TW200909005A - Lyophilized immunoglobulin formulations and methods of preparation - Google Patents

Abstract

The present invention relates generally to the field of pharmaceutical formulation of immunoglobulins. Specifically, the present invention relates to stable, lyophilized, high concentration immunoglobulin formulations. This invention is exemplified by a stabilized lyophilized formulation of the recombinant humanized anti-alpha-4 integrin antibody natalizumab.

Description

200909005 IX. Description of the invention: [Technical field to which the invention pertains] The present invention generally relates to the field of pharmaceutical dispensing of immunoglobulins. The present invention relates to a stable, freeze-dried, high concentration immunoglobulin: a formulation. The present invention is exemplified by a stable cold-dried formulation of a recombinant humanized anti-integrin antibody, natalizumab. [Prior Art]

Drugs intended to be administered to humans (4) may require stabilizers to prevent changes in the drug prior to use. Because protein f is larger and more complex than traditional organic and inorganic drugs (i.e., in addition to complex three-dimensional structures, i white matter also has a variety of functional groups), the family has a special problem with protein blending. Egg: The degradation pathway of the mass may involve chemical instability (any method involving modification of the protein by bond formation or cleavage of the chemical entity of the line) or physical instability (changes in the higher order structure of the protein). Chemical instability can be caused by deamination, racemization, hydrolysis, oxidation, cleavage or disulfide exchange. Physical instability can be caused, for example, by denaturation, aggregation, or absorption. Many protein preparations are particularly unstable at very dilute or high concentrations, and this instability often increases when storing or transporting protein f preparations. Therefore, the main challenge in the field of protein drugs is to develop formulations that maintain protein stability and activity. Antibodies (including monoclonal antibodies) differ in their manner in terms of their behavior and efficacy in the formulation. For example, the aggregation of individual antibodies with respect to isoelectric point, solubility, and individual antibodies differs from each other. Proteins vary in behavior and efficacy in the depletion, making it difficult to predict whether a compound is stable for a particular antibody. Three common problems in protein formulations include protein degradation, aggregation, dealumination and oxidation. In addition, many different reactions that affect the stability of the formulation can be performed simultaneously, making it difficult to determine what kind of reaction causes the results. See Cleland et al., "The Development of Stable Protein Formulations: A Close Look at Protein Aggregation, Deamidation, and Oxidation", Critical Reviews in Therapeutic Drug Carrier Systems, 10(4): 307-377 (1993)) = [Invention Contents] In particular, the present invention relates to stable, freeze-dried, high concentration immunoglobulin formulations. There are three steps in the preparation of the formulations of the present invention, including the preparation of a pre-freeze-dried aqueous formulation, a freeze-drying step, and a rehydration step. The present invention is directed to a stable cold-prepared formulation prepared by freeze-drying an aqueous formulation, wherein the aqueous formulation comprises from about 40 mg/ml to about 0.4 mg/ml in buffer, polysorbate and sucrose. 50 mg/ml immunoglobulin. In a preferred embodiment of the invention, the pre-lyophilized aqueous formulation comprises (4) from about 30 mg/ml to about 60 mg/ml natalizumab; (b) having a pH of from about 5.5 to about 6.5 a buffer; (c) from about 20 mg/ml to about 5 mg/ml sucrose; and from about 0.2% to about 8% polysorbate. In a more preferred embodiment, the pre-lyophilized aqueous formulation comprises (4) about 4 mg/ml natalizumab; (b) about 6 mM histidine, pH about 6.0; (c) about 41 Mg/ml sucrose; and (d) about 0.04% polysorbate 8 〇. The cold bead-dried formulation maintains the stability of the immunoglobulin and prevents the formation of aggregates and/or microparticles in the final product by immunoglobulins intended to be administered to human subjects. The freeze-dried formulation is stable for at least three months at room temperature, preferably six [and more preferably one year. The freeze-dried formulation is also stable at 2-8 ° C for 1 year, preferably 2 years. The cold-dried formulation has a short rehydration time of less than 1 minute' and is suitable for parenteral administration after reconstitution, such as intramuscular, subcutaneous, intravenous or intraperitoneal injection. The lyophilized formulation was reconstituted with a liquid to a clear solution containing an immunoglobulin concentration of about 8 〇 16 〇 mg/mi. In a preferred embodiment, the recombinant formulation comprises (1) from about 80 mg/ml to about 160 mg/ml natalizumab; (π) about 18 mM histidine having a pH of about 6.0; ) about 123 mg/ml sucrose; and (!v) about 0.12% polysorbate to brew 8 〇 β. In a more preferred embodiment, the recombinant formulation comprises about 120 mg/ml natalizumab. The pre-lyophilized formulation of the present invention can be lyophilized using suitable drying parameters. The following drying parameters are preferred: about _25. The first dry stage temperature and a pressure between about 80 mTorr and about 120 mTorr; and a second drying stage at about 20 C and a pressure between about 80 mTorr and 120 mTorr. The invention further provides methods for making and using recombinant formulations. [Embodiment] 1. Definitions As used herein, the term 'important immunoglobulins' includes, but is not limited to, antibodies and antibody fragments (such as SCFV, Fab, Fc, (Fab,) 2), and other genetic engineering of antibodies. According to the amino acid sequence of the heavy chain constant domain, the immunization 132080.doc 200909005 globulin can be divided into different types. There are five main immunoglobulin species:

IgA, IgD, IgE, IgG and IgM. Some of these classes can be further divided into subclasses (isotypes) such as IgG1, IgG2, lgG3, and IgG4; IgAl and IgA2. The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including agonists and antagonist antibodies), k-body compositions with multiple epitope specificity, and antibody fragments (eg, Fab, (Fab, 2, scFv and Fv), as long as it exhibits the desired biological activity. "Antibody" is intended to include a plurality of antibodies, early strain antibodies, humanized antibodies, human antibodies, primatized antibodies, and other antibodies produced by genetic engineering. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a modification or glycosylation variant that may be naturally occurring, except in the presence of trace amounts, comprising the population The individual antibodies are identical. The modifier "single" indicates the characteristics of the antibody as obtained from a population of substantially all of the antibodies, and should not be construed as requiring production of the antibody by any particular method. The term "monoclonal antibody" " Also includes:, chimeric "antibody (immunoglobulin)' wherein one of the heavy and/or light chains is associated with a corresponding species (4) derived from a particular species or antibody belonging to a particular antibody class or subclass. Source, and 4 hai, etc.) the remainder of the strand is homologous or homologous to the corresponding sequence derived from other species or antibodies belonging to other antibody classes or subclasses, and fragments of such antibodies, as long as they exhibit the desired biological activity The "humanized" form of antibodies to non-humans (eg, 'family rabbits, porcines, and their analogs) is a chimeric immunosphere containing minimal sequence derived from non-human immunoglobulins. a protein, an immunoglobulin chain or a fragment thereof (such as Fv, Fab, Fab, 132080.doc 200909005 F(aiy) 2 or other antigen-binding sequence of an antibody). The term "natazumab" An antibody called AN1 00226 (antibody coding number) and is the active ingredient in TYSABRI® (trade name; formerly ANTEGREN (g)). The term "natazumab " is the United States adopted name Name, USAN) (the official non-proprietary or generic name given to the drug substance). natalizumab is a recombinant, humanized anti-integrin antibody. natalizumab is an IgG4 antibody. U.S. Patent No. 5,84,G,299 describes how to use the f-synthesis and molecular biology methods to produce recombinant humanized anti-α_4 integrin antibodies including natalizumab. The term "cold; Donggan (four) y〇Phuization, lyophilized , Plus 2 is a day - the first to cool the material to be dried; and then by the method of sublimation in a vacuum environment to remove ice or cold solvent. One or more excipients are included in the pre-cooled; East Dry formulation to enhance the stability of the cold-dried product after storage. "Human pharmaceutical formulation" means a formulation which is in a manner which allows the active ingredient to be effective and which is not toxic to the individual to which the formulation is administered. J. Le Er, accepts "excipients (vehicles, additives) It is the time required for the appropriate dosage of the active ingredients to be used to provide an effective dose of the active ingredients, which is the time required to cool the solution with the solution; and the solution is clear. The particles are "stable" and the formulation is a formulation in which the protein and/or chemical stability and/or post-storage retention of the physical activity of the substrate is maintained. Various analytical techniques for measuring protein stability in 132080.doc -10. 200909005 can be used in this technology and in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, Marcel Dekker, Inc" New York, NY , Pubs. (199i) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). The stability can be measured at the selected temperature for the selected period. "Stability" The freeze-dried immunoglobulin formulation is for at least 12 months, preferably 2 years and more preferably 3 years at freezing temperature (2-8 ° C); or at least 3 at room temperature (23-27 C) Freeze-dried antibody formulations with no significant changes observed in months 'better 6 months and better. The stability criteria are as follows. No more than 1% of antibody monomer degradation as measured by SEC-HPLC Preferably, no more than 5% of the antibody monomer is degraded by SEC-HPLC. By visual analysis, the recombinant solution is colorless or clear to slightly milky white. Concentration, pH and penetration of the formulation. The pressure has a change of no more than +m〇%. The efficiency is within 7〇_13〇% of the control group and preferably in MM· It is observed that the shortening is not more than (4). Preferably, no shortness is observed. Formation of no more than 10% aggregation. Preferably, the formation does not exceed if the immunoglobulin is displayed in a visual inspection color and / or Clarity or as measured by UV light scattering, size exclusion chromatography (SEc) and dynamic light scattering, no significant increase in sedimentation, and/or denaturation, in pharmaceutical formulations": physical stability Changes in the white matter configuration can be assessed by fluorescence spectroscopy for determining the secondary structure of the protein and by measuring protein FTIR spectroscopy. If the immunoglobulin shows no significant chemical changes, then it is in the pharmaceutical formulation 132080.doc 200909005 t" maintain its chemical stability ". And 総 η wrong by the beta test and quantify the chemical changes of the protein to assess the chemical stability * buy chemical degradation 汛 mo - 4 book hang changes the chemical structure of the degradation of the hydrazine including hydrolysis or shearing (by PAGE ^ (by Such as size exclusion chromatography and SDS-Λ, Λ (by peptide mapping combined with mass spectrometry or MALDI/TOF/MS methods to gamma, '^ μ car.), deamidamine (by ion exchange) Chromatography, capillary isoelectric focusing clip, Η田* Wu Tiandong acid measurement method °) and isomerization (by measuring the isokinetic acid ^ estimated), makeup 3, peptide map, etc. To evaluate the immunoglobulins at a specific time. Β The biological activity of the poor white organisms is predicted by the biological activity exhibited in the preparation of the pharmaceutical formulation. Its biological activity to determine the biological activity of immunoglobulins, for example, can be hunted by the antigen binding test term: isotonic, meaning that the formulation of interest has a permeation pressure that is substantially visible to human blood. Isotonic formulation Generally will have an osmotic pressure of about 27 〇 328 (10). Slightly low The pressure is 25〇_269 and the slight hyperosmotic pressure is mho m〇Sm. For example, vapor pressure or ice-cold beam type permeation pressure can be used. ☆ The term 'buffer agent' is included to keep the solution staff before cold drying. These reagents may be included in the range of AI and may include histidine, succinate (sodium or potassium), phosphate (nano or potassium), Tris (paraxylmethyl)aminopyramine, diethanolamine , citrate (sodium), gluconate and other organic acid buffers. "Tensile regulator" includes salts that can be used to control osmotic pressure, such as Naci, gCl2 CaCl2, etc. In addition, 'Cryogenic protectant, bundle dry A protective agent and/or a bulking agent such as Yan sugar, mannitol, glycine, etc. can be used as a force regulator for Zhang 132080.doc 12 200909005. In the context of the present invention, immunoglobulin "therapeutically effective amount" Means the amount of a condition effective to prevent or/or therapeutically treat immunoglobulin. "The condition," is any condition that will benefit from treatment with immunoglobulin. This includes chronic and acute conditions or illness #, Including the pathology that predisposes the mammal to the condition Shaped Preferably, the disorder may be an immunoglobulin, and in connection with the recognition by α · 4 integrin (such as natalizumab) treatment and / or prevention of disease symptoms.

"Treatment" means therapeutic treatment and preventive measures. Those in need of treatment have already had symptoms and those who want to prevent it

"Preservative" is a compound that is included in the formulation to substantially reduce the action of the bacteria therein, thereby contributing to, for example, a multi-purpose formulation. Examples of possible preservatives include gasification of 18-yard dimethyl Dietary money, quaternary ammonium chloride hexahydrochloride, benzylammonium chloride (a mixture of alkyl benzyl dimethyl ammonium chloride, in which the base is a long-chain compound) and sulphonic chlorine. Other types of preservatives Including aromatic alcohols such as hexane, butyl alcohol and stilbene; hydrazine for benzoic acid, 4 such as (tetra) benzene f-acid ketone or benzoic acid; m-benzoic acid; cyclohexanol;戍 ; ; ; ; 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。. "Mammal" for therapeutic purposes refers to any animal classified as mammals, but not limited to humans, domestic animals and farm animals, and = animals or pet animals, such as dogs, sports, mammals And similar animals. The animal is a human. Preferably, the mammal or the condition is regulated when the fisherman's painful disease is treated with an effective dose of natalizumab. 132080.doc •13· 200909005 Disease Or conditions. 2. Immunoglobulin Formulations The compositions of the present invention minimize the formation of aggregates and microparticles and ensure that the immunoglobulins in solution maintain their immunoreactivity over time. The compositions comprise sterile, pharmaceutical A pharmaceutically acceptable freeze-dried formulation comprising: a freeze-dried aqueous formulation comprising a buffer having neutral or acidic pH (about 5-5 to about 6.5), immunoglobulin in sucrose and polysorbate. In a preferred embodiment, the immunoglobulin is at a concentration of from about 3 mg/mi to about 6 mg/ml, more preferably from about 4 to about 5 mg/ml, and even more preferably about mg/ml2. Pre-freeze-dried formulation Preferably, the immunoglobulin is an IgG antibody' more preferably Ig (J4 antibody, even more preferably a humanized recombinant morphine 4 antibody, and most preferably natalizumab. pH is used in the pre-freeze-dried formulation) Preferably, the buffer is about 5 5 to about 65. The pH is about 6.0. Examples of suitable buffers include histidine butanoate (such as sodium succinate) 'gluconate, Preferably, the pre-freeze-dried formulation contains histamine, preferably from about 1 to about 2 mM histidine. Even better pre-freeze-dried formulations contain about 6 mM. Histidine. The pre-lyophilized formulation also comprises sucrose. Suitable concentrations of sucrose are in the range of from about 2 mg/ml to about 5 mg/ml, preferably about ο mg/ml. The formulation also contains polysorbates such as polysorbate or polysorbate 80 (i.e., Tween 20 and Tween 8 respectively) and poloxamer (P〇l〇xamer). (e.g., poloxamer 188). In a preferred embodiment of 132080.doc -14-200909005 'polysorbate vinegar is poly _80β $ sorbitol 黯 preferably in parent volume The indifference is about 0.02 to about 0 08%, and more preferably about 〇〇4. The pre-cooled dry towel is placed in the immune ball and the egg is from about 2:1 to about 0.5.1. Preferably, it is about 1:1. The molar ratio of immunoglobulin* to sugar is about 3:1 to about 5:1, to 500:1, more preferably about 450: 1. 丄 to Γ provide a good cold to dry cake characteristics of the expansion:, glycine and mannitol) may be added to the present invention (part 2). These agents also promote the tension of the formulation, and Protect and improve long-term stability. Alternatively, the process provides a barium a + force for 5 weeks. The tanning agent can be added to the formulation to penetrate the house. The formulation may be further comprises - or a plurality of preservatives. Comparison: Pre-cooling: The dry formulation is a formulation comprising about 4 mM natalizumab about 6 mM histidine (about ) value of about 6), sugar. Above::.=Poly: Pear_ and *Dry to form a dry, stable powder 2 = wide cold to human particle-free solution. , Τ - easy to recombine into suitable for lyophilization is often used in pharmaceutical preparation to reduce drying methods. A liquid composition is prepared followed by a biologically active cold cake-like product. The method generally involves the completion of the 'to remove the ice' to leave the powder or "the sample of the cold bundle is cold; the product of the east drying can be stored at a high temperature: long intact:: a complete non-aqueous component. And can be easily recombined into a non-particulate solution by adding appropriate: 2 activity π ping d 132080.doc 15· 200909005 The diluent can be biologically acceptable and the powder of the cold coffee can be fully decomposed therein. Any liquid, water, wood, etc., is a sterile, pyrogen-free basin because it does not include salt or may affect the stability of the antibody. The advantage of cold/east drying is that The reduction in water content to the extent of various molecular events that are unstable to the product after prolonged storage is also less likely to withstand the physical stresses of transport. The recombinant product is free of particles so it can be administered without prior ethics. The pre-cooling of the present invention; the east drying of the object can be adapted to t cold; the east and the drying parameters are used for cold beam drying. For example, the parameters can include a pre-cooled bundle to maintain for about 10-30 minutes at about to about -lot. The freezing parameters may include cooling from the town to the town for a period of from about 45 minutes to about 75 minutes. Other parameters of the cooling step may be included in the cooling to the Russian; East. The drying parameter may include a first-drying stage temperature of from about -10 c to -30 °c and a pressure between about 4 〇mT°fl to about 120 mT〇n·; and from about 10 ° C to about 25 A second drying stage of pressure between about 40 mTGn^12() mT(10) is used at °C. Preferably, the total cycle time is from about 6 Torr to about 1 hr. Preferably, the east drying cycle may include pre-cooling; east step, cold; east step, first-drying step, and first drying step. Considerations for the freeze-drying cycle include freezing temperature, pressure drying, second drying, and cycle time. For example, the preferred freeze-drying cycle parameters can be as follows: First, pre-frozen, held for 15 minutes. For freezing, ramp to 60 ° for 60 minutes (rc. K_6〇t: hold for 60 minutes. In another freezing step, ramp to -5 (TC and hold for 30 minutes. 132080.doc -16 - 200909005 For the first drying, 'transformed to -15 °C in 45 minutes and the pressure dropped to 50 mTorr. Maintained at -15 〇 and 50 mT for 54 hours. For the second drying, after 35 minutes Change to 2 〇 and keep for 24 hours. The total cycle time is 82 hours. This freeze-dried product maintains the stability of the immunological activity of immunoglobulins and prevents immunoglobulins intended to be administered to human subjects in the final product. Physical and chemical degradation. The freeze-dried product is reconstituted in a diluent (for example, sterile water or physiological saline) to obtain a particle-free solution. The recombinant antibody solution is free of particles even in the lyophilized cake. After storage for a long time at a temperature, the recombinant solution can be administered subcutaneously to the individual via sputum, preferably via muscle (4).

4 An important feature of the dried product is that it is important to recombine the time or to recombine the product in order to be extremely fast and completely recombined, so that the cake has a highly porous structure. The structure of the cake varies with a number of parameters including protein concentration, excipient type and concentration, and process parameters for the freeze-drying cycle. In general, when the protein concentration is increased, the recombination time is also strong and therefore, the short recombination time is an important target for developing high-concentration cold-dried antibody preparations. The long recombination time can degrade the product quality by causing the protein to swell for a longer period of time. In addition, at the user end, the product can not be administered until the group f is produced. This will ensure that the product is free of microparticles, is administered with the correct dose, and does not interfere with its sterility. Because of this, rapid reorganization, such as reconstitution time of less than ten minutes, provides more convenience to patients and physicians. In the product of lyophilization, the desired dose can be obtained by lyophilizing the formulation at the target protein concentration and recombining the product in the same volume as the initial filling volume. The desired dose can also be obtained by lyophilizing a larger volume of the diluted formulation and recombining it with a smaller volume. For example, if the desired product dose is (10) mg of protein in the i mL formulation, the formulation can be cooled with the following liquid configuration; East Dry: 1 〇〇mg/mL protein formulation of 丄虹, 2 mL of 50 A mg/mi protein formulation or a 4 mL 225 mg/mL protein formulation. In all cases the final product can be reconstituted with 丄 mL diluent to obtain a target protein concentration of 1 〇〇. However, as the protein concentration in the pre-cooled beam dried formulation decreases, the fill volume increases proportionally. This correspondingly increases the length of the cold; east drying cycle (especially the first drying time) and thus significantly increases the cost of the product. For example, if the cold bundle material of the i mL fill volume (the height of the i-cone in the vial) takes about 1 hour to sublimate the free water, the cold bundle product of the 1 volume (1 coffee height) will be consumed. About 1 () hours - drying time. Therefore, it is preferred to have a concentrated pre-lyophilized formulation (immunoglobulin concentration of from about 40 mg/ml to about 50 mg/mi) such that the freeze-drying process will be more effective. The present invention provides a highly concentrated pre-cooled lyophilized immunoglobulin formulation (about 40 mg/ml to about 5 〇mg/nU) which is efficiently and efficiently freeze-dried to maintain an immune protein. Dry formulation for physical and chemical stability. The dried formulation is stable for at least 3 months, preferably 6 months, at room temperature. The dry formulation can be reconstituted into a particle-free solution of from about 80 mg/mi to about 16 mg/ml immunoglobulin in less than ten minutes. These highly concentrated antibody solutions are intended for parenteral administration, such as intravenous, intramuscular, intraperitoneal or subcutaneous injection. 132080.doc •18· 200909005 Preferred recombinant products comprise from about 80 mg/ml to about 160 mg/ml natalizumab, more preferably about 120 mg/ml natalizumab; about 123 mg/ml sucrose; 0.12% polysorbate 80; and about 18 mM histidine at about pH 6.0. 3. Analytical Methods Analytical methods for assessing product stability include size exclusion chromatography (SEC), dynamic light scattering (DLS), differential scanning calorimetry (DSC), iso-asp quantification, potency, at 340 nm UV, UV spectroscopy and FTIR. SEC (J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 1 1: 485 (1994); J. Pharm. Bio. Anal., 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986)) measures the percentage of monomer in the product and gives information on the amount of soluble aggregates. The potency or bioidentity of an antibody can be measured by its ability to bind its antigen. The specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, such as immunoassays, such as ELISA (Enzyme Linked Immunosorbent Assay). These methods are merely exemplary methods well known to those skilled in the art for assessing product stability. For example, the UV measurement at 340 nm measures the intensity of scattered light at 340 nm and gives information on the amount of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and gives information on protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:23 1 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm. Scien ·, 87:1069 (1998)) Measure the IR spectrum in the decyl ketone region and give information on the secondary structure of the protein. Specific analytical methods are further described in the experimental section (see above). 132080.doc -19- 200909005 4. Use of Recombinant Formulations The recombinant immunoglobulin formulations of the present invention can be administered to mammals in need of treatment with immunoglobulins according to known methods. Such methods may include, but are not limited to, intravenous administration in the form of a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracranial, subcutaneous, intra-articular, intra-sliding, intrathecal Oral, topical or inhalation routes of administration. In a preferred embodiment, the immunoglobulin formulation is administered to a mammal by intramuscular or subcutaneous administration. A typical daily dose may range from about iPg/kg to about 2 〇() mg/kg of individual weight or more preferably from about 0.01 mg/kg to about 15 〇mg/kg of individual weight, more preferably from about 0.1 mg/kg to about 1 〇. 〇mg/kg of individual weight, more preferably from about 1 mg/kg to ',, spoon 75 mg/kg of individual weight and optimally from about 3 mg/kg to about 6 mg/kg of body weight. Usually the physician will administer the immunoglobulin until the dose that achieves the desired effect is achieved. The progress of this therapy can be easily monitored by conventional methods and assays. For example, the appropriate dose of 'immunoglobulin will depend on the condition being treated, the severity and duration of the condition, whether immunoglobulin is administered for prophylactic or therapeutic purposes, prior therapy, clinical history of the patient, and immunity The response of the globulin, the type of immunoglobulin used, and the judgment of the attending physician. Typically, the clinician will administer immunoglobulin until the dose that achieves the desired effect is achieved. The progress of this therapy can be easily monitored by conventional assays. The immunoglobulin is administered to the patient as appropriate or in a series of treatments and can be administered to the patient at any time after the diagnosis has begun. The immunoglobulin can be administered in a single therapeutic form or in combination with other drugs or therapies suitable for treating the condition. As used herein, when two (or more than two) reagents 132080.doc -20- 200909005 are administered simultaneously or [single mode is administered independently so that the material #丨 will function simultaneously, it is said to combine the two reagents. Cast. For example, Benzalizumab# can be used in combination with other therapeutic agents or physic agents for the treatment of rheumatoid arthritis, polymorphism (IV), Crohn's Dis (10) and other alpha phase t diseases. The combination of therapies is administered. The invention is further illustrated by the following examples which should not be construed as limiting the invention to the scope of the specific procedures described. Instance

