzed by CEX for the quantitation of % acidic species and basic species at T0, as described above.

[0380] Initial (T0) % acidic species for all formulations are presented in Table 26. As shown in Table 26, % acidic species at T0 for all formulations were comparable. Similarly, % basic species at T0 for all formulations were also comparable (3.5-3.7%).

[0381] % acidic species for all formulations was monitored using the CEX method over time at different temperatures (up to 12 months). The rate constants for formation of acidic species, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described before. The results are summarized in Table 26.

[0382] As expected, results in Table 26 show that the rate of formation of acidic and basic species increased with storage temperature. For up to 12 months at 2-8° C., no changes were observed in acidic species formation for any of the formulations studied. However, for a 10° C. increase in storage temperature (from 25° C. to 35° C.), the rate of acidic species formation increased on average by 3.1-3.4 times.

[0383] Results in Table 26 show that the stability of the Fc multimer molecule (CSL730) at lower protein concentration (120 mg/ml) was slightly better at a storage temperature of 25° C. than the same formulation at higher protein concentration (160 mg/ml).

[0384] Results in Table 26 also show that at a fixed molar ratio of approximately 1:420, stability of Fc multimer molecule (CSL730) with regards to acidic species formation in the presence of sucrose, trehalose or proline at 25° C. was comparable. Results at 35° C. are less decisive due to fewer samples for analysis at that temperature.

TABLE-US-00022 TABLE 26 Summary of data from CEX for % acidic species comparing initial (T0), changes in % acidic species at 2-8° C. after 3 months, and rate constants for formation of acidic species k (±SE) at 25° C. and at 40° C. for 3 months of storage time Δ at 2-8° C. Form. T0 after T = 12 Code (Acidic) month k.sub.25° C. K.sub.35° C. F1 56.9 0.1 1.37 ± 0.03 4.72 ± 0.46 F2 56.8 0.0 1.33 ± 0.04  (5.63 ± 0.31)* F3 56.9 0.1 1.40 ± 0.03 4.57 ± 0.51 F4 56.8 0.0 1.32 ± 0.05  (8.65 ± 0.78)* F5 56.9 0.4 1.54 ± 0.04 4.80 ± 0.58 F6 56.9 0.0 1.44 ± 0.04 4.68 ± 0.55 *Rate estimates not representative. Not enough sample available to perform time points >2-3 months

[0385] No significant differences were observed among the 6 formulations for % basic species for the different time points and temperatures.