Example 1 A comparative study of high concentration recombinant freeze-dried formulations and liquid formulations of natalizumab was performed on stone crab macaques. The results of the study show that the recombinant lyophilized formulation of natalizal 1 produces a pharmacokinetic and pharmacodynamic profile that is very similar to the expected formulation of the liquid formulation. - Evaluate high concentration liquid formulations of natalizumab and recombinant freeze-dried formulations to compare their respective pharmacokinetic/pharmacodynamic profiles, relative bioavailability, and subcutaneous (SC) and intramuscular (IM) administration Subsequent local tolerance. On day 1 'therapeutic pharmacokinetics were assessed by administering a high concentration of liquid (丨5〇mg/mL) and recombinant cold-dried (120 mg/mL) via an extravascular route and up to day 36. Pharmacodynamic profile. On day 1, a single 3 mg dose of commercial liquid natalizumab was also administered to determine the relative bioavailability of high concentration formulations. On day 36, animals in the SC and IM dose groups were given a second injection and on the 39th day an injection site biopsy was performed to assess local tolerance. Test article, commercial liquid natalizumab, liquid high concentration natazhudan 132080.doc -21 - 200909005 High concentration of natalizumab resistant to lyophilization is supplied by Biogen Idec. Heavy = cold-dried natalizumab recombinant fluid (injected sterile water) is supplied in the form of gluten. A fresh vial of the raw material test mouth was used at each administration day and formulated to obtain a suitable concentration of the administration solution. Thirty untreated rock crab macaques (15 males and females only) were assigned to the five dose groups shown in Table 1 below: Table 1 Group No. 2 Number 1? 3- ι.η ~Έμ~ 1ν— 1 test item adjustment 1 compound 1m* dose content (mg/animal) 1〇3 volume~~ (ml/animal) H ~ concentration (mg/ml) ~ ' 20 3 Λ 3/3 — SC ΤΛ/f Γμ** Π50 ' Π50~~ 1M ~sc~~ Liquid **__ '^ Bed Drying (4)* 150 ~120 1.0 To 150 ~Τ2〇— i/3 IM Freeze-drying *** 120 1.0 120 Commercial Liquid Natizumab; ** high concentration of liquid natalizumab; *** heavy - -1-----L / v / bounds 咏 μα__ ~ Ϊ 20 - group of cold dry high concentration of that he Bead monoclonal antibody. IV = intravenous; sc = subcutaneous; sputum = intramuscular; _ = male / female. A pharmacologically relevant increase in peripheral blood lymphocytes (and, to a lesser extent, eosinophils, immunocytes, and unclassified cells) compared to pre-study baseline values was noted in all groups and considered as a test The expected pharmacodynamic effect of the article. Increased levels of affected white blood cells - generally in individual animals receiving higher sc and old natalizumab (eg, day 8 to day 36) and in receiving lower doses of commercial formulations Individual animals (usually from day 8 to day 15) last longer than the individual animals. However, there is no consistent or significant difference in the magnitude or duration of the reaction associated with the lyophilized high concentration formulation, sex or route of administration of the liquid of the test article. 132080.doc -22· 200909005 The commercial liquid ιν dose group achieved the fastest Tmax at the fastest, and the IM group was faster than the SC group for the I 纟胄 concentration formulation. When compared to the commercial liquid IV dose group, the mean Cmax value in terms of pathway was less than the dose ratio and when administered extravascularly, it was consistent in terms of high concentration formulations. Regardless of the pathway, the average u/2 value is consistent across all dose groups. Regardless of the formulation in the SC and IM dose groups, the mean AUCiast and AUCinf were greater than the dose ratio, indicating complete absorption (ie, relative bioavailability of i 〇〇%)' and when administered extravascularly, it was also comparable in formulation. Consistent. Increased counts of visible circulating lymphocyte counts and expected test items for secondary eosinophils, basophils, and unclassified cell counts were compared to pre-study baselines present in all dose groups. These results are consistent with the integrin saturation profile and attributed to the pharmacological effects of natalizumab. The duration of increase in the affected white blood cell count is usually higher than the dose-dependent and high concentration of natalizumab and the lyophilized high concentration natalizumab treatment group (eg, day 8 to day 36). A group of commercial liquid formulations received intravenously (usually from day 8 to day 15). No consistent differences in the changes in leukocyte populations were observed between the high concentration of natalizumab and the recombinant freeze-dried formulation, or the absence of the proposed route of administration (subcutaneous or intramuscular injection) or animal sex Any differences related. In conclusion, the high concentration of natalizumab formulation was well tolerated and produced peripheral blood lymphocyte counts after subcutaneous and intramuscular injection of 120 mg (reconstituted lyophilized formulation) into the stone crab macaque. Pre-132080.doc -23* 200909005 The pharmacologically relevant increase, straight, as early as an intravenous dose of 3 〇mg commercial liquid preparations, but slightly longer than its persistence. Example Currently 1 to 2 hours 卩 卩 main #. This requires the delivery of natalizumab in the form of a 〇Μΐν infusion. This requires the patient to go to the hospital wow 蛊萦 TM / professional infusion center. In order to make that he beads, two convenient: need to subcutaneously. It is better to follow the higher concentration I. And the lower level of the bulk drug substance development freeze-drying formulation The study described here is designed to screen the effects of various initial and final protein concentrations on the stability of the use of recommended sugar as an excipient. (Research A). The second part of the study was designed to screen for other well-known effects on protein stability; East Dry Protectants and Excipients (Research... Also try to test for manufacturability based on fill volume and recombination volume Solution 0 In Study A, four formulations were prepared to test the effect of initial and final protein concentration on real-time and accelerated stability for pre-freeze-dried cakes and freeze-dried cakes. The cold-baked formulations all exhibited stability sufficient to be used in the form of a pre-freeze-dried cake for a retention time of up to one month. The two freeze-dried formulations exhibited sufficient growth for further development. The formulation characteristics and stability of the candidate. One of the formulations consisted of a starting concentration of 40 mg/mL and a recombinant concentration of 1 〇〇mg/mL and a weight ratio of protein to sucrose of 1:1. The two candidate formulations contained a starting concentration of 50 mg/mL and a recombinant concentration of 2 mg/mL and a weight ratio of protein to cane to 2: 1. Both formulations contained histidine buffer and polysorbate. Ester 80 132080.doc -24· 200909005 A high-concentration liquid formulation with the same excipient profile as the current natalizumab (TysabriTM) formulation is also available. Under the 乞, this formulation is also considered sufficient The aggregates of the further developed candidates formed a steady rate of release of the amine faster than the freeze-dried formulation. At acceleration/twist, the formulation showed a tendency to form low molecular weight degradation products, which was frozen The same degree was not observed in the dry formulation. The experimental design material was used for the preparation of the natalizumab of Biengenide, which was prepared by Bi〇genide (^# in February 003). This material has been formulated and bottled so the material needs to be mixed and the polysorbate 80 is removed prior to use in such formulation studies. In short, this is by diafiltration to low ionic strength, high pH (10 _ tris, 10 mM NaC pH 8.5) was achieved in a buffer. Under these conditions the material was bound to a DEAE-Sepharose column and then dissolved using 10 mM sodium sulphate, 140 mM NaCl (pH 6). Next, diafilter the column elution solution to 6 mM histidine (PH 6) Concentrate to between 7 〇 mg/mL and 100 mg/mL for further formulation. Chemicals and Reagents All chemicals and reagents used in this study (except for notes) are constructed from VWR and are ACS grade or More preferred grades "When USP grade reagents are available, they are used as excipients. Polysorbate 80 is purchased from sigm<p. P6474) and is derived from plants and has a low peroxide value. Formulations The following formulations were prepared by diluting the stock solution of the excipient and active substance 132080.doc -25.200909005 to the desired concentration as indicated in Table 2: Study a Formulation Parameters. All formulations were prepared at pH 6. Table 2: Study A formulation parameters. Formulation ID ^ lyophilized concentration recombinant concentration " 3944-18A 20 mg/mL Ab, 20 mg/mL strict sugar, 6 mM his > 0.02% PS80 100 mg/mL Ab, 100 30 mMhis, 0.1% PS80 3944-18B 40 mg/mL Ab, 40 mg/mL sucrose, 6 mM his, 0.02% PS80 100 mg/mL Ab, 100 mg/mL strict sugar, 15 mM his, 0.05% PS80 3944-18C 50 mg/mL Ab, 25 mg /mL strict sugar, 6 mM his, 0.02% PS80 200 mg/mL Ab, 100 mg/mL 24 mM his, 0.08% PS80 3944-18D 75 mg/mL Ab, 40 mg/mL strict sugar, 6 mM his, 0.02 % PS80 225 mg/mL Ab, 120 mg/mL strict sugar, 18 mM his ^ 0.06% PS80 3944-18L 100 mg/mL, 10 mM sodium phosphate, 140 mM NaCl (pH 6) '0.02% PS80 NA " ' ------ Abbreviations: Ab = antibody, his = histidine, PS80 = polysorbate 80. Formulations for Study B The following formulations were prepared by diluting the stock solutions of excipients and actives to the desired concentrations as shown in Table 3: Study B Formulation Parameters. All formulations were pH 6. Table 3: Study B formulation parameters. Formulation ID Pre-freeze-drying concentration Recombinant concentration... 3976-4C 50 mg/mL Ab, 25 mg/mL sucrose, 6 mM his ' 0.02% PS80 200 mg/mL Ab, 100 tnM his ' 0.08% PS80 3976-4G 50 mg /mL Ab, 25 mg/mL sucrose, 15 mM NaCl, 6 mM his, 0.02% PS80 200 mg/mL Ab, 100 mg/mL tnM NaCl, 24 mM his, 0.08% PS80 3976-4H 50 mg/mL Ab, 25 mg/mL recommended sugar, 6 mM his ' 0.02% PS20 200 mg/mL Ab, 100 mg/mL by mM his ' 0.08% PS20 3976-41 5ϋ mg/mL Ab, 25 mg/mL trehalose, 6 mM his 0.02% PS80 '200 mg/mL Ab, 100 mg/mL sea 24 mM his, 0.08% PS80 3976-4K 40 mg/mL Ab, 25 mg/mL sucrose, 6 mM his ' 0.02% PS80 160 mg/mL Ab, 100 mg/mL mM his ' 0.08% PS80 Abbreviation: Ab = antibody ' his = histidine, PS 80 = polysorbate 80. 132080.doc * 26 - 200909005 Method Preparation of the formulation The solution was aseptically filled and filled into sterile glass vials as indicated. All samples for pre-freeze-drying analysis and 3944_181^ were filled into 2 cc vials with 〇 5 ^^, plugged with stoppers and capped. The formulation to be freeze-dried was filled into a 5 cc Kimbie vial in the following volume: formulation W44_ 18A-2.5 mL, formulation 394448^.25, formulation 3944_i8c_ 2 mL, formulation 3944_18ΕΜ 5 . All formulations for study b were filled in 2 mL into 5 cc Kimble vials. Freeze Drying Cycle For Study A, the vials were frozen at k 2 〇 °c. At the weekend, the freeze dryer failed and the temperature dropped to _7〇. Hey. An attempt was made to restart the freeze-drying cycle, causing the temperature to rise to 3-4 t and then re-freezing. Restart the freeze dryer and continue the cycle. The first drying was carried out at 1-2 (rc) under vacuum for 20 hours. Then, the temperature was ramped to 20 C over 3 hours and held at 1 〇〇mT rr for 30 hours for the second drying. For study B, the vial was at -5 〇. (: 2 hours under freezing to ensure that the sentence was cold; East. Then, under a vacuum of 100 mTorr, the temperature reached _4{rc for 20 min. Then '2 〇 In a minute, the temperature was changed to _ 25 ° C with a vacuum of 1 〇〇 mT 〇rr and the first drying was continued for 20 hours. The storage temperature was slowly changed to 20 ° C over 1 〇 hour to start at 1 〇〇 rrTorr. The second drying was followed by 2 hours at 2 ° C. The study set the pre-freeze-dried liquid, high-concentration liquid control and cold-dried 132080.doc -27- 200909005 cake vial placed in 5 〇, 30 ° C and 40. < 3 times. Weekly test at 4 (TC stored samples. At 3, at 24, 8 and 12 ° y and 12 weeks at 3 (TC storage) Samples. Samples that were inaccurately sampled at 4, 8 and 12 weeks. For some formulations, other vials at (10) and 5 °C were analyzed at 6 months and i years. All formulations were assayed for zero time after zombie, sputum/end cognac and lyophilization. The samples were assayed by the following methods. Not all assays were performed at all time points except for samples used for pre-freeze drying. Outside the zero time point, the same two vials were sampled for each test. Visual inspection of all samples was performed according to the visual inspection and the appearance was recorded. Check the color, uniformity, stability and remelting of the freeze-dried cake Evidence. Check the color, clarity and presence of microparticles in the liquid and recombinant samples. Residual moisture and reconstitution time at zero time, use Karl Fischer to verify the residual moisture of the freeze-dried cake (BOP 000-01290). The appropriate volume of DI water, followed by gentle rotation to measure the reconstitution time. Record the time when the cake is completely dissolved (EOP 〇〇〇-〇 1292). Concentration measurement of the concentration of all samples. Use natalizumab placebo The sample was diluted to 1 mg/mL. UV scanning was performed at 400 nm to 240 nm at 200 nm/min using a Varian 3 00 Bio spectrophotometer and a 1 cm pathlength colorimetric tube. Luminosity. The absorbance at the λ maximal value is recorded and the concentration is determined by dividing the value by 1.498 (the absorption coefficient of 1320.000.doc -28 - 200909005 benzumab) and adjusting it by appropriate dilution. The turbidity is 10 mm. Small volume colorimetric tubes (Starna Cells Inc., Cat. No. 16.160-Q-10\Z20) measure the absorption of pure samples from 300 nm to 400 nm. Reported values recorded at 360 nm. Size Exclusion Chromatography The amount of monomer, high molecular weight, dimer, and low molecular weight material was determined using a modification of Biogen SOP 22d.505. The sample was loaded onto the column in pure form and the load volume was adjusted so that the mass on the column was about 400. The quality of the reference material load is also adjusted to be comparable. Debt measurements were recorded at 215 nm and 280 nm, whereas the main peak exceeded the gauge at 2 15 nm, so the 280 nm trace was used for the calculations reported here. Cation exchange chromatography Perform cation exchange chromatography. Due to the difference in the chromatography system, the gradient was slightly modified so that the main peak was dissolved between 9 and 12 minutes. Since the system used was a binary pump, high salt washing was performed only after each group of analyses. There is no evidence of any peaks in the recent residence time leaching in high salt washes. Since the high salt wash was not performed after each sample, the rebalancing time was shortened to 7 minutes. Efficacy The efficacy of selected samples was analyzed by VCAM lysate assay (AAM 00 00965) and by Jurkat cell assay (AAM 00 1-00700). Results of Study A This study was designed as a screening study to examine the initial protein concentration and the effect of the final 132080.doc -29-200909005 protein Handu on freeze-dried cake parameters, reconstitution time and stability. Preliminary studies have shown that sucrose provides adequate short-term lyoprotectant properties for natalizumab, while mannitol is not available. For this, use sucrose

\ Perform the initial screening as a lyoprotectant. Early studies have also shown that the optimal pH for natalizumab is PH 6. Phosphate, citrate and histamine S-type buffers are most commonly used in this 1) 11-value buffer. Phosphate buffers are not optimal for use in freeze-dried proteins due to their pH shift after freezing. The citrate buffer is associated with injection pain in some subcutaneous formulations. Therefore, histidine is selected as a preferred buffer material. The initial concentration was determined to be 6 paws "to prepare the formulation and the final concentration after reconstitution was maintained at or below the blessing. For study 3A, the polysorbate 80 concentration in the pre-lyophilized solution was maintained at 〇·〇2 % 'Because this has been shown to give adequate protection to the protein and also maintain a final concentration equal to or low 1%. Select the sugar concentration to maintain the protein to sugar weight ratio between 1:1 and 2:1 while still maintaining close The final solution of the isotonic solution. The table in the appendix of the pre-freeze-dried formulation and the liquid formulation contains all the results recorded for all formulations. Upon visual inspection, all formulations were colorless and clear to milky white & Visible particles. + As expected, milky white increased slightly with increasing protein concentration. No sample showed changes in protein concentration in the analysis change and no tendency was observed. For example, the turbidity was not measured by the absorbance at 360 nm. No change was observed at 6C for 6 months or under paper for 12 weeks. Peach pre-cooled beam dried samples showed turbidity for samples of 4〇mg/mL, 5() mg/mL The time was slightly increased. μ mg/mL and 75 mg/mL pre-cooled 132080.doc -30- 200909005; East dried samples and high concentration liquids did not prove to be present in 4 (rc samples. Monitoring of all formulations) The formation of dimers, high molecular weight aggregates and low molecular weight substances. The table results are shown in the table in the appendix. Figures 1 and 2 show the formation of high molecular weight substances at 30C and 40 ° C. There was no change in the content of 'high molecular weight substances in any of the formulations for 6 months.

The ί fc material is shown in this article. 3 (Results under rc (Figure 丨) shows that for any pre-freeze-dried formulation, there is no tendency to form a high-molecular shelf after 12 weeks of storage. High-concentration liquid formulations are shown at 30 ° C. The monthly storage period of high molecular weight substances has a clear increase. At 4 ° C, the formation of high molecular weight substances in the pre-freeze-dried formulation does not seem to start until 4 weeks after storage. Then, the amount of high molecular weight substances continues to increase. High-concentration liquid formulations showed lower initial percentages of high molecular weight material, but after 3 months of study under thieves, this amount appeared to increase at a constant rate. =3 and 4 are shown at (10) and 40. Under pre-cooled bundles The formation of low molecular weight substances in the dried solution. Under 3 (rc, there is only a small change in the content of low molecular weight substances during storage for 3 months', but there is a clear tendency to rise. Only high concentration liquids are determined at 6: month. There are a large number of low molecular weight substances, almost 6%. During the storage at 40C, there is a slight concentration-dependent formation rate of the formation of low molecular weight substances, because the sample: The faster the formation. Again, at 3... and 4. (: The formation of lower molecular weight ruthenium appears to occur at a faster rate than the formation of high molecular weight substances. ^ 132080.doc •31 - 200909005 _ This becomes a liquid sample Important degradation path. For the α σ stored in the + 崤 崤 , , , , , , , , , 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低 低矣矣- No. All pre-freeze-dried formulations are G.15% from the initial inclusion of about 0.6-υ·9/〇 to 6 months. High-concentration liquids are not even displayed in 6 j' Any low molecular weight. Quality and 7 are shown at various temperatures due to the high monomer and low molecular weight base too κ 之 total monomer loss. Under 2_8t:, monomer percentage A East: change & this will indicate this Any of the formulations will be properly stable in the form of pre-monthly formulations at wc. Up to 6 significant liquid formulations are also shown to show no change under 2_ generation. 3〇t sample formation: reduction Very little. This change is due to the formation of low molecular weight substances, which also indicates such a blending. Any of the above-mentioned 缰 缰 缰 + k will be filled with stability at ambient temperature to allow processing. Ancient, curved low eight boats are not skillful, and the liquid is displayed in the month of the month and the low knife The increase in sputum material at the same time the bottle-recognition 4 Λ. The early body was lost. All the formulations were not tested at 40 C: r, 单体 monomer loss during the month. The rate of loss seems to be roughly the same as protein The squeaky right I λ box saponin, medium 100 mg/mL liquid and 75 mg/mL pre-freeze-dried formulation show the formation of a molecular weight material into the degradation machine. Stabilization. @进-step study can be used to analyze the degradation path by cation exchange chromatography to show a more acidic substance f (c in all cases, knot, tiansheng push " 々 / 々 amine Displacement, where time and seed pre-cooling; there is no difference in the East Dry formulation. Since acetamidine is generally affected by pH, heterogeneity and certain buffer materials 132080.doc -32- 200909005, this result will be expected by us. Under the generation, the degradation of the high concentration liquid formulation was not significantly different from the material secret formulation. However, it showed significantly faster degradation at 3G C and 4G C. This is most likely due to the presence of high ionic strength and phosphate buffer. Freeze-dried formulation

Table 5 12 is not in the form of a freeze-dried formulation at $3, (3). The result of the stability of the sample ασ stored under ◦. All formulations were stored for 12 weeks at 30. 〇 and 5. Store under your arm for 6 months. In addition, in the thief and 5. [After 1 year, the formulations were analyzed for 3944-18Β and 3944-18C. The residual moisture of the formulation was analyzed at the initial time point. Residual moisture is higher than the required residual moisture' This may be attributed to the experience experienced during the cold; east drying cycle. For these samples, the residual moisture is in the range of 5-6. The recombination time is measured and is directly related to the initial and final protein concentrations. =mg/mL protein solution cold to dry the cake cost average μ] "Lilai recombination. The sample from 50 mg / mL freeze-dried average 6_7 points: 〇 mg / mL cold special drying cost "minutes and from 2 〇一冷珠=It takes 4_5 minutes. The values can vary, but do not show any tendency for inter- or temperature. : Check the appearance of the reconstituted vial. All samples were clarified to milky white, no light::: r: two formulations stored under the thief for 1 year - some: color! The turbidity is measured as a function of the absorbance at 36 〇 nm. high. 2 The average of the turbidity rises when the storage is under the sail for 12 weeks. The formulations show that the turbidity threshold is not visible when stored under (10) for up to 12 months. All formulations were stored* when stored under the generation as much as 132080. (ΐ〇ς -33 · 200909005 The turbidity increased slightly in one year, except for the formulation 3944_18C. (See Appendix E-Η in the table). No changes in protein concentration were observed in the standard changes inherent in manual filling and recombination. The formation of low molecular weight and high molecular weight degradation products of recombinant samples was analyzed by size exclusion chromatography. Low molecular weight degradation was evident at some time points. However, there does not appear to be a tendency to form. The amount of all time points is less than 0.2% in any sample, which is considered as the detection limit of the Bi〇genIdec assay. The main degradation pathway is via dimers and higher-order aggregates. The formation of monomer loss over time is shown in Figures 8, 9 and 1〇. At 4〇. There are some monomer losses in the underarm storage, where the losses in the formulations 3944-1 8D and 3944-1 8C Formulations 39944-1 8B and 3944-1 8A showed a less pronounced tendency to decrease. The same propensity sequence was observed at 30 C, where 3944_18D and 3944_i8c showed no faster monomer loss. No formulation was shown at 5. : the next time Up to 6 months for 3944-18A and 3 944-18D and up to one year for 3944_18B and 3944·l8c. Selected samples by impotence exchange chromatography. Appears in the formulation There is no difference in it. Under 5, there is a displacement from 71% main peak to 66% main peak. The percentage of acid peak remains fairly constant with time, but the percentage of basic peak increases with time from 8_1〇% of the initial time point to 1% after one year. The 30°c and 40°c samples showed the same tendency, with the percentage of basic peaks reaching 18-19% after one year at 30 °C and reaching 22- after 3 months at 401. 26%. Other work will be required to characterize this reaction pathway and determine the source of the degradation material. Results of Study B 132080.doc -34- 200909005 Selection of Formulations Study Study B to study excipients other than sucrose for Nata The effect of the stability of the mAb and the results seen in Study A. Based on the results of Study A, the initial concentration of 40-50 mg/mL was determined to be optimal for good cake formation and recombination characteristics. The ratio is determined to be a 2:1 weight ratio with an initial concentration of 50 mg/mL and The recombinant concentration target was 2 〇〇 mg/mL. In addition, a formulation was tested at a starting concentration of 40 mg/mL and a target recombinant concentration of 16 〇 mg/mL. The formulation also had a protein weight ratio of 16:1. Ratio to sugar. Formulation 3976-4C contained the same formulation as 3944_18C in Study A. The stability of the pre-lyophilized solution was tested in Study A and was not repeated in b. Reports from the literature have indicated addition to high concentrations Low levels of sodium chloride in the protein solution can help reduce the viscosity of these solutions. Formulation 3976-4G 15 mM NaCl was added to the pre-freeze-dried formulation to examine the effect of NaCl. Polysorbate 2(pS2〇) is often used in protein formulations to replace polysorbate 80 (PS80). An equivalent amount of PS20 was substituted for 0.02% PS8 in formulation 3976_4h. In the formulation 3976_41, sucrose was replaced with an equivalent amount of trehalose. Pre-freeze-dried formulations Analyze the appearance of pre-freeze-dried formulations. Regardless of the storage temperature, no significant change in appearance of any of the solutions occurred. They are colorless and slightly milky white with no particle appearance. There was no change in the protein concentration in any of the formulations. The turbidity was measured at all time points. The initial turbidity metric was higher for the formulation 3976-4G, but remained fairly constant at subsequent time points. No change in turbidity was observed in any of the 132080.doc -35-200909005 formulations, although the values measured for 3976_4G were still higher at all temperatures than at other formulations. The loss of monomer caused by the formation of dimers and higher molecular weight aggregates and caused by low molecular weight sheets was monitored. As previously seen, the monomer percentage of any sample did not change at 5 ° C for up to 3 months. Formulation 3976-4K was also analyzed at rc for 6 months and did not show any percent change in monomer. Figures 11 and 12 are shown at 30, respectively. (: and 4 (low molecular weight species forming each formulation under rc. As previously seen, there is a substantial formation of low molecular weight species in all formulations at 4 (TC), with a lower protein concentration in 3976-4K The rate of formation is slightly lower. These results can be compared to the results seen in study a. In addition, it appears that the excipient has no effect on the reaction. At 30 ° C, the low molecular weight substance increases slightly. It can be between 0.2 and «.4«/0 after 12 weeks. Compared with 40. Under the armpit, the high molecular weight substances of all the formulations are slightly increased, except for 3976-4H. It contains PS2〇 (Figure 13). The high molecular weight of any formulation does not change at other temperatures. It shows that all the formulations are stable to form aggregates, and the main degradation path is to form low molecular weight substances at high temperatures. Either of them will be stable enough to develop into a pre-freeze-dried block drug. Perform cation exchange chromatography on all formulations at the initial time point, but only for 3976-4K at 5〇C Execute after 6 months of storage. As seen in a, there is a shift to a more acidic substance during storage. This degradation is driven by pH and temperature. Freeze-drying formulation 132080.doc -36- 200909005: Analysis of moisture and cake of all samples at initial time point Appearance and reconstitution time. The appearance of all cakes is acceptable. The moisture content is slightly lower than that of research. # Between 3% and 4%, except for the formulation 3976-41. , /, with 5.4% moisture. At all time points and temperatures, 'recombination time of all samples is generally acceptable and less than 10 minutes'. A few exceptions take between 10 and 14 minutes. The initial protein concentration of 3976_4 ft also showed a faster recombination time. Once reconstituted, all samples were colorless and slightly milky to milk at all temperatures.

The white calendar lasts for 12 weeks, except for 3976_41 and 3976-4G. At 40 ° C and after 12 weeks ' 3976-41 is completely milky white. 3976_4G; ^3〇C)c i. After 6 weeks and at 5 7 and 4 (circle 8 weeks, it is completely milky white after 8 weeks. The self-weight, and the tendency of v-bubble and bubble formation is visible, add (10) It seems to reduce the viscosity of the self-adhesive substance. However, it also shows that the white light of the solution is significant, and the formulation is 3976_4C at 6 C and 1 year after the knife is added. 3976-4C at 5t; After one year, it showed a slight yellow color. The analysis was carried out at 5 〇 and at 30 ° for 6 months and 1 year, the formulation was 3976_4K. The formulation 3976_ 4k showed a slight yellow after storage for 6 months at 30C, but at 5〇c This is not the case after i years of storage. At 40 C, the sample was analyzed by size exclusion chromatography for up to 3 months. Samples were analyzed at 5C and 30C for up to 3 months, and then at one year. Analysis. In addition, the formulation 3976_4 (: and 3976-4K was also analyzed at 6 months. The amount of low molecular weight substances was less than 0.2% in all samples. The main degradation pathway was attributed to dimer and higher molecular weight. Formation of the substance. Figures 14, 15 and 16 show the formation of aggregates at various temperatures 132080.doc -37- 200909005 Monomer loss. Obviously, the monomer loss in the trehalose-containing formulation 3976-41 is faster than other formulations at all temperatures. Formulations containing PS20 and NaCl are shown to be compatible with formulations containing sucrose and PS80. Equivalent degradation. Formulation 3976-4K showed the least unpleasant monomer loss at all temperatures. This formulation has a lower initial protein concentration, a lower recombinant concentration, and a higher ratio of sucrose to protein. The selected samples were analyzed and analyzed. The results of this assay may vary slightly, but all formulations appear to maintain a steady change in charge distribution at 5 ° C. At 40 ° C, all formulations show a decrease in main peak and acidity. The alkaline substance was increased. This tendency was also observed in the samples from the formulation 3976-4K, and the samples were analyzed after storage for 1 year at 30 ° C. At the 8 week time point, the VC AM lysate was used. And Jurkat Cell Assay The efficacy of the formulation 3976-4K stored at 5 ° C and 40 ° C was analyzed. The results are shown in the table below. Table 4: Benchmark Results Sample / Assay VC AM Lysate Jurkat 3976-4K - 5 °C 126% 98.2 for Screening study for the formation of high concentration freeze-dried formulations of fatalizumab. 3976-4K - 40°C 丨124.3% 丨104% Conclusion The pre-freeze-dried block formulation lasted up to 6 at 5 °C. Months, there was no monomer loss in the high concentration liquid formulation, however there was an increase in the amount of acidic substances that may be caused by the deamination of the amine 132080.doc -38 - 200909005 plus. At 3 〇〇 under 6 months and at 4 〇. ^ /V a For 3 months, this formulation shows monomer loss due to the formation of inter-knife and low molecular weight degradants. High protein concentrates in the formulation may not exhibit long-term stability sufficient to be suitable as a commercial formulation without further optimization. Pre-cooled East Dry formulations are shown to be stored under the generation (8) for any significant monomer loss. The change in the amount of the decylamine of the 箄 τ ‘, ,, and the like can be comparable to that seen in the high concentration liquid.

On the evening of the day, the formulation stored at 3 ° C showed little or no increase in dimer and high molecular weight aggregates over time. However, there is an increase in the amount of low molecular weight fracture material formed. At 4 〇C, the increase in low molecular weight material is faster than the increase in high molecular weight material. : Min: The rate of increase in the amount of matter seems to be related to the protein concentration. Body: 5, containing a protein concentration of 50 mg / mL or less and histidine, Yan sugar and PS8G formulations seem to prove that the pre-cooled granules have sufficient stability. The lyophilized formulation is dried from 50 mg/mL or less and reconstituted to 1 〇〇 to 2 〇〇 胤 胤 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示 显示Accepted restructuring day (4). Formulations containing histidine, and ps8 oxime were used for up to one year without monomer loss & Some of these formulations were slightly stored - a slight increase in post-test substances; however, this increase was difficult to quantify in the case of a hypothetical assay variability. The main degradation pathway of proteins at high temperatures is the formation of polymers and higher molecular weight aggregates. The addition of NaCl reduces the viscosity but results in an increase in the turbidity of the solution. Substitution of PS80 appears to have no effect on stability, while substitution with trehalose increases aggregation. 132080.doc •39· 200909005 Body formation rate. Freeze-dried formulations containing sucrose, histidine and polysorbate 80 and natalizumab proved sufficient for inclusion in preclinical and early clinical studies. Additional studies will be performed to optimize starting protein concentration, sucrose to protein ratio, recombinant protein concentration, and freeze-drying cycles. In addition, the stability of the recombinant sample will be examined. r 132080.doc -40- 200909005

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:Hee Β^^^κ : 锄鹓 bond acid peak % main peak % basic peak % 18.1 69.6 12.4 18.6 70.6 10.9 N/AN/AN/A 24.0 63.9 63.9 σν cN rn rn cn <N ^ 2 S sister < ΓΓ in RS007-001 3448 RS007-001 33690 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 0.06 0.06 0.09 0.09 Ο·11 1 0.07 1 0.09 | 0.08 | 0.14 0.15 Aggregate% 0.63 0.63 0.49 0.49 0.47 0.45 0.42 0.43 0.36 0.37 Dimer% 1.01 S 1 ·Η J 0.99 0.98 0.98 0.97 Η 〇τ—Η Ο 〇0 1 i Monomer % 98.29 98.43 I 98.44 98.43 98.50 98.48 98.48 98.49 98.47 Turbidity at 360 nm 0.041 0.035 0.037 0.021 0.020 "0.025 1 0.027 1 0.035 1 0.033 Concentration (mg/mL) 21.0 | ________________1 21.0 N/AN/A 20.7 20.9 20.5 20.5 Appearance N/AN/A 13 Λ 〇13 (3⁄4 ο <&] ¥CD clarification, colorless clarification, colorless clarification, colorless clarification, colorless clarification, colorless clarification, colorless vial η CN CN '< (N 1—Η <Ν rH CN time point / temperature 00 inch 12 12 weeks, 5° C π P 132080.doc -41 - 200909005 〇〇ο

Acid peak % Main peak % Basic peak % ^ vq 〇0 0\ ΓΑ VsQ 18.6 70.6 10.9 1 N/AN/AN/AN/A 2 $ | ϊ Τ « RS007-001 3448 I RS007-001 33707 RS007-001 33758 RS007 -001 33757 RS007-001 33897 LMW% 0.06 0.06 0.13 0.12 L〇_n" 0.16 1 〇"9 | 019 1 [0.24 | 0.23 Aggregate% 0.63 0.63 0.48 0.48 0.49 1 0.48 0.43 0.43 0.39 0.40 Dimer % ι— Η rH s 0.93 0.93 0.94 0.95 0.93 0.93 0.99 0.99 Monomer 98.29 98.30 98.46 98.48 98.40 98.42 1 98.45 98.45 98.37 98.38 Haze at 360 nm 0.041 0.029 0.044 0.028 0.027 1 0.034 0.031 0.018 0.024 Concentration (mg/mL). 21.0 21.0 N/AN/AN/A 21.0 20.8 Appearance N/AN/A λ Dh o Ί3 Oh Ο > ^:: Clarification, colorless clarification, colorless clarification, colorless clarification, colorless clarification, colorless clarification, colorless vial (N * ' < (Ν (N (N Η time point / temperature o 3 weeks, 30 ° C 6 weeks, 3 (TC 9 weeks, 30 ° C 12 weeks, 30 ° C -42-

132080.doc 200909005

Acid peak % Main peak % Alkaline peak % 18.1 69.6 12.4 18.6 70.6 10.9 N/A Ν/Α Ν/Α 63.6 20.7 15.8 64.7 19.2 16.1 ί ^ Τ « CO RS007-001 3448 RS007-001 33834 RS007-001 33690 33693 RS007- 001 33891 LMW% 0.06 98.30 0.22 0.22 0.35 0.44 3.81 1 3.63 5.83 5.22 Aggregate % 0.63 21.0 0.49 0.49 0.45 0.49 0.65 1 1 0.64 j 1.04 1.03 Ί 1.01 0.06 0.96 0.95 1.00 1.04 2.01 1.76 Monomer % 98.29 0.63 98.33 98.34 98.20 98.03 1 93.93 1 94.15 1 91.12 92.00 Haze at 360 nm 0.041 0.060 0.045 0.040 0.043 "0.060 1 0.057 1 0.068 0.055 Concentration (mg/mL) 21.0 98.30 N/A Ν/Α N/A 20.8 20.5 Appearance N/A 21.0 13 Cl, Ο ¥墉13 〇Ία *&) > 4= 13 〇, 〇In <fi] 13 (3⁄4 澄清 Clarification, colorless clarification, colorless clarification, colorless clarification, colorless vial (N (Ν (N ( N time point / temperature 〇 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40 ° C 12 weeks, 40 ° C 132080.doc • 43· 200909005

s tTi o lin m ..^ M Acid peak % Main peak % Authentic peak % 19.0 70.9 10.0 inch 00 ^ 〇 *—Κ **-**. Η—Η < I <Ν S . A inch 25.2 62.8 11.9 25.5 63.0 11.5 ί $ UJ </1 RS007-001 3448 RS007-001 33690 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 〇〇〇〇c5 so ο Ο Ο g 〇o 1 1 aggregate % in Ό 〇〇CN d in O 吞o 妄ο ο o 卜 O ΓΛ 他 o tamizumab, 40 mg/mL sucrose, 6 mM histidine, 0.02% PS-80 荽Ί SS 〇\ 〇\ 〇Os o 1—H 00 Ον Ο y A s ί—H o C) o Monomer % 98.26 I 98.26 j 1 98.38 98.37 98.42 98.44 98.43 98.45 98.47 98.71 Turbidity at 360 nm 0.053 0.054 0.054 0.050 0.038 0.059 0.043 0.055 0.052 Concentration (mg/mL) 〇ο << m cK to m 〇\ cn »—H Os m ITi 〇< m female< 澄清 clarification, sl opal, colorless clarification, sl opal, Colorless (5) Disc 13 λ ο 13 Ο ΊΆ V. sl opal, colorless V· sl opal, colorless v. sl opal, colorless v· sl opal, colorless (Ν (N CN T—H (N TH (N 佑gis time point) /temperature〇P inchΡ 00 P Wl (N 6 months, 5t 132080.doc -44- 200909005

Acid peak % Main peak % Alkaline peak % 19.0 70.9 10.0 in (inch 00^0 1 - (1 face < 46.3 8.0 45.7 45.8 46.5 7.7 2 $ T ® U ^ 00 RS007-001 3448 RS007-001 33707 RS007-001 33758 RS007-001 33757 1 1 RS007-001 33897 LMW% 〇〇cn 〇m Τ"·Η ο 〇〇CN (Ν 〇<N <N 〇(N 〇(N 〇Aggregate% ο 〇〇妄ο 〇 〇 inch inch om 〇mo 荽Λ s ρ»·Η S ON Ο 〇\ ο σ\ 〇〇\ 〇ON as d 00 〇\ os T-H monomer% 98.26 98.26 98.45 1 98.45 1 98.41 1_____ 98.42 1_ 98.38 98.40 98.33 98.33 Haze at 360 nm 0.053 0.042 0.055 1 0.056 0.053 | 0.058 0.060 0.042 0.042 Concentration (mg/mL) Ο Ο < 1 C Bu o Appearance 1 Clarification, v. si opal, colorless clarification, ν· Si opal, colorless clarification, colorless clarification, colorless si opal, colorless si opal, colorless v. si opal, colorless v. si opal, colorless 1 —— (Ν CN (Ν T _< (N CN time point / temperature ο (5) ^ p V〇^ ^ b 12 weeks, 30°C -45- 132080.doc 200909005

Acid peak % Main peak % Basic peak % 19.0 70.9 10.0 ΙΤ) τ—( 00 ^ ο Γ*-» Face 1 69.2 16.5 14.4 69.2 17.2 13.6 2窣f ϊ T VS RS007-001 3448 RS007-001 33834 RS007-001 690 RS n Τ n n n n n n n ο oo v〇o CN 〇o (N r—H Ί s Η S ο ο T—( TH rn oo monomer % 98.26 98.26 98.30 98.30 97.92 97.90 93.68 93.56 89.97 90.02 turbidity at 360 nm 0.053 0.071 0.062 0.065 0.065 1 0.084 0.080 0.100 0.103 Concentration (mg/mL) Ο Ο << 1 00 00 m cK m Appearance << Clarification, ν· si opal, colorless clarification, ν· si opal, colorless clarification, sl opal, Colorless clarification, sl opal, colorless sl opal, colorless sl opal, colorless ν· sl opal, colorless v. sl opal, colorless I (Ν (Ν <NT—H (N (N time point / temperature ο 4 <N Inch 酹〇 inch inch ^ p 鳑〇00 inch 12 weeks, 40°C -46- 132080.doc 200909005 ®N^fi2 : Stepping on B Benzene

:M 08-sd %(Nod two swi 9 , t3sbbs iss inch issnsns Cao? Acid peak % Main peak % Quantitative guide % 18.6 71.0 10.4 18.4 71.2 10.4 N/AN/A ON (N Os t-H 1—I (N ' ^ —(N 卜 'O <N — ί fN — 26.1 61.9 12.0 2 $ f ϊ τ « ζΛ RS007-001 3448 RS007-001 33690 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 0.07 0.08 1 0.10 j [o.io 1 0.09 1 o.io 1 0.09 0.09 0.14 0.15 ! Aggregate % _1 0.71 0.71 0.57 0.56 0.50 0.52 0.45 0.46 1_ 0.38 0.38 荽Ί 1.03 1.03 1—Η > 1 Η 1 1.01 ] 1.00 1.01 1.04 1.04 s 1.03 Monomer % 98.19 98.18 98.31 98.33 98.40 98.37 98.42 98.41 I 98.44 98.44 Turbidity at 360 nm 0.074 1 0.070 ] 0.068 0.062 0.056 0.054 0.057 jr 0.064 0.060 Concentration (mg/mL) 52.1 51.5 Ν/Α N/A 50.3 50.6 1 51.9 50.6 Appearance 1 ———— ____I N/AN/A Clarification, si opal, colorless 1 clarification, sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless 1 ______ . 1 sl opal, colorless sl opal, colorless vial (N <N τ-H (N (N 1'"< Interval / Temperature 〇 Inch 00 12 weeks, 5°C π P Ό 132080.doc -47- 200909005

Acid peak % Main peak % Alkaline peak % 18.6 71.0 10.4 18.4 71.2 10.4 Ν/Α Ν/Α Ν/Α, inch... Ό 13⁄4 T « of RS007-001 3448 RS007-001 33707 RS007-001 33758 RS007-001 33757 RS007- 001 33897 LMW% 0.07 0.08 0.13 [0.08 1 °·17 [0.26 jj 0.25 j 0.36 0.36 Aggregate % Buddy Buddy 0.49 1 0.49 1 0.44 0.46 rH Inch Ο Inch Ο 0.37 0.37 荽Λ SS 0.96 1 0.98 1 ON Ο j 0.99 1 0.99 1 0.99 s 1 Monomer % 98.19 98.18 98.42 98.40 98.55 98.37 98.34 98.35 1 98.21 98.21 Turbidity at 360 nm 0.074 [0.057 1 0.058 j 0.059 0.067 0.064 1 0.063 j 0.052 0.055 Concentration (mg/mL) τ- Η (N 51.5 Ν/Α Ν/Α Ν/Α 52.6 Appearance N/A Ν/Α Clarification, si opal, colorless clarification, si opal, colorless clarification, colorless clarification, colorless si opal, colorless si opal, colorless si opal, Colorless si opal, colorless vial (Ν (Τ CN Τ-Η CN F-Η (Ν Time point / temperature Ο 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 ° C - 48-

132080.doc 200909005 005

Acid peak % Main peak % Alkaline peak % 18.6 71.0 10.4 18.4 71.2 10.4 ! N/AN/AN/A 73.7 14.8 11.5 2 $ τ « ω ΐ- m RS007-001 3448 RS007-001 33834 RS007-001 33690 RS007-001 33693 RS007-001 33891 LMW% 0.07 0.08 in OO OO 3.34 6.29 m 9.60 Aggregate % <1 < Buddy 〇oo L—0:47...” o 0.89 1_ 0.89 00 荽Ί SS o 0.99 | —丨― g Cn vr> rn monomer% 98.19 98.18 | 98.16 I | 98.18 | 95.11 | 95.33 | 91.46 | 91.44 | 86.90 Haze at 360 nm 0.074 0.081 0.080 j 0.071 0.080 0.095 0.095 0.116 Concentration (mg/mL) <N in N/AN/AI NIA 49.8 Appearance N/AN/A Clarification, si opal, colorless clarification, si opal, colorless clarification, si opal, colorless clarification, si opal, colorless si opal, colorless si opal, colorless si opal, colorless vial ,丨丨< (N (N 1—^ CN CN time point / temperature Ο 2 weeks, 40°C 4 weeks, 40°C 8 weeks, 40°C 12 weeks, 40°C -49- 132080.doc 200909005 〇〇¥^Hns ςο: SB 垵 垵 茛 铋趔 铋趔 铋趔 铋趔 oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo oo , , , , , , , , , , , , Main peak % Basic peak % 18.5 72.3 9.2 17.9 71.9 10.1 N/AN/AN/A 26.2 62.0 11.8 ON inch (N ^ CN ' miscellaneous V0 g, medicine 00 pin ~ RS007-001 3448 RS007-001 33690 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 0.09 0.09 0.13 0.12 | 0.13 | | 0.12 I 1 Q·11! T—H o 0.15 0.15 1 Aggregate % 1.15 inch rH 0.90 0.89 0.83 1_____ 0.81 0.75 0.75 0.59 0.59 '1 # 1.53 1.53 r丨< t—^ in rH in t1 ^ 1.56 1.56 | 1.53 <1 m Monomer % 97.23 97.23 97.46 97.47 97.54 | 97.55 | 97.58 97.58 97.72 97.73 Turbidity at 360 nm 0.097 0.089 0.089 0.071 | 0.071 | 0.072 0.070 | 1 ! 0.081 0.078 Concentration (mg/mL) 77.2 77.6 N/AN/A rn !_Τ5ΛI 1 75.9 75.9 Appearance N/AN/A Clarification, sl opal, colorless clarification, sl opal, colorless sl Opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless vial Η CN <N τ-H (N (NH CN time point / temperature 00 00 12 weeks, 5 ° C π P学"η 132080.doc •50- 200909005 ροε 132080.doc oxo#-^i

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Acid peak % Main peak % Alkaline peak % 18.5 72.3 9.2 17.9 71.9 10.1 << 1 CN '―i 00 I> ^ »~H 68.6 13.7 17.7 SEC reference standard and average area count RS007-001 3448 RS007-001 33834 RS007 -001 33690 RS007-001 33498 RS007-001 33891 LMW% gdsd IT) od inch οό On (N 00 11.57 10.41 aggregate % inch rn o (N 〇oo 卜 ο m m rn 00 oi OO Μ m m in tH m in m ui m oo ri § i H s (N CN monomer% 97.23 97.23 97.31 97.33 93.12 93.00 88.74 | 88.57 83.49 86.55 turbidity at 360 nm 0.097 0.092 0.111 0.104 0.098 0.136 0.135 0.165 0.174 concentration (mg/mL) CM vo 1 sense 1 inch ON appearance < clarification, sl opal, colorless clarification, sl opal, colorless clarification, sl opal 'colorless clarification, sl opal ' colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless Vial r-H (N < N r - < CN 1 (N (N time point / temperature o P o < NP o inch OO 12 weeks, 40 ° C 132080.doc -52- 200909005 〇 \ 08-SDH ./.1.0, roaring a fwg 0S, 窭槌Ί日/οί日001 00- ®w^*qul Σ.ο: Lai B driving quilt: 铋 铋 acid peak % main peak % alkaline peak % 1-H 〇〇τ-Η cK ο 〇f***·· 2 Ν / Α Ν / Α Ν / Α 00 TH ^ vd CN (N β — 21.2 66.6 12.2 Bee V0 00 RS007-001 3448 RS007-001 33720 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 1_ 1 1 1 1 II 1 1 I 1 0.09 0.08 1 1 1- Aggregate% 0.78 1_ 0.79 0.70 0.69 I 0.69 1 1_ 0.68 0.77 0.76 0.64 0.66 , 1 Fan 0.97 0.98 0.90 0.89 〇 Ο ΟΝ Ο Ο g tH os r"H monomer % 98.24 98.23 98.41 98.41 98.40 98.41 98.04 98.07 98.35 98.33 t B ^ 0.111 0.100 0.146 0.164 0.136 0.138 0.135 0.134 0.150 0.153 Concentration (mg/mL) 95.7 96.7 Ν/Α Ν/Α 94.2 'd 96.3 96.2 KF% of moisture 〇; Ν/Α Ν/Α Ν/Α N/A Recombination time (minutes) 〇in ro (N — <Ν o CTn (N appearance clarification, si opal Ια ψ*, C3 la & clarification, sl opal clarification, sl opal disc two cd & Disc II β gn m II β cd & Η #- & H disc II e c3 Oh disc II β cd Ο vial CN (N τ — Η <Ν CN »—H (N time point / temperature 〇 inch (5) οο 12 weeks, 5°C Ό \d 132080.doc •53 - 200909005 ^ v〇^ 婶· τ-Η 〇〇ι-^ 0^0 0 i~H »—K ^ <Ί Ό 2卜Z i < P ^--' Tf (NV〇rH ^0^0 Τ"Η 4 — (Ν Ό — butterfly V0 will Γ柘 4吟 · female s enzyme § black in oop psi § 〇〇 1- Pi t—H o卜έ" ζΑ ^ Ρύ o 00 ^ ¢^1 g|- a^l S 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 〇o 1 1 1 I 韬OO o 〇rn ΙΟ o 〇〇?; 〇mov〇Ό 〇o CN oo 寸ο M# σ^ o 00 Os o 00 ON O 00 o S <N r-H 〇I^H vo cn SS T-< m ν〇 Τ—( 韬1^-inch CN od 〇\ <N 00 ON m 00 ro od et 00 ON oo On <N (N 00 〇\ 艺 00 Os κη oo 00 s; 00 m 00 〇V ^ oo &gt ; Ή oor*H IT) TH 〇 o o CN 〇 VO in O Ό o CN inch oo inch o ON o Ο concentration (mg/mL) t> V/S 卜^o 〇s < face Z 'O ^o 00 〇 \ o *〇G\ Γ〇ΟΝ Φ^χ- 卜 Z 1 ZI i I|i .Ifh.t CD®. ψ*Ί om ΓΠ l〇cn o vd (N rn p ui m 'O 'Tj Ro (N ^Ο ΓΛ Ί/2 a yoke gn ' 13 t—4 ^s3 you ** 〇r base Si o ^ «3 & alkali Ίλ Ί3 &lt ;fi3 &墉«) #. c 13 Ο ^sD a 13 &<s3 磲13 Oh Ο , CN ^-H (N TH CN r-H <N CN i-Η CN S(4) ^,Ά o ?^〇V〇^ "p 痹〇〇[ ^ ^°〇(N cn Μ " 〇° ^ nc:^ 132080.doc -54- 200909005 aoTr Acid bee% main °°/〇 alkaline peak % 1 —1 00 1—« cK ο 〇τ-Η Γ^«· Τ-Η S 3 ^ 1 face < 辕V0 you + female RS007-001 3448 RS007-001 33791 RS007-001 33720 RS007-001 33693 RS007-001 33891 LMW% IIII Ο sd J 1 1 1 1 1 1 d CN d Aggregate% 00 Ο On Ο Ο On 〇^T) O' O' »r> o S O' CN O' <N 〇Μ荽ο Οο ο m cn t—H 00 CN rH o 00 卜r—4 <N inch (N monomer% 98.24 1 98.23 97.83 97.82 98.07 98.04 97.86 "97.79 97.19 97.16 § γ-^ύ , rp g昶他 0.111 1 0.100 1 1 ... 0.153 1 0.181 1 0.160 0.159 0.168 0.210 0.230 0.238 Concentration (mg/mL) 卜 in ON ο ΟΝ < z Os <N Moisture KF% inch · ζ i Recombination time (minutes) ο 00 rn r -^ r^i rn (N (N On Bu in cn appearance clarification, si opal Ί λ " 13 1 clarify ι Cheng Clear, si opal ^ 13 ^ & opal, colorless two e Λ墉ο opal, colorless two e a dish o 1 I vial τ-Η (Ν CN <N <N CN time point / temperature Ο 2 weeks, 40°C 4 weeks, 40°C 8 weeks, 40°C 12 weeks, 40°C ^Λ乱〇·T4.lli=ls ·砘璁=.Λ) 132080.doc -55- 200909005 φ| 钵ο m •. Pan acid peak % Main peak % Basic peak % 18.9 71.0 10.1 19.5 69.8 10.6 N/AN/A 19.3 68.9 11.8 19.4 68.7 11.8 21.3 66.7 12.0 21.1 66.8 12.0 20.4 65.5 14.1 19.7 66.2 14.2 %' Lai RS007-001 3448 RS007 -001 33720 RS007-001 33245 RS007-001 33881 REF025 (RS007- 001)33759 REF025 (RS007- 001)36185 0.08 0.08 1 1 1 1 1 1 1 1 0.08 0.09 1 Face I 1 I 1 1 1 韬0.79 0.75 0.63 0.64 0.62 | 0.63 | 0.71 0.67 0.60 0.60 0.67 0.68 >1# 1.04 1.07 0.93 »—H 〇\ o 0.92 | 0.92 | rH 〇.丨< Ο 1.00 0.99 t, 15 mM histidine, 0.05% PS-80 Ϊϊ- 98.10 98.10 98.44 98.46 [98.46 | 98.46 | 98.13 98.01 98.39 98.40 98.34 98.33 The turbidity at 360 nm 0.095 0.109 0.139 0.133 CN 〇 inch o 0.115 0.138 0.127 1 _ 0.132 I 1 0.125 0.126 Concentration (mg/mL) 96.0 95.9 N/AN/A 93.1 93.1 93.5 1 93.4 93.3 92.8 OS N/AN/AN/AN/A Ν/Α m 獬1 B Mound «w 00 ro 00 <N CN 〇\ 卜^(N On i〇卜oi <N 2 ks玲t2_味a 崁Ί Jm>| W〇巍2 clarification, sl opal clarification, sl opal 7/: -13 target & ^ 13 _§ m S· o 13 a opal, colorless opal, colorless opal, colorless 1 opal, colorless opal, colorless opal, colorless i cs (N CN CN H (Ν r·^ (N time point / temperature ο 00 00 12 Week, 5°C Ό month 1〒, c 132080.doc •56- 200909005

_ 18.9 71.0 10.1 19.5 69.8 10.6 Face 1 1 23.5 66.3 10.2 22.6 68.3 9.1 22.7 62.2 15.1 23.0 62.0 15.0 22.9 58.1 19.0 22.9 58.4 18.7 Bee but coffee 1 1 RS007-001 3448 1 1_ RS007-001 33707 RS007-001 33758 RS007-001 33757 ι_ RS007-001 33897 REF025 (RS007-001) 33774 J REF025 (RS007-001) 36185 LMW% 〇Ο 1 1 1 1 1 1 1 1 1 1 I 1 Ο ί—Η Ο 〇o rH o yn o Ο黩S4 On Ο Ο CN VsD O' CN v〇O' CN IT) o 00 00 Ο 00 ίη Ο ο 〇in CN o CN 〇o rn ο Μ荽S » Η H o T—H s to ΓΛ rn »— Η ζΙ »—Η rn CTn l—H inch (N in (N (N (N CN 锲5S(- 1 98.10 1 98.10 98.37 98.33 98.13 98.14 98.08 1 97.99 1 1 96.62 1 97.58 97.45 97.33 96.82 96.90 at 360 nm Haze 0.095 0.109 0.137 0.123 °·121 0.123 1 0.160 1 0.147 1 0.159 0.126 0.188 0.195 0.230 0.226 Concentration (mg/mL) Ο Ό · OS Ο Os z Z < Bu Os 00 inch iri OS 〇〇rn 〇\ 〇\ m· 〇\ << i < i οο ΓΟ m rn o in inch 00 00 m CN — CN 00 in <N (N \〇00 clarification, si opal "Ί3 澄Clear, sl opal, colorless clarification, sl opal, colorless dish 13^3 Oh Ίλ m Ί3<β3 Gh α opal, colorless" opal, colorless 1 opal, colorless opal, colorless opal, colorless opal, slightly yellow opal, slightly yellow' μ <N <N <Ν (Ν (N CN CN time point / temperature ο 3 weeks, 30 °C 畛0〇"ρ 鳑ο ^°〇(N m M ^ P 132080.doc -57- 200909005 P? Acid peak % Main peak % Alkaline peak % 18.9 71.0 10.1 19.5 69.8 10.6 Surface 0 — 0 τ—1 ^ CN <Ν 21.8 55.8 22.4 Miscellaneous V0 Γ 碱 Alkali 4 + 友 ^ ^ ^ RS007-001 3448 RS007 -001 33791 RS007-001 33720 RS007-001 33693 RS007-001 33891 LMW% 〇g Ο o ο (Ν 00 00 Η ο Ο ο Ο Ο Ο 韬 韬Os Ο jn ο 宕o 〇\ ο Ο ο ο T*~ H Η Ο ο cn (Ν Ο ο # Ί 卜 r- (N ιη Ο) (Ν 00 (Ν CN SS (Ν 〇 CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN ^ t ^ 0.095 0.109 0.142 0.149 1 0.189 0.195 0.227 1 0.226 1 0.161 0.173 Concentration (mg/mL) Ο 〇\ 〇\ as <<<< (Ν Ο) KF% of moisture ON 2 ί Time (minutes) 00 CO <T) inch m 00 ν〇Ο \〇〇\ cn 1 4 Appearance clarification, si opal clarification, sl opal clarification clarification, sl opal clarification, sl opal disc 13 α ο Λ Λ α ο m 13 α ο Dish 13 Dh ο (N τ·^ (Ν (N 1—Η <Ν ι 1 (Ν time point / temperature ο ^ ρ ο < Ν inch • P f wear " ρ 尔 ο 00 Inch 12 weeks, 40. . (βφ-J卿 IBdo·Art I.HIS.衅璁ΙΛ) 132080.doc -58- 200909005

Acid peak % Main peak % Basic peak % 19.1 70.9 10.0 N/AN/A 18.6 63.8 17.7 19.9 68.9 11.1 21.2 66.4 12.4 21.0 66.6 12.4 20.0 65.5 14.5 inch V〇ON cK in rH SEC reference standard and flat 4 area count RS007-001 3448 RS007-001 33720 RS007-001 33245 RS007-001 33881 REF025 (RS007- 001) 33759 REF025 (RS007- 001) 36185 Manufactured: Test start date: 0.5 mL filled i LMW% 0.08 0.08 0.13 0.13 1 1 1 1 0.08 0.09 1 1 Coffee 1 1 1 1 1 Aggregate% 0.86 0.86 0.79 0.80 0.72 I 0.66 | 0.76 0.77 0.66 0.66 0.73 0.74 '1# 〇τ—Η 1.10 1-16 丨118 1 110丨113 1 1.25 00 <N CN 1.40 1.33 § 〇. Monomer % 97.96 97.96 97.91 97.89 | I 98.18 | 98.21 | j 97.90 97.88 98.07 98.08 97.86 97.93 Haze at 360 nm 0.095 0.106 0.157 I 0.153 | 0.141 I 0.152 | 0.154 0.150 0.167 0.167 0.156 0.152 1〇〇mg/mL sucrose, 24 mM histidine, 0.08% 1 concentration (mg/mL) 172.0 173.2 N/AN/A 154.5 1 1 170.6 174.9 175.3 178.1 172.2 KF% of moisture οο in N/AN/AN /AN/AN/A Reorganization time (minutes) Os 10.5 OO 00 in in tr^ om \o 'O 00 rn Appearance clarification, si opal clarification, si opal clarification, si opal 13 g. opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless opal , colorless reconstituted 200 mg / mL nata beads j vial tH 1 _ (N CN < N τ - H CN (N time point / temperature o inch 00 12 weeks, 5 ° C 12 months, 5 ° C 132080. Doc -59- 200909005

^r^od i—( C^« m 70.9 10.0 1 face 1 20.0 59.0 21.0 3^00 (N —iTiiN oomo CNVOr-. 2Γ8 63.3 15.0 20.9 61.1 17.9 61.2 18.0 ooSi- , 4748〇〇1 RS007-001 33707 RS3 〇3°7758001 RS007-001 33757 RS3°3°8797001 REF025 (RS007-001)33774 REF025 (RS007-001) 36185 LMW% g 〇o 1 1 1 1 1 1 1 1 1 1 1 1 gogo 1 1 1 1 1 1 1 1 Aggregate% oo o 00 o rn o ΓΟ 00 00 l〇o ό 〇 d d 〇dgoo cn 〇〇 og »»H '1# o rH o 00 t—H 00 VO •η <q — Η 50 Γ—H 70 m <N <N (N 卜 in rn in ΓΛ rn monomer % 97.96 97.96 97.79 97.77 97.74 >* 卜 CN in o 1 97.41 | 96.88 96.93 96.16 96.13 95.35 95.26 at 3 60 nm Mixed shoulder degree 0.095 0.106 0.165 0.169 0.157 m <0 d Ό 00 o κ> r-Η d 1 0.178 0.183 0.237 0.227 0.221 0.220 f^oii 172.0 173.2 < 1 174.5 177.6 173.2 174.1 171.2 168.8 OO < i 1 &lt ; <σ; m 〇(N m — »—H — >0 rn ON 4 inch ivS o C) inch ' o 'O appearance clarification, si opal clarification, sl opal -.•ϊώ Sl ο r碟ο c : οίδ3 〇&g t; 潍g 3 "3 o «3 o opal, colorless carbon 13 ao carbon 13 Oh 〇41 13 ao 13 Oh o Φ(Κ 13 α ο production (N Η CN CN r-H ΓΝ1 (N (N 1- H (Ν Is o ?^o CNr〇tH 2〇学rn vo πςΡ Painting O 尽 m (N 132080.doc •60- 200909005 \

s$|i$ ^ ^ · Ον Ο < ί < ί < ί yri \〇ίΓί ν〇Ο 2 sd 〇< Ο Ο κ t- ζ cn »—< (Ν (Ν Ό <Ν SEC reference standard and average area count RS007-001 3448 RS007-001 33791 RS007-001 33720 RS007-001 33693 I 1 RS007-001 33891 gg inch (N j Ο y—^ |~3 Ο Ο Ο Ο Ο Ο 〇Ο Ο 00 00 % ιη 00 00 00 Gs 00 m Os S ο ο ο ο ο ο ο ο Ν ίΝ CN CN rn r〇rn id ΟΝ 〇\ m ΟΝ 〇\ 00 Ρ; 芝 00 'O 00 Ον <Ν ΟΝ s®S- νο ΟΝ Os On ιη Ον 茇•360 turbidity ITi s Ι^ Η g α\ r^i 宕 inch (N 0.208 Os (Ν ^ 1 ^ ο ο Ο ο ο Ο 〇Ο S ο (Ν < f < ζ < c 〇'Si) CN ΓΛ Ζ r KF% 00 水分 < 1 face 1 recombination time (minutes) ο τ-^ m 00 〇 cn ΓΟ 寸 oi — (Ν 00 13 Οη 13 Οη &Η Ί3 Ph <e3 Ο o #.端41 σ3 α ο 13 α ο 13 Οη ο 13 & Labuan-Η <Ν r-Η (Ν <N (Ν Η (Ν time point / temperature 1 Ο 瘠 (Ν 40 ° C inch | 40°c 1 轵00 1 40°c 1 12 weeks, 40°C 132080.doc -61 - 200909005

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¥'^fiTHloo:«B Benzene 妓 茛 : Identify! I趔 Bromine Pier Acid Peak % Main Peak % Authentic Peak % 19.7 70.3 10.0 ^ N/AN/AN/A 20.9 66.6 12.5 20.8 66.7 12.5 Drug V0 Γ Γ 4 4 4 4 Female RS007-001 3448 RS007-001 33720 RS007-001 33245 RS007-001 33881 REF025 (RS007-001) 33759 LMW% 0.08 0.09 1 0.14 0.13 0.12 o 0.10 0.08 1 1 1 1 Aggregate % 1.39 1.39 1.32 1.33 1.25 L28 1 (N t1 i L24 1 1.09 1.08 Μ # m 1.64 1.72 1.67 1-69 1 1.69 1.85 1.80 00 1.79 Monomer % 96.90 96.88 96.82 96.87 96.94 | 96.91 | 96.82 96.88 | 97.10 97.13 Haze at 360 nm 0.093 0.097 0.136 0.143 0.143 | 0.142 j 1 0.145 | | 0.134 j 0.149 0.152 1 1 Concentration (mg/mL) 194.7 _ 184.1 1 j N/AN/A "224.7 j 222.3 | 196.9 197.1 KF% of water 19.0 20.5 r N/AN/A 1 N/A 1 N/A Recombination time (minutes) ( N \6 rn — 18.0 m 18.8 15·3 27.0 15.5 Appearance clarification, si opal clarification, sl opal clarification, sl opal clarification, sl opal opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless vial 1丨丨< (N (N CN r-H CN (N time point / temperature ο inch 〇〇 12 weeks, 5°C π P 132080.doc • 62- 200909005

Acid peak % Main peak % Basic peak % 19.7 70.3 10.0 ^ o N/AN/AN/AN/A 21.6 63.3 15.2 21.6 63.4 15.0 Disadvantage V0 ^ ^ a ^ ^ RS007-001 3448 RS007-001 33707 RS007-001 33758 RS007- 001 33757 RS007-001 33897 REF025 (RS007-001) 33774 LMW% 0.08 0.09 1 1 1 1 0.14 0.14 1 1 1 1 0.09 0.09 1 1 Aggregate % 〇 \ 1-H rn 1—H 〇\ CN 1-H 00 CN 1-H TH »-Η CN 1—t AT—< 1 1.26 I VO (N r—^ m rn rH 1.27 Μ荽m S 2.12 (N 2.28 2.67 2.84 | 2.40 ] 2.83 | 2.80 3.57 3.57 % by monomer 96.90 96.88 96.48 96.49 96.41 96.04 | 95.91 | | 96.39 | | 95.81 | | 95.86 | 95.10 95.16 〇W tt?l 0.093 0.097 0.173 0.168 0.143 0.166 0.178 0.170 0.174 "0.173 0.238 0.225 concentration (mg/mL) 194.7 184.1 N/AN/ AN/A [211.6 223.5 | 181.3 183.7 KF% of moisture (NN/AN/AN/AN/AN/A Recombination time (minutes) o cK 20.5 m od Os CN O) 27.6 21.6 22.4 | ON 00 24.4 22.9 1 Appearance clarification , si opal clarification, sl opal clarification, sl opal, colorless clarification, sl opal, colorless sl opal, colorless sl opal, colorless opal, colorless opal, colorless op Al, colorless opal, colorless opal, colorless opal, colorless vial (N τ-Η CN (N CN CN time point / temperature o 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 °C 6 months, 30°C 132080.doc •63- 200909005

Acid peak % Main peak % Accurate peak % 19.7 70.3 10.0 °® n & N/A 1 i N/AN/AN/A 1 ^ RS007-001 3448 RS007-001 33791 RS007-001 33720 RS007-001 33693 RS007-001 33891 LMW% 0.08 0.09 0.07 0.06 1 0.16 0.15 1 1 0.15 1_ 0.07 0.10 | 韬cn On rn t—H rn 1 丨崤m rn tH On ΓΠ o rn (N Bu, 1荽m 3.40 1_ 2.74 | 3.19 3.22 3.77 4.34 | 4.54 Monomer % 96.90 96.88 [95.17 I j 95.87 | 95.26 95.23 94.86 j [94.10 93.87 [94.12 Haze at 360 nm 0.093 0.097 0.161 | 0.153 0.180 0.179 0.210 0.211 0.248 0.235 | Concentration (mg/mL) 194.7 184.1 N/AN/AN/A 194.2 | 215.4 | KF% of moisture (NN/AIN/A : N/A i_ N/A Recombination time (minutes) o Os 20.5 inch | 15.0 | inch 〇 On cn 00 rH I 20.8 I 16.4 | Appearance clarification, sl opal clarification, sl opal clarification clarification, sl opal clarification, sl opal opal, colorless opal, colorless opal, colorless opal, colorless I <N (N , 丨丨H <N (N < N time point / temperature o 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40 ° C 12 weeks, 40 ° C -64- 132080.doc 20090900 5 εκ φ| 钵m ο step on ra ·· ^ acid peak % main peak % probability peak % 20.8 68.0 11.3 N/A 1 N/A 1 1_ J | N/A i |ι ^ <ΰ* oo RS007-001 33834 RS007-001 33626 ” | RS007-001 33670 REF025 (RS007-001) 33608 LMW% 0.07 〇0.12 » 1 j 0. 10 t—H o Aggregate% 0.57 0.54 0.54 r 0.45 0.44 i_ 0.47 0.47 § C/3 Ph % S 〇, cd 1 1-H f B f Μ ! a 佑 a Ί T Η I 0.96 0.96 monomer % 98.31 98.31 98.31 98.59 98.60 98.36 98.35 turbidity at 360 nm 0.181 1_ 0.105 0.093 1 0.103 0.098 0.098 0.107 Concentration (mg/mL) 50.0 50.7 N/A i- 50.0 50.0 N/A Appearance, pH 1 Clarification, si opal, colorless i sl opal, colorless sl opal, colorless 13 «3 sl opal, colorless 13 <03 &amp Alkali sl opal, colorless vial (N (N time point / temperature 〇 P inch P to 00 12 weeks, 5 ° C -65- 132080.doc 200909005 /. \

Acid peak % Main peak % Alkali peak % 〇〇O rn Ο 00 (NN/AN/AN/AN/A ί $ 00 RS007-001 33834 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 LMW% 0.07 0.06 0.14 0.10 0.10 0.16 1_ 0.16 0.47 0.47 韬sP ψ(\ ©S 0.57 0.15 0.45 0.41 0.42 0.35 0.35 0.37 0.38 >1 # 1.04 0.95 0.99 0.95 0.95 0.99 0.99 1.12 1.10 Monomer % 1 98.31 98.54 98.38 98.54 1 98.53 1 98.50 1 98.50 98.04 98.05 Turbidity at 360 nm 0.181 0.038 0.034 0.109 0.108 0.104 0.102 0.102 0.106 Concentration (mg/mL) 50.0 50.7 N/AN/AN/AN/A Appearance clarification, si opal, colorless sl opal , colorless sl opal, colorless Ί3 <θ3 o'^ sl opal, colorless sl opal, colorless sl opal, colorless 丨sl opal, colorless 13 <&>& 墉 vial CN CN r H <N CN time Point / temperature Ο 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 ° C 132080.doc -66- 200909005 $

Acid peak % Main peak % Alkaline peak % 20.8 68.0 11.3 N/AN/AN/AN/A 卞S minus U ^ RS007-001 33834 RS007-001 33732 RS007-001 34124 RS007-001 33852 REF025 (RS007-001) 33590 LMW % 0.07 0.48 0.48 3.25 1 3.30 ! 8.29 1_ 8.59 10.41 10.72 1 uiil^ 'sfD W 0.57 0.46 0.46 0.43 1 0.42 0.62 0.65 1 1.39 rn 1-H >1 # 1.04 〇〇00 Ο r—Η i 1.08 1.34 1.22 1.47 1.47 Monomer % 98.31 98.05 98.05 95.23 1 95.20 89.74 89.53 86.74 86.46 Turbidity at 360 nm 0.181 0.105 0.109 0.147 0.106 0.121 0.116 0.128 0.131 Concentration (mg/mL) 50.0 丨50.7 N/AN/A j 52.0 54.0 N/A Appearance clarification, si opal, colorless clarification, si opal, colorless clarification, si opal, colorless sl opal 9 colorless 13 <sD o墉sl opal, colorless 13 «3 o' 4= Ίλ sl opal, colorless sl opal, colorless vial CN T—H CN (NV «< CN time point / temperature 〇 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40 ° C 12 weeks, 40 ° C 132080.doc -67- 200909005

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Prepared: Test start date: 0.5 mL Filling amount Acid peak % Main peak % Alkaline peak % 21.0 67.9 11.1 Ν/Α N/AN/A 妒踅柘氐 · · RS007-001 33834 RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 LMW% 0.07 0.03 0.03 1 1 1 1 0.12 0.12 Aggregate % 0.65 0.59 0.58 0.46 0.45 0.50 0.49 S 〇f B f $ ! S 4 ssf by S ' s , 1荽Ο 0.99 0.95 0.96 r —< monomer% 98.22 1 ! i_ 98.38 98.39 98.59 ;98.59 1_ 98.31 98.31 Haze at 360 nm 0.050 1 1 1_. _ 0.058 0.065 0.060 0.068 i_______ 0.066 0.065 Concentration (mg/mL) 53.7 51.7 Ν/Α o 49.8 i N/A Appearance, pH clear, si opal, colorless, 6.11 v. si opal 5 colorless v. si opal > colorless sl opal 5 colorless sl opal, colorless sl opal, colorless sl opal, | colorless vial 1- H CN (N time point / temperature Ο Wfa^ inch 00 12 weeks, 5 °C 132080.doc -68- 200909005 ροε 132080.doc %#si迤i^s O ON ^ C · Τ-Η CN — inch ε8Γηεso. ^00^

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PS Acid peak % Main peak % Alkaline peak % 21.0 67.9 11.1 1 < 1 < τ « Contains: ST RS007-001 33834 RS007-001 33732 RS007-001 34124 RS007-001 33852 REF025 (RS007-001) 33590 LMW% 〇 7 c5 3 Ο oo oo rn oo ON OO OO (N aggregate % Ο ο ο o $ ogo 〇s 00 ON 〇M荽»—Η ssg <τ—H 〇\ (N τ" ·Η (N 00 o § o Monomer% 98.22 98.06 98.08 95.41 95.25 90.07 89.83 89.27 89.09 Turbidity at 360 nm 0.050 0.102 0.098 0.099 0.081 0.104 0.097 0.101 0.097 Concentration (mg/mL) cn IT) Bu 1 1 1 in OS Appearance 〇• W v. si opal, colorless v. si opal, colorless 1 si opal, colorless si opal, colorless si opal, colorless si opal, colorless si opal, colorless si opal, colorless—"Η (N tH CN (NT·^ ( N time point / temperature Ο 2 weeks, 30 ° C 4 weeks, 40 ° C 8 weeks, 40 ° C 12 weeks, 40 ° C -70- 132080.doc 200909005 si 132080.doc

08-sd%so, 毽绪^^日9, 窭澳碰, 1日/§日53,^蚌#荤蔌1661)日01〇植物楗蛘命 IK ¥机械姊Is SO : SB Benzene : 铋趔 铋趔 acid peak % main peak % alkaline peak % rn 〇 Cn i - < ν, Ο τ -1 Ν / Α 1 N / A 1 1 N / A 1 1 will sister + female g, no co RS007-001 33834 RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 LMW% 0.07 0.10 1 S 〇1 1 1 1 0.10 o Aggregate% 0.68 0.60 ID Ο 〇0.46 0.48 0.48 Ί S 1-^ 0.99 0.96 0.97 g TH TH monomer% 98.19 [98.26 1 98.44 98.59 | 98.58 | 98.35 | 1 ! 98.34 Haze at 360 nm 0.035 0.056 0.050 0.076 0.093 0.063 0.057 Concentration (mg/mL) 1 51.0 ^Τ) Ν/Α ( N iT) N/A appearance, pH clear, sl opal, colorless, 6.13 ν. sl opal, colorless v. sl opal, colorless I sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless vial Η r -Η <N 1-H (N ^ - 1 CN Time point / Temperature 寸 P P 00 P iTi 12 weeks, 5°C • 71 - 200909005 ροε Acid peak % Main peak % Alkaline peak % 19.7 69.3 11.0 Ν/Α N/AN/AN/A Miscellaneous* U ^ RS 007-001 33834 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 LMW% 0.07 in Ο Ο 〇o 0.32 | 0.32 | o 0.42 Aggregate % 〇〇VO 〇r—Η in ο r -Ή yn ο 0.47 0.44 0.38 0.40 0.39 00 m 〇 Gong S4 ^ , 1 ο ο o ON c> sg in rH monomer% 98.19 98.33 98.33 98.32 98.51 98.21 98.23 98.12 98.06 turbidity at 360 nm 0.035 0.006 0.010 0.061 | 0.060 0.059 0.056 0.069 0.067 Concentration (mg/mL) 51.0 in Ν/Α N/AN/AN/A Appearance clarification, si opal, colorless, 6.13 si opal, colorless si opal, colorless si opal ' colorless si opal, colorless si opal , colorless 丨sl opal, colorless sl opal, colorless sl opal, colorless vial τ-Η (N (N (N time point / temperature Ο 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks) , 30°C -72- 132080.doc 200909005

Acid peak % Main peak % Basic peak % 19.7 69.3 11.0 Ν/Α Ν/Α 1 i N/AN/A τ « in RS0007-001 33834 RS0007-001 33732 RS0007-001 34124 RS0007-001 33852 REF025 (RS007-001) 33590 i LMW% 0.07 0.42 0.42 2·94 3.08 7-91 1 8.04 9.78 | 10.72 Aggregate% 1 i_ 0.68 0.48 0.48 0.48 0.46 0.82 0.81 r—H in 1.49 丨1 Gong 1.06 1.03 1.02 丨1.09 I 丨1.09 1 1 1 -32 | 1.27 Ό m ψ—i 1.63 Monomer % 98.19 98.07 98.08 95.49 “95.37 1 89.95 89.89 | 87.14 86.16 Turbidity at 360 nm 0.035 0.065 0.069 0.061 0.060 0.085 0.081 0.099 0.110 Concentration (mg/mL) 51.0 51.5 Ν /Α Ν/Α 52.9 (performance=78.1%) | 53.4 j N/A Appearance 1_ 3 α 0 ' ^ 7 W 1 Clarification, sl opal 'Colorless clarification, sl opal, colorless sl opal, colorless ν· sl opal, colorless Sl opal, colorless sl opal, colorless 1 __ sl opal, colorless sl opal, colorless 1 vial ι~Η Η (N CN (N CN time point / temperature 〇 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40°C 12 weeks, 40°C •73- 132080.doc 200909005 Preparation: Test start date: 0.5 mL fill Sex peak % Main peak % Alkaline peak % 〇s 〇ν η τ-Η τ-Η Ν/Α Ν/Α N/A 26.4 62.5 11.0 m oo 〇vd oi “ rsj v〇 — 2 $ T « sister 00 RS007- 001 33834 RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 33633 LMW% II 0.03 0.03 III io 0.10 1 0.16 0.16 Aggregate % ί j 1 0.60 0.53 0.53 0.44 0.45 1 0.46 0.46 0.39 0.40 ^ mAb, 25 mg/mL sucrose, 6 mM group citric acid, 0.02% PS-80 dimer% 0.98 0.98 0.99 0.95 1 0.96 j 1.06 | 1.06 1.07 s monomer % 1 98.41 98.45 98.45 98.61 98.59 1 98.37 | 98.39 98.39 98.39 Turbidity at 360 nm 0.035 0.049 0.049 0.051 1 0.057 1 0.054 0.052 0.065 0.076 Concentration (mg/mL) 41.8 41.8 Ν/Α Appearance, pH 13 S祀3勹〇Η ^ v. sl opal, colorless sl Opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless 1 sl opal, colorless sl opal, colorless ^ g 'Bb next day vial τ-Η (N (N (N CN time point / temperature 寸 inch 00 12 weeks) , 5°C ^ Oo Lifting heart vo 132080.doc -74- 200909005

Acid peak % Main peak % Alkaline peak % ; 1〇On On ^ N/AN/A i N/AN/AN/A 2 $ U -Τ' RS007-001 33834 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 LMW% 1 1 0.14 0.14 0.10 0.10 0.29 0.30 0.38 0.37 Aggregate % 0.60 T—Η κη ο 0.49 0.43 0.42 0.40 0.38 0.37 0.38 Dimer % 0.98 0.99 0.99 0.93 0.93 s rH 1.07 1.12 1.09 Single Body % 98.41 98.39 98.37 98.54 98.55 98.28 98.25 98.13 98.16 Turbidity at 360 nm! 0.035 0.018 0.013 0.060 0.067 0.047 0.054 0.074 0.063 Concentration (mg/mL) 41.8 41.8 N/AN/A 1 | 1 N/AN/AN/ A Appearance, pH 13 〇> — 7 phase 3乞sl opal 5 colorless 13 <θ3 *"3 sl opal, colorless Ί3 & dish S -«3 & dish 1A sl opal, colorless sl opal, colorless Sl opal, colorless vial r—<(N <N (N r-H CN time point / temperature 〇 3 weeks, 30 ° C VO 〇 \ 12 weeks, 30 ° C 132080.doc -75- 200909005

Acid peak % Main peak % Alkali peak % On On N/AN/AN/AN/A 2 s ϊ ί ^/1 RS007-001 33834 RS007-001 33732 RS007-001 34124 RS007-001 33852 REF025 (RS007-001) 33590 LMW% 1 1 0.38 0.24 2.40 2.60 6.41 6.30 7.55 7.35 Aggregate % 0.60 0.48 0.45 0.47 0.72 0.70 1.35 1.38 Dimer % 0.98 1.00 0.93 1.06 1.23 1.26 1.58 H rH Monomer % 98.41 98.14 98.39 96.06 95.90 91.64 91.75 89.51 89.66 at 360 Turbidity at nm 0.035 0.058 0.054 0.054 0.055 ! 1 0.065 0.064 0.081 0.083 Concentration (mg/mL) 41.8 41.8 1 N/AN/A 41.3 (Efficiency = 80.0%) 43.1 N/A Appearance, pH 13 d, 〇t — M clarification, sl opal, colorless clarification, sl opal, colorless 13 ·«] o #. V. sl opal, 1 colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless vial T-H (N <N r-H CN y-M (N time point / temperature o 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40 ° C 12 weeks, 40 ° C 132080.doc -76- 200909005

LK Acid peak % Main peak % Alkaline peak % 1>' m 〇〇6^ (N^sO — 20.4 69.0 10.7 20.1 64.7 15.2 < z 22.1 63.9 14.0 22.9 62.9 14.2 ί 21.8 66.2 12.0 00 (Ν Ο r^V〇 (N (Ν^Ο — 20.5 65.3 14.2 «Kt! $ίΓ RS007-001 33834 RS007-001 33791 J RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 33633 REF025 (RS007- 001) 36188 LMW% 〇c5 o CN r-H d (Ν d I 1 II Ο Ο ο ο II 1 1 Ο Ο wish m ··« View d CTn VsO o Os Ό d 〇\ VO d Ch ό d 00 V ) 00 00 ^Τ) Ο s ο ο in ο m in d 00 ο § zh CL, 京g Ο f key S af 錾 錾 一 一 1 s曰 « ITi 〇〇 2 watts, rose oftL tnJ 〇®Γ ·^ 气3 Sg 23⁄4 Wo Ο # Ί ί» H i H r—H ot <r—< t—H ο ^•Η Production "Η S ▼ _Η 00 (Ν ,丨η 00 CN Τ—Η CN ” i 00 CN »—Η \〇r—Η 5®!- 98.28 98.14 98.14 98.03 98.03 1 98.32 98.33 97.99 1 97.99 98.24 98.19 97.76 Haze at 360 nm 0.028 0.160 0.134 0.131 j 0.140 0.131 0.135 0.144 1 0.147 0.133 0.157 0.113 concentration (mg/mL) 〇r Η in 183.2 169.8 180.0 185.4 < 176.0 172.6 1 222.8 Page ί < Ι | ί Ifti.t biH. τ〇π < in cn o IT) 00 C^) οό Ο »嶙Ο; inch· 00 〇\ 00 (Ν rn back to ffi & H 13 〇〇〇 clarification clarification 133⁄4] & dish 13<e3 Ίο opal, colorless opal, colorless ο disc 13 Oh Ο grave 13 α ο Ί Ί 3 Ρ ο I- opal, slightly yellow vial rH (N rH (Ν CN (Ν (Ν rΗΗ time point / temperature pre-drying o P in inch Ρ ίη 00 ν〇<Ν ^ rH * sigh.戚^冢验篥^嗒阳^0-毕-§4-^^荽荽^麦*^. ^令衾^^婼七厚11了龊^:^》^:画^1食* 132080.doc -77- 200909005 \ Acid peak % Main peak % Alkaline peak % 20.4 69.0 10.7 20.1 64.7 15.2 N/A j N /AN/AN/A τ « RS007-001 33791 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 LMW% 0.06 0.07 0.14 0.14 0.13 0.13 o 0.10 o 0.09 Aggregate % 0.69 0.69 0.68 0.69 0.68 0.67 | 0.24 0.25 0.73 0.72 荽Ί 1.10 1.68 1.64 OO 00 1.89 | 2.71 2.75 2.49 2.47 Monomer % 98.14 98.14 97.50 97.53 | 97.31 I 97.31 | 96.94 96.91 96.68 96.71 Turbidity at 360 nm 0.160 0.134 0.165 0.169 0.175 0.168 0.181 0.169 0.175 0.184 Concentration (mg/mL) 183.2 169.8 N/AN/AN/A 1 N/A KF% of moisture Ο N/AN/AN/AIN/A Recombination time (minutes) in rn inch · CN 00 OO Appearance clarification ,sl opal clarification,sl opal 13 <a3 o'^ a <e3 opal, colorless opa, colorless" opal, colorless j opal, colorless opal, colorless opal, colorless vial. (N (N i _H (N (N CN time point / temperature o 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 ° C -78 - 132080.do c 200909005

Acid peak % Main peak % Basic peak % 20.4 69.0 10.7 20.1 64.7 15.2 I < 24.6 55.6 19.8 26.5 58.8 14.7 2 $ U ^ RS007-001 33791 RS007-001 33732 RS007-001 34124 1 1 ! RS007-001 33852 LMW% 1 1 〇oo IT) 〇in 〇o 1 1 1 1 Aggregate% CTn Ό Ο Ό o Buooo 00 oso in (N 00(N 00 o Ί rH of—( (N (N (N g <N 落· Rn 00 cn monomer % _1 98.14 98.14 96.83 96.88 96.21 96.17 95.82 95.80 Haze at 360 nm 0.160 0.134 | 0.179 | 1 0.165 | 0.172 1 1 0.184 0.203 0.214 Concentration (mg/mL) 183.2 _ 169.8 ί 168.0 162.6 179.0 179.4 KF% of moisture Ο inch ' i << reorganization time (minutes) IT) ro m rn (N rn in 〇 \ inch in σ; appearance clarification, si opal clarification, si opal "clarification, sl opal 1 clarification, si Opal si opal, colorless: sl opal, colorless & media vial (N (N r*H (N r - Η < N time point / temperature o 2 weeks, 40 ° C 4 weeks, 40 ° C 8 weeks, 40 °C -79-

132080.doc 200909005

慽m ••Loading flooding_Female W Acid peak % Main peak % Alkaline peak % 1 Bu (Nj οό \ό »—Η ^-Η 20.7 62.4 16.8 Surface ^sO m Η 4 inches · (NO — 22.0 64.0 14.0 < ζ medicine τ « 00 RS007-001 33791 RS007-001 33626 RS0G7-001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 36412 LMW% Ο g Ο Ο rH Ο g ο 1 1 II Ο Ο ο Ο r·^ Ο CN Ο Aggregate% ο rH Ό Ο ί-Η ο (Ν Ό Ο Ο ^Τί ο 00 m ο οο ο s ο 00 ο Sugar, 24 mM histidine, 60 mM NaCl, 0.08% PS- 80 t积荽Ί ο ▼ < rH *—Η m »-Η Η g S , _·Η ΓΝ| 1—^ Ό <Ν Τ"Η Τ—Η in \q monomer% 98.21 98.20 98.16 1 98.14 J 98.37 98.35 98.06 98.05 97.86 97.66 Turbidity at 360 nm 0.269 0.273 0.291 1 0.510 1 0.287 ! 0.509 0.471 0.485 < 2 Concentration (mg/mL) 184.2 182.0 1 183.7 184.0 << KF% of moisture < &lt S «η § S »—Η, ami ami ιτΗ 耠5 4! «is Reorganization time (minutes) inch · Ο 00 CN οο (Ν rn 00 卜 00 inch · < * Μ si opal, colorless Opal, _ colorless ν. opal, Color 1 ν. opal, colorless ν. opal, colorless ν. opal, colorless < ζ (Ν (Ν &Ν;Ν <Ν τ—Η (Ν time point / temperature Ο P in inch Ρ οο Ρ 12 months , 5°C 132080.doc -80- 200909005

Acid peak % Main peak % Alkaline peak % 18.1 65.7 16.2 20.7 62.4 16.8 N/AN/A i- N/AN/A ! N/A 2$ u ^ RS007-001 33791 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 REF025 (RS007-001) 36412 LMW% 〇g 〇(N 〇0.12 〇o 〇o 0.14 inch oot—H oo 0.14 Aggregate% Ό HIH VO o 0.62 cn VO ov〇o 0.64 0.59 oo — 0.67 0.90 On 〇 Η Η Η Η r·^ rH rH 2.00 | O) 2.20 2.19 2.34 2.47 00 OS cn Monomer % 1 98.21 98.20 97.65 1 97.60 I 97.24 | 97.31 | 97.07 97.07 96.91 96.75 94.99 95.19 Turbidity at 360 nm; degree 0.269 _i 0.273 | 0.376 | [0.444 I 0.436 1 0.373 0.443 0.402 0.401 0.460 N/A Concentration (mg/mL) 184.2 182.0 N/AN/AN/A NIA N/A KF% of moisture N /AN/A 1 N/AN/AN/A Recombination time (minutes) 〇oo o (N (N inch CN (NN/A appearance clarification 1_ opal, colorless opal, colorless v. opal, flawless: v. opal , colorless v. opal, colorless v. opal, colorless v. opal, colorless v. opal, colorless N/A vial CN rH (N (N r-^ (NT~H (N <N time point / temperature o 3 weeks, 30°C 6 weeks, 30°C 9 weeks, 30°C 12 weeks, 30°C 12 mo 30°C 132080.doc -81 · 200909005 k

Acid peak % Main peak % Alkaline peak % '-< ^ (N 〇〇in \〇20.7 62.4 16.8 sac 1 m ι—> CN od <Ν ^ — 〇in in tj1 “ inch · CN (N < $ $ m RS007-001 33791 RS007-001 33732 RS007-001 34124 RS007-001 33852 REF025 (RS007-001) 33590 LMW% g Ο g Ο m ο m η ο in rH o ο ι I 1 1 (N rH oo体 Ν Η Η 00 ο 00 00 00 00 00 00 00 00 00 00 ♦ ♦ ♦ ♦ ♦ ι ι ι ι Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Body % 98.21 ι 98.20 I 96.97 97.06 96.32 96.53 96.21 95.55 94.92 94.31 Haze at 360 nm 0.269 0.273 1 0.322 1 0.328 1 0.428 0.332 1 0.339 0.364 0.359 | 0.397 Concentration (mg/mL) 184.2 182.0 169.0 174.2 164.6 180.0 < KF% of water M rn 1 1 Recombination time (minutes) 'sO inch · Ο CN «τΐ ON oo 00 〇\ (N (N in CN r-^ female clarification, si opal clarification, si opal si opal, colorless sl opal , colorless ν. opal, colorless v. opal, colorless 1 dish 13 ao <e3 dish 13 Oh 〇r—< (Ν (Ν CN (N time point / temperature 〇 〇 (Ν inch 酹〇 inch inch Ό 〇〇 inch inch 12 wk 40°C 132080.doc •82- 200909005

Dim•• 苯 故 故 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 I 2 $ V VR 00 RS007-001 33791 RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 36412 1 LMW% 〇C> om 〇1 1 1 1 S 〇O o 〇r H oo 1—H o Aggregate% 00 〇o 00 oo 〇〇ΓΛ 〇 (N oor^· o sugar, 24 mM histidine, 0.08% PS-20 t accumulation «4 ^ Ί CN <NT- H 卜r-H CN TH 00 CN t-H 卜r-H rH monomer% 98.04 98.05 [ 97.95 97.92 98.25 98.24 [ 97.89 1 97.90 "97.69 97.68 turbidity at 360 nm 0.169 0.161 1 0.143 0.160 0.154 f 0.154 1 0.163 Concentration (mg/mL) 173.3 165.4 180.0 178.5 KF% of water 1 1 ^璨4^ a ^ S ® Form, late pnit γτγΙ # >W ^ 5 ,|| >w 〇2 Recombination time (minutes) 00 in (N ¢50 r-HT^H 'o VO inch · Clarification clarification a a <e3 C/3 >: l<e3 ° medium> opal, colorless 1 1 瑞13 a 〇碟13 a 〇&lt ;〇3 dish 13 a 〇 R—Η (N t-H (N CN T—^ CN »—H <N time point/temperature Ο P inch P 00 shout P Ά ^ 12 months, 5°C 132080.doc 83 - 200909005 0〇0£

Acid peak % Main peak % Basic peak % ^ in 1-H On On rH 1—Η »—Η 20.7 62.9 16.4 Face < Face 1 Production 2 $ RS007-001 33791 RS007-001 33758 RS007-001 33506 RS007-001 33775 REF025 (RS007-001) 33586 REF025 (RS007-001) 36412 LMW% Ο Ο inch Ο ^ ^-Η Ο CS oo 1 1 1 1 S 〇Ο Ρ" Η Ο Ο o TH o aggregate % oo ο ο 00 ο ο 00 oo 卜Ο 1-H 〇 (N 00 〇—_·Η 00 ο VO <N »—H Ό r"H i1 1 H 荽Ί (N (Ν ^Η r—Η ο ^-Η 〇 〇\〇$ C\ r—K inch (Ν (N ( (Ν 〇 s rn 00 q — monomer % 98.04 98.05 ί 97.65 1 1 97.41 1 97.18 | 97.12 | 97.15 1 97.04 96.55 96.64 94.62 94.67 on 360 Haze at nm 0.169 0.161 0.162 1 0.175 J 0.176 j 0.202 | 0.196 0.195 0.203 0.195 <Concentration (mg/mL) 173.3 165.4 2; 2: KF% of moisture ΓΟ -^r < Recombination time (minutes) 00 in >10* 1 m in iTi 00 〇\ inch· 00 rn 〇\ in si opal, colorless si opal, colorless dish 13 & dish 13 ao carbon 13 Dh dish 13 a ^s3 矶 13 Oh Ο & (Ν τ -Ή <N (N CN r—Η (Ν T—^ (N time point/temperature ο 'Ο 鳑ο ^ p er〇 \〇 ^ • P 12 weeks, 30°C 12 months, 30°C 132080.doc -84- 200909005

Acid peak % Main peak % Alkaline peak % 19.4 69.5 11.1 20.7 62.9 16.4 N/AN/A 23.2 57.7 19.2 22.8 58.8 18.5 N/A ί $ Τ vs W Ϊ~ GO RS007-001 33791 RS007-001 33732 RS007-001 34124 1 RS007-001 33852 REF025 (RS007-001) 33590 LMW% II 0.07 0.06 1 0-14 | 〇_13 1 0.15 0.14 1 1 1 1 0.10 0.10 Aggregate % ! 0.78 0.77 Τ·_ H 00 o 1 0.97 0.97 0.89 ! 1 0.88 1.14 1.12 Review (N rH CN | 2.25 | 2.24 I 2.76 2.76 3.28 3.28 4.06 4.01 Monomer % 98.04 98.05 96.80 96.82 96.12 96.13 95.83 95.84 94.71 94.77 Turbidity at 360 nm 0.169 0.161 | 0.185 | | 0.184 | 0.189 0.228 0.217 0.257 0.255 Concentration (mg/mL) 173.3 165.4 N/A 165.6 151.5 177.3 175.2 i N/A KF% of moisture ΓΟ N/AN/A 1 N/AN/A Recombination time (minutes) 00 in On ,· ι in IT) Appearance clarification clarification, sl opal clarification, sl opal v. sl opal, colorless carbon 〇 > opal, colorless opal, colorless opal, colorless opal, colorless vial <N CN CN H CN Time point / temperature ο 2 weeks, 40°C 4 weeks, 40°C 8 weeks, 40°C 12 weeks, 40°C 13 2080.doc 85· 200909005

• • Benzene acid peak % Main peak % Alkaline peak % 20.8 63.5 15.6 20.5 64.4 15.2 Ν/Α 22.4 63.3 14.3 23.2 62.2 14.5 J Ν/Α Ν/Α 2 $ τ « oo RS007-001 33791 RS007-001 33626 RS007- 001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 36412 LMW% 〇Ο r—Η Ο 11 1 1 1 0.10 Ο Ο Ο r—^ t—K Ο Aggregate % 〇ο Ον Ο 0.77 0.64 m Ό Ο 0.70 ο ο r^i Ο ο : Alginose, 24 mM histidine, 0.08% PS-80 t 荽Ί inch iTi (Ν cn (N Ν cn r-^ 00 < Τ) monomer% 98.03 98.05 97.86 97.89 98.17 98.16 f 97.80 97.82 97.39 1 197.38 Haze at 360 nm 0.152 _ 0.150 0.142 0.140 0.156 0.151 0.160 1 0.171 Ν/Α Concentration (mg/mL) 167.2 157.4 I Ν/Α 180.0 183.4 Ν/Α 1 I Ν/Α KF% of water in in Ν/Α N/A Ν/Α Ν/Α ^ fI turn a ^ §窆- 纠, mei ii oml rid open condensate single master ^ Q 41 iltml σ · W\ 〇〇 Reorganization time (minutes) in Ο ο6 ΟΝ 卜 Os inch 'Ο rn Ν/Α Appearance clarification clarification ν· sl opal, colorless V. sl opal, colorless opal, colorless opal, colorless opal, Color opal, colorless Ν / Α vial CN t - Η (N rH (Ν Ν Ν η (Ν time point / temperature ο Ρ in inch Ρ tn 00 12 weeks, 5 ° C 12 months, 5 ° C 丨 132080.doc - 86- 200909005

Acid peak % Main peak % Alkaline peak % 20.8 63.5 15.6 20.5 64.4 15.2 N/A — Ν/Α I Ν/Α N/AN/A ί $ τ « Includes £ RS007-001 33791 RS007-001 33758 RS007-001 33506 RS007 -001 33775 REF025 (RS007-001) 33586 REF025 (RS007-001) 36412 LMW% 0.07 1 0.06 Lal3.. 0.13 1 ο ΓΟ Η Ο 0.12 m ο 0.10 0.10 oo Aggregate % 0.77 0.76 0.82 J 00 ο 00 ο 0.80 0.87 0.87 0.95 0.97 vo (N \q 荽Ί inch i inch 丨 2.19 | 2·! 8 1 2.62 2.66 1 1 3.22 1 3.29 j 3.74 3.74 6.74 monomer % 98.03 98.05 96.87 96.88 96.44 96.41 95.79 1 95.71 95.21 95.19 91.45 91.53 at 360 Haze at nm 0.152 1 0.150 0.177 | 0.173 0.219 0.200 0.227 0.240 0.255 0.268 N/A Concentration (mg/mL) 167.2 1 I _1 157.4 N/A Ν/Α ι_ Γ 丨Ν/Α N/AN/A Moisture KF% inch N/A Ν/Α Ν/Α 1 N/AN/A Recombination time (minutes) 〇\ 〇〇CN 〇α\ 00 Ι> 00 inch Ο in cK 卜 N/A Appearance clarification sl opal, colorless Slopa Bu colorless opal, colorless opal, colorless opal, colorless 1 opal, colorless opal, colorless opal, colorless N/A vial (N < Ν (N <N (N time point / temperature 〇 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 ° C 12 months, 30 ° C 132080.doc • 87- 200909005 f

Acid peak % Main peak % Basic peak % 20.8 63.5 15.6 20.5 64.4 15.2 < i 28.9 49.9 21.1 00 ΓΟ rn On (Ν ^―' 1 it τ « in RS007-001 33791 RS007-001 33732 RS007-001 34124 RS007-001 33852 REF025 (RS007-001) 33590 LMW% 〇〇〇ο in ο inch ο 1 1 I 1 oo Aggregate% 〇〇〇\ 00 〇(Ν 〇\ Ο 〇\ inch (Ν Ο cn <N 00 T— H r—H Ί inch F—Η inch Η r—H inch rn 芩rn IT) 00 CN — ΙΤί OS vo ON monomer % 98.03 98.05 95.50 95.41 93.80 93.91 92.75 ! 92,70 90.63 90.67 Turbidity at 360 nm 0.152 0.150 0.239 0.245 0.295 0.301 0.424 0.428 0.546 0.580 Concentration (mg/mL) 167.2 157.4 Sense 164.1 1 [175.0 173.2 (Efficacy = 122.4%) 1 171.7 KF% of moisture 1 1 < Recombination time (minutes) in ON 00 〇 (Ν Ν Ν Ν VO VO VO VO VO VO VO VO VO VO VO si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si si (N τ - Η (Ν H (Ν (N » - H < N time point / temperature 〇 " ρ Ο Ν (Ν inch, P comparison 〇 inch inch "P 酹〇 00 inch 12 weeks, 40°C 132080.doc -88 - 200909005

Acid peak % Main peak % Basic peak % CNCN^O 0s! 〇^\ TH 21.0 63.4 15.6 < 22.1 65.3 12.5 22.6 63.7 13.7 i Cr;<N On V〇iH 21.9 66.2 11.9 20.9 65.2 13.9 20.2 65.6 14.2 Si If RS007-001 33791 RS007-001 33626 RS007-001 33670 REF025 (RS007-001) 33608 REF025 (RS007-001) 33633 REF025 (RS007-001) 36188 S 〇gd inch H o inch « < ο I 1 II gogo 1 1 1 1 1 1 1 1 ••°» 韬rn c> oo ο 〇\ ο 00 Ο o 00 VO o ON κη 〇CTs d 〇〇, sugar, 24 mM histidine, 0.08% PS-80 r"H (N (Ν ί—Η SS (N Ov r—H <N m (N N 98.11 98.11 98.00 98.00 98.38 98.39 98.04 | 98.02 98.21 98.20 98.13 98.09 turbidity at 360 nm 0.186 0.153 0.138 0.149 0.146 0.163 I 0.160 | 0.158 0.168 1_ 0.164 0.155 0.154 Concentration (mg/mL) 119.3 124.5 154.7 (Efficiency = 133%) 150.7 Surface 147.9 155.2 145.6 142.9 cn i Surface SS B Sn , Rose BftL nri Pieces 1 5 ° Reorganization time (minutes) oo (N ΓΟ 卜 (N inch (Ν ο rn 卜 寸 · rn (N ON cn inch cn 卜 clarification 碡 1 /93 o ^oi > difficult ^ι > opal, colorless opal, colorless -3⁄4] o opal, colorless opal, colorless opal, colorless opal, colorless opal, colorless (N CN t Η CN <N (N &lt ;N Λβί彐 inch · 〇 time point / temperature o P inch Ρ WI οο 举 ^ sigh CJ learning Vi CN 132080.doc •89- 200909005 / \

Acid peak % Main peak % Basic peak % 19.2 69.2 11.6 21.0 63.4 15.6 N/AN/AN/AN/A 22.1 63.7 14.2 22.2 63.6 14.2 21.0 62.6 16.5 21.0 61.7 17.4 if RS007-001 33791 RS007-001 33758 RS007-001 33506 RS007 -001 33775 REF025 (RS007-001) 33586 REF025 (RS007-001) 33633 REF025 (RS007-001) 36188 LMW% 0.08 0.08 1 1 i 0.18 1 1 1 1 0.12 I 1 1 1 1 1 1 1 1 t 1 1 1 1 1 Aggregate% 0.73 1 0.74 0.65 1 0.74 0.64 I 0.64 | | 0.71 I 1 0.65 I 0.27 0.27 0.35 0.39 0.94 0.94 Ί 1.08 1.08 1.38 1 1.56 1 1-59 1-94Ί [1.80 | 2.41 2.41 3.03 3.01 2.99 2.98 Body % 98.11 98.11 97.97 97.58 97.80 | 97.76 97.23 97.55 | 97.33 97.32 96.63 96.60 96.07 96.08 Turbidity at 360 ran 0.186 0.153 | 0.176 1 0.141 1 0.195 1 I 0.175 j 1 0.188 | 0.204 0.188 0.180 1 0.252 0.259 0.218 0.203 Concentration ( Mg/mL) 119.3 124.5 N/AN/AN/AN/A 144.6 143.9 140.8 145.0 KF% of water rn N/AN/AN/AN/AN/AN/A Recombination time (minutes) 00 (N rn rn (N m rn oo rn cn rn (N (N rn o inch appearance clarification clarification Islopa b colorless | ·ί〇3 g- 1 opal, colorless | 1 opal, colorless | 1 opal, colorless | 1 opal, colorless I 1 opal, colorless | 1 opal, colorless opal, slightly yellow opal, slightly yellow opal, slightly yellow opal, slightly yellow vial (N < N r*H CM CN 1-H (N (N (N time point / temperature 〇 3 weeks, 30 ° C 6 weeks, 30 ° C 9 weeks, 30 ° C 12 weeks, 30 ° C 6 months, 30 ° C CN 132080.doc -90- 200909005

Acid peak % Main peak % Basic peak % 19.2 69.2 11.6 21.0 63.4 15.6 Meaning _ 23.6 58.7 17.7 vn (N ro d cK S (Ν a — ί ί υ f RS007-001 33791 RS007-001 33732 Γ RS007-001 34124 1_ RS007 -001 33852 REF025 (RS007-001) 33590 LMW% 〇Ο (Ν 00 00 ο Τ Η 1 1 1 1 1 Ο τ-Η Ο g Ο Aggregate% Ο Ο Ο ο ο ο (Ν 00 d 00 ο <N 〇m (N 〇ο ο Dimer% g 00 00 t-Η 1丨丨< CN ο CN rn <N CO 卜(Ν rn 卜 (Ν ΓΟ monomer % 98.11 98.11 97.29 1 97.32 96.70 :96.72 I 1_ 96.63 96.66 95.69 1 95.70 Turbidity at 360 nm 1- ! 0.186 1_ 0.153 0.198 0.171 0.195 0.185 0.228 0.202 0.225 0.220 Concentration (mg/mL) 119.3 124.5 1 149.7 146.5 145.5 (potency =144%) 148.0 KF% of water 1_ 卜ΓΛ ί i 重组 Recombination time (minutes) 00 (Ν rn ΓΛ (Ν' 00 O rn 寸 ^Τ) (Ν Appearance clarification clarification, si opal, clarification, sl opal 13 <s3 &amp ;墉Ια :sl opal 5 i colorless 13 D, 〇<e] 13 13 & ·ί〇3 Cu Ο P ο Ϊ τ·^ (Ν ί-Η CN τ—Η (Ν 1-H (Ν <Ν time point / temperature ο ^ ρ Ο Ν Ν Ν & 〇 〇 〇 〇 00 inch 12 weeks, 40 ° C 132080.doc -91 · 200909005 Example 3 As demonstrated below, Three formulations with slightly different initial protein concentrations were successfully lyophilized and showed excellent stability when stored at 5 ° C for 6 months. The stability of these formulations was observed at 8 weeks storage at 40 ° C. The stability seen previously was comparable. The stability of the pre-freeze-dried formulation stored at 5 ° C for 6 months and the recombinant solution stored at 5 ° C or 25 ° C for one week was also excellent. The initial concentration of 40 mg/mL showed slightly better stability and better recombination characteristics than the other formulations. However, recombination of this formulation allowed for a 1 mL deliverable dose at 150 mg/mL. Materials The following table lists the materials and their sources. Table 22 Article manufacturer block natalizumab Elan (77.4 mg/mL) sucrose (96 mg/mL) Elan 1% PS80 Elan 60 mM histidine Elan 6 mM histidine Elan 0.22 micron cellulose acetate filter paper Corning 5 cc vial of Kimble Grey Butyl, 20 mm; bed dry stopper, 2-slot Kimble 20 mm open cover Wheaton Table 23 _Μ_ _HPLC_ HPLC —

Uv-Vis Spectrophotometer ^

Karl Fischer Coulometric Tkrator _2H|t_ Natalizumab was used in this study. The natalizumab is the same as the 132080.doc -92- 200909005 tamizumab used to study 3 A and study 3B (AQS-2 190). The natalizumab used in these studies was prepared from natalizumab supplied by Biogenldec. Polysorbate 80 included in this material that has been formulated and filled into vials is removed prior to use in this formulation study. Briefly, the material was subjected to diafiltration into a low ionic strength, high pH (10 mM tris, 10 mM NaCl, pH = 8.5) buffer. The material was bound to a DEAE-Sepharose column under these conditions and then dissolved using 10 mM sodium phosphate, 140 mM NaCl (pH 6). Next, the column lysate was diafiltered into 6 mM histidine (pH 6) and concentrated to between 70 mg/mL and 100 mg/mL, followed by further formulation. Chemicals and Reagents All chemicals and reagents used in this study (except for notes) were purchased from VWR and are of the ACS grade or better. When a USP grade reagent is available, it is used as an excipient. The polysorbate 80 line was purchased from Sigma (Cat. No. P6474); and was derived from plants and had a low peroxide value. Formulations The following formulations were prepared by diluting the stock solutions of excipients and actives to the desired concentrations. All formulations were pH 6. Table 24: Study 4 Formulation Parameters Formulation ID Pre-freeze-drying concentration Target Recombinant Concentration 4089-1L 40 mg/mL Ab, 24 mg/mL Severe Sugar, 6 mM his, 0.02% PS80 150 mg/mL Ab, 90 mg/ mL sucrose, 24 mM his, 0.08% PS80 4089-1 Μ 45 mg/mL Ab, 24 mg/mL sucrose, 6 mM his, 0.02% PS80 4089-IN 50 mg/mL Ab, 24 mg/mL sucrose, 6 mM his, 0.02% PS80 Abbreviations: Ab = antibody, his = histidine, PS80 = polysorbate 80. 132080.doc -93- 200909005 Method Preparation of Formulations Sample formulations were sterile filtered and filled into sterile glass vials as indicated. All samples for pre-freeze-drying analysis were filled into 3 cc vials in 〇5 mL, stoppered and capped. The formulation to be lyophilized was filled into a 5 cc Kimble vial at 4.0 mL. Freeze Drying Cycle During the freezing phase, the vial containing the sample was first cooled to 5 for 30 minutes. Next, the temperature was changed to 2 minutes and it was kept under it for 60 minutes. For the final stage of freezing, the vial was ramped to -40 over 45 minutes. . The temperature was maintained at this temperature for 2 hours, and a vacuum of 100 mT 〇rr was applied for the last 2 minutes. Next, the first drying was carried out at a temperature of 1 〇〇 mT rr under a vacuum of 2 Torr for 2 hours and maintained at the temperature for b hours. The storage temperature was slowly ramped to 2 (rc) for a second drying and then at 2 Torr for 2 hours. The underarm was kept for 6 hours. The study set the pre-freeze-dried liquid and the freeze-dried cake. The vials were placed at 5 C and 40 C. They were tested at 4 2、 at 2, 4, and 8 weeks. (: Samples stored under the test. Samples stored at 5t: at 4 weeks and 6 months. At week time, 4 vials from each storage temperature were reconstituted, then 2 vials were placed at 5 ° C and 2 vials were placed at 25 ° Ct for a week-long reconstituted stability. All formulations were assayed at zero time after lyophilization. The samples were assayed by the following methods and the results are presented in Table 27_3. Not 132080.doc -94- 200909005 Checks were performed at all time points except for pre-cooled bundles The same two vials were sampled for each test after drying and cold > East dried samples. Visual inspection of all samples was performed according to visual inspection and the appearance was recorded. Check the freeze-dried cake Color, uniformity, stability and remelting Check the color and clarity of the liquid and the recombinant sample and the presence of the particles. Residual moisture and reconstitution time at zero time, use Karl Fiseher reservoir titrator to check the cold; residual moisture of the east dried cake. The appropriate volume of DI water was then gently swirled to measure the reconstitution time. Record the time at which the cake was completely dissolved. Concentrations were diluted to i mg/mL using natalizumab placebo to measure all samples. Concentration. UV absorbance was scanned from 400 nm to 250 nm at 200 nm/min using a Varian Cary 3〇〇Bi〇 and a 1 〇 claw light pathimetric tube. The absorbance at the maximum was recorded and the value was divided by Concentration was determined by adjusting the concentration of 1 498 (the absorption coefficient of natalizumab) by appropriate dilution. The turbidity was measured using a 10 mm small volume colorimetric tube (starna CeUs Inc., catalog number 16.160-Q-10\Z20). Pure samples are absorbed from 300 nm to 4 〇〇 nmi. Reported values recorded at 360 nm. Size exclusion chromatography for the determination of monomers, high molecular weight aggregates, dimers and low molecular weights in samples by size exclusion chromatography The amount of matter. The sample was loaded on the column in pure form 132080.doc -95- 200909005 and the load volume was adjusted to allow the mass on the column to be approximately 400 叩. The mass of the reference material load was also adjusted to be comparable. At 215 nm & The readout was recorded at 280 nm, however the main peak exceeded the gauge at 215 nm and was therefore calculated using the 280 nm trace. The results are now in Table 27_3. Results Pre-freeze-dried formulations

Tables 27-30 contain visual inspections for all formulations recorded, and all formulations were colorless and slightly opalescent without any visible particles ▲. At 5. (: or (d), the appearance does not change with time. No sample shows changes in protein concentration in the analysis change and no tendency is observed. For example, by measuring the absorbance of (10), the turbidity of all the formulations is: After 5 CT^ months, there was a slight increase. The thief was pre-cooled; the East dried sample showed that the turbidity increased with time for all the formulations. Monitoring the dimer, high molecular weight (aggregate) of all the formulations. And the formation of low molecular weight substances. The results are shown in the table and in Figure 17_19. Formulation of molecular formation of the substance. Pre-cooling like drying 妒 '咼 molecular weight substance formation does not seem to start until 4 weeks after storage. At 8 weeks, The small increase in high molecular weight material is more pronounced. The high molecular weight material in any formulation stored for 6 months under human C: = change. This information is shown in the appendix, but not shown here in Figure 18. The lower molecular weight is in the form of a negative aid. It is a clear tendency to form a low molecular weight material during storage under 401. The formation of a substance seems to be more than a polymer. The rate at which the formation of matter is fast enters 132080.doc -96- 200909005 仃' makes this an important degradation path in liquid samples. For the percentage of 柹σ 储存 stored at 5 °C, the low molecular weight substance is white for 6 months. There is a very small change in the knife ratio, which is shown in the appendix of the figure, but it is not shown in the chart. The initial content of all pre-^, the material is from about 11.11% when it reaches 6 months in the suburbs. 〇·18/〇. Bl0_ec has been shown to be used for this 杈疋 ϊ 下限 & & & & & & & & & & & & & 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向 倾向The total (4) loss caused by the formation of high molecular weight substances and low molecular weight substances is 45t: the lower 'monomer percentage is basically changed. This will indicate that any of these formulations will be in the form of a pre-cooled dry formulation. 2 Stable storage for up to 6 months. Analysis of the selected sample by cation exchange chromatography. In all cases, the results showed a shift to a more acidic material (de-alcohol), based on time and temperature. Storage. Various pre-freeze-dried formulations There are differences. Because of the ionic strength and the influence of certain buffer materials, the amide is generally pH-dependent, so this result will be expected by us. This data is shown in the table below. All of the pre-cooled/east-dried formulations are Results are shown similar to the pre-lyophilized formulations in studies 3a and 3B. Freeze-Dry Formulations Table 25 below shows the recombinant volumes tested to achieve a protein concentration of 150 mg/mL. Next, for the remainder of the study The recombination volume closest to the target is not determined. After each sample is completely dissolved, the concentration is checked. The recombination volumes for 4089-1L, 4089-1M, and 4089-1N are 〇85 mL, 1·〇mL, respectively. And 1.1 mL. 132080.doc -97· 200909005 Table 25: Recombinant volume formulation vial Recombination volume (mL) Degree of resolution (mg/mL) 4089-1L 1 0.8 2 0.9 —---/ 143 9 3 0.85 151 8 ~ 4089-1Μ 1 0.8 168 2 2 0.9 162.5 3 1.0 158 5 4089-IN 1 Γ 0.9 179.4 2 3 1.1 146.1 159.9 154.4 153.5 1.1 160.9 156.8 Table 26 below shows the ability to remove the dose from the recombinant sample. This is only possible for 4〇89-1N, and its system is reorganized. This data indicates that a final transferable volume of i mL at 15 〇 mg/mL will be required, a 4 mL fill volume and a protein concentration of 50 mg/mL will be required. Table 26: 1 mL dose evaluation formulation recombination volume (mL) _ im vial removal vessel (mL) 4089-1L 0.85 21 standard specification, ratio 0.71 0.75 2 0.75 a 〇r 4089-1M 1.0 21 standard specification , 11⁄2 to 1 U.〇5 0.9 2 Γ 0.8 4089-1N 1.1 20 standard size, 1 % is called 1 Uo j 1.0 2 1.0 3 1.0 Stability After visual inspection, all formulations seem to have acceptable freeze-drying The cake was verified to verify that the 4 mL volume was successfully lyophilized in a 5 mL vial. After reconstitution, all formulations were colorless and slightly opalescent, while 132080.doc -98- 200909005 did not contain any visible particles. At 5 or 4 (rc, the appearance did not change with time. No sample showed a change in protein concentration in the analysis change and no tendency was observed. For example, by measurement of absorbance at 36 〇 nm, all formulations were The turbidity was shown to increase slightly from about 〇.〇99_〇1〇4 to 〇111_ 0' 116 after 6 months at 5〇c. 4. The pre-freeze-dried sample showed turbidity of all the formulations. The percentage of moisture in the freeze-dried cake is in the range of 4.3 to 5.6, as measured by the Karl Fischer Coulometric Titrator. The average of 4089-1L, 4089-1M and 4089-1> The moisture percentages were 45, 5.2, and 4.9. The water in these samples was relatively high. The recombination time for this study was less than 丨7 minutes. Figure 4 shows the recombination time at 4 (rc). It seems to be at 40〇c. There was a slight increase in reconstitution time during storage. Samples with higher initial protein concentrations also showed a tendency to longer recombination time. This was also indicated in earlier studies (Studies A and B). Monitoring of all formulations of dimers , formation of high molecular weight aggregates and low molecular weight substances. The results are shown in the following table. Figure 5 shows the formation of a high molecular weight material at 4 (the storage period at this temperature). There is a clear tendency to form high molecular weight species. The rate of formation of aggregates appears to be in the formulation 4〇89- lL is slightly slower. Without wishing to be bound by theory, this can be attributed to the lower protein/length of the starting formulation or a slightly higher ratio of sucrose to protein. Under VC, in storage 6 There was no change in the high molecular weight material 3 in any of the formulations for a month, which showed Hmw〇/0 at various times. Figure 6 shows no more than 40. Formation of low molecular weight substances under the armpit and exhibits low molecular weight 132080.doc -99 - 200909005 The formation of a rapidly increasing amount of pre-freeze-dried sample. The lyophilized sample showed no change for 8 weeks. For 5t: the sample stored under Lv, there was no percentage of any low molecular weight material over a 6-month period. This information is shown in the appendix but is not shown here in the graph. Figure 7 shows the total monomer loss caused by the formation of high molecular weight materials and low molecular weight materials at 40t. At 5t: the monomer percentage is essentially unchanged. This will indicate that any of these formulations will be in the form of a freeze-dried formulation at 2 - 8. (: Stable storage for up to 6 months) And longer, the sample was also analyzed by cation exchange chromatography. In all cases, the results were shown at 40. (: shifted to a more basic substance after the next 8 weeks. Very little charge in the assay variability The distribution changes. The selected sample also shows a slightly more acidic shift at this point in time. This data is shown in the table below and is expressed in the last column as acid peak %, major peak %, and basic peak %. After storage for 4 weeks at 5C or 40C, the samples were reconstituted and then placed at 5 and 25t for 1 week of recombinant stability. The test was performed at the initial time point and at 1 week. The table in the appendix includes all the results recorded for all formulations. Upon visual inspection, all formulations were colorless and slightly milky white. All samples were free of particles, but two were not. Two vials containing "Liansong white particles" were stored at 4 在 before reconstitution and stored for 1 week under 25. The analyst may contaminate the vial when opening the vials for initial time point testing. No sample display There was no tendency to observe changes in protein concentration within the variability. After 5<t or 25. After i week 132080.doc -100-200909005, the turbidity of all formulations was not shown in the analytical variation range. The internal changes were measured by absorbance at 360 nm. The formation of dimers, high molecular weight (aggregates) and low molecular species of all formulations was monitored. The results are shown in the table below. And after 5 weeks under 5, the content of the high molecular weight substance in any of the formulations did not change. The content of the low molecular weight substance appeared to increase slightly after the reorganization, and at 5 (: The results were essentially the same after recombinant storage. These results show that all formulations are reconstituted and then at 5t or 25. (: will remain stable after 1 week of placement. = Recombination stability of the stored sample is also Cation exchange chromatography. The results of all the two formulations showed a slight increase in acidity after 1 week, no residual temperature before reconstitution. Samples stored at 5 C before reconstitution were also Not stored} After a week, it was slightly more versatile. The sample stored at 4 rc before recombination showed less displacement to more specific substances after 5 weeks of storage. The changes were minimal and verified. The signs of variability are better than the actual changes in protein. Conclusions The above study 4 has shown that the 4 mL volume can be successfully freeze-dried in a 5 Kimble vial with an acceptable cake structure. It was determined to give 150 mg/mL. The recombination volume of the target protein concentration was 0.85 mL for 4089-1L (40 mg/mL pre-cooled; East dry concentration), 1.0 mL for 4089-1 M (45 mg/mL pre-freeze dried concentration) and for 4089- 1N is 1.1 mL (50 mg/mL pre-freeze-dried concentration). For the only formulation capable of delivering a 1 mL dose, the minimum recombination volume of M m 132080.doc -101- 200909005 is displayed. These results can be used to guide the filling Volume, total solids and recombination volume Final Description of Development. In general, samples with lower initial protein concentrations and higher sucrose to protein ratios show shorter recombination times and slightly better stability with respect to monomer loss. According to fill volume and recombination volume' this indicates Lower final protein concentrations produced a more desirable product. Recombinant stability showed that all formulations were stable after 1 week of storage at 5 and 25 Torr without monomer loss. All two pre-freeze-dried formulations were 5 It remained stable for 6 months. However, 'at 40. (: After storage, all formulations showed a tendency to increase the amount of low molecular weight substances. The formation of low molecular weight species appears to be an important degradation pathway in liquid samples assuming its rapid rate of formation. The freeze-dried formulation also demonstrated stability for up to 6 months at rc. After storage at 4 ° C, there is a clear tendency to increase the amount of high molecular weight material, while the amount of low molecular weight material remains the same. This shows that the shape of the high molecular weight material is a more important degradation path in the freeze-dried sample. Other optimizations of residual moisture content and the ratio of sugar to protein should improve stability. 132080.doc 200909005 \ ®w-^fnm ς.ο:琛at benzoquinone: face m趔ooo-sd %so^毽建鹿WS 9 ,窭难口 S/SXU inch (Ν,^_·# ^^Ίε/ω)ε 0 inch acid peak % main peak % basic peak % 0>__〇N__rj ^ ^ 00 ^ ^ (N ON ^ inch inch 00 1 On vn v〇ί—; <Ν 00 o t> ^ (N ^ ^ ^ ^ t- Ο ΓΛ (N v〇— ν〇^―< ri (Ν ν«ο — u ^ Γτΐ REF025 (33849) REF025 (33582) <N 〇g S s $ 03⁄4 W REF025 (33838) REF025 (33851) C/3 spider 1—Η Ό CN vn <N in 〇00 r—^ 00 s 〇od ,· t> dd h-3 韬ΓΊ Ό rp (Ν 00 1—Η 〇 \ 〇〇ooo ο ο oo Ί CN >'"<> H KT) r—H ON H rH (Ν Η ο rn cn in (N s jn jn S 〇\ mm •"丨零 & quot quot & t t & & t t 湛 湛 湛 § § § § § § O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Oo Η 1—Η r-^ inch〇«] ^3 &lt ;s3 ffi VO #. 41 #. 碟• • • #- 15 ao 13 Oh 〇13 a 〇13 ο & 〇13 a 〇15 a ο 13 ao 13 Oh 〇"w ψ7λ "μ "w节η妹ΊΆ Seven time points / temperature o wf <NP o inch Ρ ο 00 40°C π Ρ -103· 132080.doc 200909005 / Prepared: Test start date: 0.5 mL Filling acid peak % Main peak % Alkali Sex peak % v〇IT) ON oi · Η α — in (Ν rn '―1 inch inch — η勹m 5沄&< 1 26.2 60.8 13.0 26.3 60.6 13.1 Life ® 2 ^ -CT> ^ 〇 2.泛 t REF025 (33849) REF025 (33582) IT) s CN 〇 £ S 9 ^ REF025 (33838) REF025 (33851) LMW% Ο 〇ΓΛ 〇ON 00 — (Ν m 〇00 t '·Η Ο Ό 聚集 Aggregate % m νο ο 〇〇00 inch ο ο τ-Η r-H 吞ο 卜 ο g cA CL, s c5 a , 窭h-3 1 a spider dimer% m CN Τ—Η ψ ·Η 00 T —Η (Ν (Ν &Ν;Ν (Ν rH (Ν rH in CN monomer% 98.02 _i 97.66 97.67 93.41 93.64 90.14 90.46 98.09 98.11 turbidity at 360 nm 0.041 0.058 0.063 0 .081 0.079 0.108 0.161 0.066 0.068 Concentration (mg/mL) cn Ο <Ν Os (N oo τ-Η Ο Appearance / pH si opal, colorless / 6.07 Extreme si opal, colorless extremely si opal, colorless extremely sl opal, Colorless and extremely sl opal, colorless and extremely sl opal, colorless and extremely sl opal, colorless and extremely sl opal, colorless and extremely sl opal, colorless TH <N <N · < (Ν (N time point / temperature 鳑Ο inch 鳑Ο inch inch &quot ;p window 〇o inch π P Ό 132080.doc •104- 200909005

Prepared: Test start date: 0.5 mL fill amount Acid peak % Main peak % Basic peak % 21.7 67.6 10.8 ^ 00 inch 卜 r·^ inch inch > 9 gas ΙΟ N/A N/A 26.5 60.6 13.0 . Inch β 2 H csj v〇 — ω ί xn w REF025 (33849) REF025 (33582) REF025 (33610) REF025 (33838) 1 REF025 (33851) LMW% _1 0.12 0.79 0.80 4.60 5·27 1 8.26 | 8.86 0.19 0.19 Aggregation Body % 0.63 0.43 0.43 0.55 0.48 ^L13^1 inch 0.48 1 ! 0.48 Grinding, 6 mM histidine and 0.02% PS-80 M荽1.24 1.17 1.17 1 ί·25 1 | 1.22 I 丨1-42 1 1.40 1.26 1.25 1 Monomer % 98.01 97.61 97.60 93.60 93.04 89.19 88.61 98.08 98.08 Turbidity at 360 nm 0.053 0.062 0.062 0.087 0.084 | 0.118 | 0.128 0.072 0.067 Concentration (mg/mL) 52.6 55.9 54.5 55.8 55.9 | 55.3 53.9 51.9 52.3 B k ° 4· Appearance/pH I -1 ! sl opal, colorless /6.08 Extreme sl opal, colorless extremely sl opal, colorless extremely sl opal, colorless extreme sl opal, colorless extreme sl opal, colorless extreme sl opal, colorless extreme sl opal, Colorless extremely sl opal, colorless vial | τ··Η τ—Η (N CN r—^ (NT—< (N time point / temperature Ο 2 weeks, 40°C 4 weeks, 40°C 8 weeks, 40° C π P m ^ VO 132080.doc •105- 200909005 ο 9Hd - 08-sd%80O^^^^sst^ :¢8 嬷H 黎Ί £ s -o-'of-f^3⁄4 崩一^IaJsv8O阳Is inch ζ , 錾 έιέ — 〇6 , 辉蚌#^-ΊεΫ 〇ς! _ Acid peak % Main peak % Basic peak % 21.1 65.1 13.8/ inch— νη—〆CO (NVO – inch oc »-H ίη ΓΛ (N Ό 19.7 63.5 16.9 20.1 68.3 11.6 20.2 67.4 12.5 20.2 67.2 12.5 On ^ O cnoooo cs...— 21.8 60.0 18.2 OOOCS ' iri rn (NO — O Bu 04 ^ in rn ω impurity +'° REF025 (33709) 1 1 1 REF025 (33582) REF025 (33610) REF025 (33838) REF025 (33851) LMW% 0.14 0.13 1 m V·— o 0.21 1- 0.23 0.24 0.20 0.16 0.17 0.15 0.16 髅崃S4 0.61 0.61 0.61 0.60 1 ! 0.60 0.63 0.63 0.69 0.67 0.59 0.59 二钤1.38 00 cn r— cn 2.19 2.18 2.63 2.60 3.39 3.37 Bu 1—^ 1.47 韬ssi- 1 97.87 97.87 97.89 96.99 1 97.00 96.50 96.56 95.76 95.78 97.83 97.83 Haze at 360 nm 0.102 0.099 _i 0.103 0.109 0.111 0.136 I 0.135 0.156 0.155 0.113 0.113 Ak (mg/mL) 164.8 164.5 153.7 161.9 157.2 159.2 159.1 166.4 164.1 153.7 152.3 — inch · ro — N/AN/AN /AN/A (N CN (N fN inch rn — rn On rn bur 00 rn ON oi m 〇 < inch πβΡ sl opal, colorless _1 sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless Vial cs m (N (N CN CN time point / temperature Ο 2 weeks, 40 °C \o 132080.doc •106- 200909005). 05.蟑》1? inch you Li Bu 0. 0 inch 衾柘 灭 side ^: ± 1 ^ 漤 domain side Ι ε < . # s \

Acid peak % Main peak 0 / 〇 basic peak % 20.2 67.4 12.5 20.2 67.2 12.5 23.0 66.4 10.6 22.4 69.0 8.6 < 20.6 69.6 9.8 20.3 70.2 9.5 SEC reference standard (average area count) iTi (N 〇g S 9 ^ W w REF025 (33604) ΙΛ) CN 〇g S 03⁄4 W REF025 (33604) LMW% ! 〇Ο (N 〇Ο 〇»—H (N 〇as <N o 卜CN 〇Aggregate% so cn Ο § o § ο cn ο m 〇in o in 〇>1 # m ^s〇CN (Ν § <N Ό in (Ν νο (N ( monomer % 96.50 96.56 96.57 96.61 96.55 96.52 96.72 96.70 turbidity at 360 nm Degree 0.136 _________________! 0.135 0.132 0.132 0.137 0.135 0.150 0.125 Concentration (mg/mL) 159.2 159.1 154.6 159.2 1 150.1 153.6 Recombination time (minutes) CTn rn Bu ^r{ 1 C) Inch · 1 Appearance si opal, colorless sl opal, colorless Sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless, & fluffy "white particles sl opal, colorless τ**Η (N (N m inch inch D3 initial storage after initial reorganization 25 〇C 132080.doc -107- 200909005

%Acidic Main peak % Basic peak % 20.2 69.2 10.7 〇〇m Γ^* cK ο H i-Η 22.6 60.2 17.2 inch — cn ^}- (N (N Ό — 1 N/A 00 α卜do od (NO — —〇〇O CN od (NO — SEC Ref Std -1 REF025 (33620) REF025 (33604) N/A REF025 (33604) LMW% i 0.19 <Ν Ο 0.22 0.23 N/A 0.25 0.27 Aggregation Body % 0.63 0.63 0.60 0.60 N/A 0.55 0.55 Ί # 〇m· C\ rH On rn r—HJN/A rn l1"·^ 00 m Monomer % 97.78 _i 97.77 97.80 97.79 N/A 97.83 97.80 at 360 nm Turbidity! 0.106 0.106 0.107 0.109 N/A 0.109 0.112 Concentration (mg/mL) 160.9 161.7 150.6 157.6 N/A 162.8 153.9 Recombination time (minutes) 00 00 cn N/A 1 Os — inch N/A Appearance 13 < aD si si si opal, colorless Ί 3 <fi3 & dish 13 o disc sl opal ' colorless sl opal, colorless sl opal, colorless sl opal, colorless vial _1 CN ".H CN ΠΊ inch CO initial initial reorganization Storage 25 °C 132080.doc -108- 200909005 I wish m ·« 〇〇〇lH»—(〇〇-—Jiricn CN^O — Γ^ — CN ri〇0〇3N〇nC MH-卜(N 〇K(N 50(NO 3^rni^ 1—H ^-H CN inch inch OC^(N CN'vO—· Bu CN — rnr^a! os inch p-iodas γ-ηΌγ〇 CN^一—〇〇—l/S inch· :ΝΌ— CN史圆δ ss nn <N〇ITi^—s CN 乂〇cn Lu〇0 D<w 〇^n Γχ,ΟΟ [3^ So - 5 inch Ο inch inch inch 1—H o (N 〇o On 〇〇\ o VO o 卜τ—H oot—H o 黩ο \oof H oo § o \〇\oo VO o 〇ro r^ o S o iT) o - by-cold) East dry '150 mg^nlj 卩 beizumab, 90 mg/mL sputum, 24 mM histidine and 0.08% PS-80, pH 6 reconstituted with 1.0 mL DI water to obtain 0.8 9 mL volume, 1荽ON cn τ-Η 〇\ ΓΛ ^1 io CN inch(N s cn 莩(N CN ON rn »—H ON CO (N 黩tml jinp in 00 s; 00 s; m 00 VO vd Os vb Os (N \〇On CN <N ^d as Os ON 00 r—H in On 00 gM«r 1. C\ sos »—H os rH o S o CN o P; r·&quot ;^ o (N cn O o ίΓ> in H o T-H rH o \or-< T-H o Fresh S f^Sb VO o (N ί〇〇\ L〇CN tn 00 s rH \〇cK r·1 i Bu CN smmy 00 m iri <〇< 1 face 1 <〇(N rn 卜 00 inch In oo m rn 00 iri o 卜·41 IS ao disc 15 Oh o 4i w * cd a 〇^s3 "S a 〇^a3 13 a 〇13 ao -H CO #. 13 & ^¢0 13 a 〇 Disc 13 o, o <s] 媸 13 ao disc IS ao ?-h (N m CN CN T*~H (N <N || system o learning '〇νοπςν-) • 109· 132080.doc 200909005 . 05.蟑 wi? inch 4Φti 3. 0 inch restaurant> _^:^^漤¥|« €£< /%

Acid peak % Main peak % Basic peak % 19.8 63.2 17.0 20.2 67.4 12.4 00 Η H inch · (N β — 22.7 68.3 9.0 Z 20.1 70.5 9.4 5 inch · oo 2 SEC reference standard (average area count) ^ (N 〇 £ S m γλ 2 0 REF025 (33604) l〇/-N (N 〇g S nj rn s ^ REF025 (33604) LMW% 〇\ ψ·^ 〇ON fH ooo G\ H oo <N 〇00 < N Ο aggregate % νο ο oso (N v〇〇o Ό 〇00 o ο δ4, 1 gong s rn (N Ov (N 00 00 (N s Ό C\ CN s CN oo CN monomer% 96.12 96.22 96.16 1 - 96.27 96.11 1_ 96.17 96.30 96.37 |With 〇^ t Υ- ^ 1- 1 0.137 1 1_ 0.132 0.131 0.131 0.136 0.138 0.132 0.130 Concentration (mg/mL) 160.8 159.6 153.7 152.2 < 148.9 1 1 147.5 Recombination time (minutes) 00 rn 1 〇T—H 1 Appearance sl opal 5 Colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless λ <fi3 &磲#丝4二趔寒<63 13 <e3 o ^4l vial CN < N m inch m inch S® initial τ-H storage after initial P丨丨 distortion recombination 25〇C 132080.doc -110- 200909005 t£4 f.

Acid peak % Main peak % Detective peak % 20.3 68.8 10.9 23.8 59.6 16.6 22.6 60.8 16.6 ! N/A ! 〇〇〇sm O (N CN < 20.2 61.8 18.0 SEC reference standard (average area count) REF025 (33620) REF025 ( 33604) N/A REF025 (33604) LMW% 0.18 i 0.19 0.21 0.22 N/A 0.25 0.25 Aggregate % 0.62 0.62 0.59 0.59 N/A 1 0.55 ! 0.54 Ί ^ 1.40 ! 1.40 1 1 * 1.40 1 N/A 1.37 1.37 Monomer % 97.79 97.78 97.80 97.79 N/A 97.84 97.84 Haze at 360 nm 0.108 1 i 0.106 0.112 0.111 N/A 0.112 0.111 Concentration (mg/mL) ----- 1 150.1 _1 149.2 145.2 145.8 N/A 141.8 140.0 Recombination time (minutes) 00 N/A m rn N/A Appearance sl opal 5 Colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal, colorless "3 ¢3 sl opal, colorless Vial CN CN inch m inch CO initial r»H Storage after initial reconstitution 1 25〇C 132080.doc -Ill - 200909005 f.

Prepared: Freed #干完求: Test start: Ι | ί r^lOOCN, ν〇 — .卜CJ inch 'tNO — ΖΓ5 64.8 13.6 Xi—(»—( JSOOCA 2DD 63.7 16.2 20.8 62.2 17.0 d<N(> r^v〇—XJOSCN rn'OON 2377 56.8 19.5 ΚΣ9 65.7 13.3 -^〇N〇gl ^ 709) ^582) REF025 (33610) REF025 (33838) REF025 (33851) -1 0.14 0.14 0.14 0.21 0.21 0.19 0.20 0.17 0.18 0.15 0.15 韬0.62 0.62 0.61 0.62 0.62 0.69 0.69 " ! 0.78 0.78 0.60 1_ 0.60 1 >1# 1.41 1.41 *—Η 2.62 2.63 3.25 3.21 ” 4.26 4.25 1.56 in T — ?.〇m®if, 24 mM histidine and 0·08%_, ρΗ6 韬iejL JIBp 97.84 97.84 97.84 96.56 96.54 95.87 95.90 94.79 94.79 97.75 97.76 rp卜贤0.099 0.100 0.099 0.116 0.115 0.135 0.135 0.157 0.155 0.115 0.114 Η bl) 161.3 168.6 170.3 164.0 159.3 163.3 159.6 162.7 166.5 149.1 149.1 inch · 卜m iri N/AN/A 1 N/AN/A Brewing Ό οο wS cn ^ S〇t·^ Os in CN 16.1 o 卜cK i^Z ^)Τ®Ί I3<a3 '"όο I3<e3 13<S] 13<s3 Fine: •«<63 〇#. !3< B3 13<s3 细: I3<e3 »*"H (N m »-H CN r-^ CN τ-^ CN CN Ο Vsoccy^ 132080.doc -112 - 200909005 .〇5.蟑>South inch哞哞^3.〇寸念柘^0|«^:^^鹚 Bromine «9£< 132080.doc Acid peak % Main peak % Alkaline peak % 1 1_ 20.8 62.2 17.0 »—· uo c> oi K (NO — 22.8 66.6 10.5 ^ CN gas 9 core 20.1 70.5 9.4 SEC reference standard (average area count) in ^ (N 〇 £ S S3 w 1 REF025 (33604) 1 1_ in ^ (N 〇g S Oh w REF025 (33604) 1 LMW% Os Ο oom (NO (N CS oor^) Ο CN 〇Aggregate% On 〇Ov VO o S oo 〇\ VO o Os o Os Ο o , 1 match In CS cn T—H (N cn 〇\ T—1 rn rn (N rn (N rn s rn S rn monomer % 1 95.87 95.90 95.92 i_ 95.97 95.86 95.87 96.06 96.07 turbidity at 360 nm 0.135 0.135 | 0.133 0.132 0.136 0.136 0.131 0.128 Concentration (mg/mL) 163.3 159.6 159.1 155.9 1 150.5 149.9 -53⁄4 Ss S Φ ^T) CM 1 00 Bu 1 Appearance sl opal 5 Colorless sl opal, colorless sl opal, colorless sl opal, colorless sl opal , colorless sl opal, colorless sl opal, colorless sl opal, colorless τ-H (N CN inch inch ca initial Wl initial storage after reconstitution 25〇C -113 - 200909005. Ton 蟑w iFiz you 袈iοας矣柘W deer side: ^^漤崁 side ls¥ /.. a\

Acid peak % Main peak % Detective peak % ΓΟ —... ο 〇 (SJ VO — 20.4 69.0 10.6 | 23.6 59.7 16.7 00 inch 00 CN CN — 21.2 61.9 16.9 inch m (N ο (N (N so – SEC reference standard ( Average area count) REF025 (33620) REF025 (33604) REF025 (33604) LMW% (Ν CN Ο 〇\ T—4 o <N 〇CN 〇(Ν Ο (N o aggregate % CN o ro 〇ON in o § o yn ο l〇o Ί # m 寸 f—^ i ο 1—Η o Monomer % 97.72 97.74 97.75 97.72 97.80 97.80 Turbidity at 360 nm 0.107 j 1_ 0.105 1 0.114 1 0.109 N/A 0.111 0.109 Concentration (mg/mL) 162.0 164.7 162.3 161.8 1 155.0 153.4 Sa S >W Φ 1 face appearance! 1 sl opal, colorless sl opal, colorless 1 sl opal, colorless sl opal, colorless sl opal, , colorless 3 ·ί〇3 &carbon Ία sl opal, colorless sl opal, colorless rH (N 1-H (N ΓΟ inch cn inch ca initial 1 storage after initial recombination 25〇C 132080.doc -114- 200909005 natalizumab re-distribution development Table 37 Pre-freeze-dried amount Name Antegren (mg/mL) Sucrose (mg/mL) Histamine (mM) PS-80 (%) A 40 2 7.7 6 0.04 B 40 46.2 6 0.04 C 40 9.2 6 0.04 Table 38 Target after freeze-drying name Antegren (mg/mL) Sucrose (mg/mL) Histamine (mM) PS-80 (%) A1 160 110.8 24 0.16 A2 100 69.3 15 0.1 B3 160 184.8 24 0.16 B4 40 46.2 6 0.04 AS 100 69.3 15 0.1 A6 40 27.7 6 0.04 Cl 100 23.0 15 0.1 C8 40 9.2 6 0.04 C9 160 36.8 24 0.16 A10 100 69.3 15 0.1 Bll 100 115.5 15 0.1 Example 4 The purpose of this study was to use experimental design to determine the effect of final protein concentration and optimal protein to sucrose ratio on freeze-dried natalizumab to prevent aggregate formation and minimize it. Experimental Design Parameters The final protein concentration of 40 132080.doc -115 - 200909005 mg/mL to 160 mg/mL was tested at a ratio of protein to sucrose of 1:100 to 1:500 to 0.01% protein per 10 mg/mL protein. The amount was kept constant at 1 mM per 40 mg/mL protein. Polysorbate 8 〇 contains 'and histidine will. The starting protein concentration (pre-cooled; Dongganyu) will be 40 mg/mL. The vial was filled at 2 mL fill and then reconstituted to the desired final protein concentration. Short-term stability was tested at 40 ° C at 0, 2, 4 and 6 weeks Procedure: f. 1. Diafiltration of the current formulation against one of the following buffers. Then ultrafiltration to a protein concentration greater than 40 mg/mL. The protein concentration was measured and diluted to mg/mL. a. Formulation SA: 1〇〇: 丨 sucrose: protein ratio _ final formulation 7, b. formulation SB: 300:1 sucrose: protein ratio · final formulation ι, 2, 5, 6, 10; c. Formulation SC: 500:1 Sucrose: Protein ratio - final formulation 3, 4, 11 β 2. Fill 2 vials of each formulation in 2 ml/vial. This allows 1 vial for stability at 40 degrees and other vials for Tg, moisture, sputum analysis, osmotic pressure, etc. The following amounts are required: a. Formulation S A : 120 mL; b_ formulation SB: 200 niL; c. Formulation SC: 120 mL. 3. Freeze the vial using a conserved loop to reduce moisture and reconstitute to the desired protein concentration. 132080.doc -116- 200909005 a. Formulations 4, 6, 8 - Recombination of 2 mL with a final indifference of 40 mg/mL; b. Formulations 1, 2, 5, 6, 10 with 0.8 mL Recombination to produce a final concentration of 100 mg/mL; c. Formulations 1, 3, 9 - Recombined with 0.5 mL to produce a final concentration of 160 mg/mL. 4. At 2, 4, and 6 weeks, stability was checked by turbidity, concentration, appearance, and S E C at 40 degrees. Other assays can be performed at time zero and at 6 weeks, such as IEX, non-reducing SDS-PAGE, and oxidation. Table 39 Formulation number Final initial final protein protein: sucrose recombinant volume 1 A 160 300 1 2 A 100 300 1.6 3 B 160 500 1 4 B 40 500 4 5 A 100 300 1.6 6 A 40 300 4 7 C 100 100 1.6 8 C 40 100 4 9 c 160 100 1 10 A 100 300 1.6 11 B 100 500 1.6 Formulation A: 40 mg/mL protein, 6 mM histidine '0.04% PS80, 27.7 mg/mL for 5 formulations. Formulation B: 40 mg/mL protein, 6 mM histidine '0.04% PS80, 46.2 mg/mL for 3 formulations. 132080.doc -117 - 200909005 Formulation C: 40 mg/mL protein, 6 mM histidine, 0.04% PS80 > 9.2 mg/mL for 3 formulations. Each vial was filled with 4 ml = 160 mg/vial as above and reconstituted to the desired volume. Total protein used = 4 mL * 40 mg/mL * 11 formulations * 30 vials 350 vials. 52800 Formulation A 600 mL 80 vials DF vs 3 It Formulation B 360 48 vials DF vs 2 It Formulation C 360 48 vials DF vs 2 It Ultrafiltration to approximately 1/2 volume. 132080.doc 118- 200909005 ο 腐_1日80 Yangshuo you 荦 杷 friends * ^ «lso.lKfo = l ^ : Lai B ^ ^ etflfiK: frame invitation.绪,[,回工弑茛ssi岔宁# 08-SCU 0/0910, bite 璁 lAt day inch<N-窭 pants day/ss 8011, sa)^uv qm/Sm 〇9一

I CN Acid peak % Main peak % Alkali peak % 15.8 66.7 17.6 13.6 71.2 15.2 〇\ inch · ci boat f**"» 15.2 70.5 14.3 SEC total peak area count 33274 39516 41343 40153 LMW% 1 1 Ο Ο Ο Ο Aggregate Body % Coffee 1 Ο c> I 1 1 1 Μ荽〇〇in ο οο ο 〇 monomer % 99.50 99.27 99.22 99.21 Haze at 360 nm 0.126 0.101 0.110 0.108 Concentration (mg/mL) 126.9 150.3 159.5 J 141.4 KF% of moisture rn Recombination time (minutes) 00 rn — inch^6 Appearance, pH value <a3 > HH IV, ΒΥ7 6.37 卜CQ > ^ ^0 CO Ο 00 00 CN 132080.doc 119- 200909005 Acid Peak % Main peak % Alkaline peak % »0 ΠΊ (N ^O 00 O On »-h <N Os On Calving Γ**, ON '«O TH σν tri v-H ^ oo in t-H r- H V0 ro m <N 卜絮犮m ON m OS O^ m 00 On mm U ifc C/1 g T—H go cJ oo 1 Sun 1 1 Ί s cn r—H (N 〇rH o oo s ( N 00 ON 00 ON 00 OV oo ON i solution 1 0.132 1 0.111 ] 0.116 0.119 B "5〇布寸m OO 旦 in (N cn r—H » H t—h ^.Se§ 00 00 CN t> s·^ rn 'pH value ifi】 CQ BY6 27 CQ appearance, >> ^ v〇>> za inch\〇OO 12 weeks-120- 132080 .doc

Acid peak % Main peak % Detective peak % 15.1 66.4 185 ^—* Γ^Ι ^ —ν〇(Ν 14.8 65.9 19.3 减减T—Η CN (Ν ν〇OS ro et m 00 ΓΛ 1 c\ Ο ο % ο Ο 韬崃1 1 I 1 τ·^ (Ν Τ 荽Ο Ί (Ν CN ^Τ) 97.81 00 (Ν 00 ο ΟΝ | Rebel 0.124 0.113 0.113 3 ^ ε Ο rH τ—Η rH Recombination time (minutes) τ-Ή — τ—Η Colorless ΒΥ7 BY7 >>> CS (Ν寸 VO 200909005 对.鹿«1m e. 一阳swxa^^杷^* Bromine* 口日9.1 昍哲昍=一念: step 01 Drive through ^茛: 3 storage. ¥i f21B draw $"0i ooo-sd %ΙΌ ,毽$3sslol ,#_ qm/3m e.69 , §bo31uvlm/§m 02

Acid peak % Main peak % Alkaline peak % 15.7 67.6 16.8 14.0 71.0 15.0 15.3 70.7 14.0 寸 Inch (NW ci inch · r~H f*·»». vH SEC total peak area count 36736 35806 r^sT 35037 LMW% 1 1 Ο goo aggregate % 1 1 Ο d 1 1 1 1 丨1荽r-Η ο ο o CN 〇 monomer% 99.49 99.28 99.25 99.21 turbidity at 360 nm 0.095 0.111 0.107 0.107 concentration (mg/mL) 00 vS 00 106.6 1 104.1 Moisture KF% Bu (Ν ζ z < Recombination time (minutes) Bu <Ν Inch CN Bu r4 (N Appearance, pH> 41 III, BY7 6.36 Bu HS? Jl〇CS Ο W! 00 Gift CN 132080.doc 121 - 200909005

Acid peak % Main peak % Alkali peak % Bu oo in 4 ci inch · f-*»· Η 00 00 in vS —"Η 14.9 69.3 15.8 15.4 68.9 15.6 SEC total peak area count 35825 35581 35055 34771 LMW% Ο Ο g Ο go Aggregate % IIII 1 1 1 1 Dimer % S (Ν Η m (Ν 00 cn H monomer % 98.90 98.81 98.67 98.55 turbidity at 360 nm 0.101 0.106 0.101 0.105 concentration (mg/mL) i_ 106.2 103.8 I ! 102.3 vd 〇\ Ο rn 00 CN Os rn Appearance, pH plate 1-1 b m > III > BY7 6.34 b ffl HH time point Ό 00 comparison (N acid peak % main peak % alkaline peak % 15.5 66.5 18.0 15.3 64.2 20.5 15.1 66.5 18.4 SEC total peak area count 1 _I 35468 35751 35788 LMW% 1 1 0.08 0.09 Aggregate % 1 1 II 0.21 Dimer % 1 _1 1.47 2.16 2.38 Monomer % 98.53 97.76 97.32 at 360 nm Turbidity 0.106 0.110 0.111 Concentration (mg/mL) 105.0 104.7 104.5 Recombination time (minutes) m ΓΠ 00 (Ν &<Ν Appearance III, colorless III, ΒΥ7 IV, ΒΥ7 time point η 5 1 I32080.doc •122 · 200909005 Luhan, 80 years old, s£f矣茫-鹿鹿1S0.I阳岔0=1念: step on 01 benzene break ^茛-"0ί. 绪七学1^茛跋迗赍学# 08-Sd 0/09ΙΌ , 绪绪摩ss 17<N ,窭维Is/S曰8·inch8 S3JS31UV 1ιχν§ε 〇9-acid peak% main peak % basic peak % ΓΛ <Ν mS ΓΟ ΓΟ rn r-Η Γ*-'· 14.9 70.8 14.3 15.1 70.6 14.3 SEC total peak area count I 31624 37676 37764 丨 fine LMW% 1 1 Ο Ο ο s ο aggregate % 1 1 0.10 IIII , 1 Gong Ο ο 0.57 S Ο monomer % 99.55 99.37 99.34 99.32 turbidity at 360 nm 0.112 0.124 0.105 0.116 Concentration (mg/mL) 121.4 148.1 145.3 138.2 KF% of water CN Ν/Α Ν/Α Ν/Α Recombination time (minutes) inch rn inch inch Appearance, pH IV, colorless III, colorless III, ΒΥ7 6.36 1 III, ΒΥ7 5# Ο I 00 12 weeks 132080.doc -123 - 200909005 r

(N Acid peak % Main peak % Alkali peak % Bu P inch Bu 9 h (Ν ^ 00 inch α ν〇y—^i Γ**"*· ' '* 4 ci inch · 1—H f·-^ Ι—^ m ο ^ 1—^ 1"*·«· 1—Η U") 〇> ^* ι—Η 37627 cn ν〇 inch m ® mm Ό mgg 〇〇ο ο 锲1 1 I 1 荽〇 Tn <Ν 00 Ί 〇ο Ο ο (N (N οο 99.11 ο 〇 as as σ < σ < 0.093 0.109 0.099 0.118 system S (N 40.8 inch · Η CO CN , , 〇 〇 ^ sd Ο ♦ ΙΙ Φ \ό 〇〇'pH value<a3 卜ffl Μ Ρ; CQ appearance, HH |—Η ΗΗ > S? wf wt CO inch 00 -124- acid peak % main peak % basic peak % 'so inch a 00 On O in V〇c^ in uS 00 iri 00 v〇00 CnJ 璨t 3742! 3764i U * WS: m Ha t> cn 1 ggoo 韬1 1 1 荽Ί r- og »—HT—^ Monomer % m (NS CTn 00 o 00 ON o inch §跶T—H oo ^H o part position 0X) 卜o inch inch.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Ifi] CQ Bu CQ Appearance> HH j—j 03 <N inch v〇132080.doc 200909005.刍«Ί日6.£阳?15£当爱刺杷^-迪_1£0. inch阳赍0=1 read: SB benzoquinone: I wish a few blessings «Life. Le seven learns one tooth tooth single 碓: miscellaneous 5Wοοοέ % inch 00, 毽绪鹿 i 9, 窭 pants IS/MSS inch, Ssb ^uvlul/&CUI J inch J_mu 09s poo-rsl Acid peak % Main peak % Alkaline peak % 15.9 67.5 16.6 Ο I—* Ον ^ r—Η < 15.2 70.5 14.3 «η ο — Bu u V0 S 33960 34758 35571 34456 LMW% 1 1 Ο 聚集 Aggregate % 1 1 ο 1 1 (Ν r—Η Ο Μ Gong〇ο 00 Ο ο Monomer% 99.56 99.35 99.34 99.35 Haze at 360 nm 0.054 0.041 ι____________ 0.046 0.051 Concentration (mg/mL) 〇〇^0 C^) Ο 00 m <Ν 00 m Ο Ο KF Moisture % m (N < 重组 Recombination time (minutes) Ο) 卜CN ΟΝ rn Ο) Appearance, pH value < 03 #- ΗΗ •ifi] 4ί |"H II,ΒΥ7 6.37 Carbon ΜΗ Time point Ο 00 <Ν 132080.doc -125· 200909005

Acid peak % Main peak % Alkaline peak % 〇〇ι~Η ι~Η t"-·* 14.9 70.6 14.5 15.4 70.2 14.4 15.5 70.0 14.5 SEC total peak area count 34667 35075 34925 35107 LMW% Ο o 〇o Aggregate % 1 1 1 1 1 1 1 1 Ί CN Ο to o § 〇m 00 o Monomer % 1 ! 99.22 99.19 | 99.12 99.10 OW 0.040 0.044 0.053 0.052 Concentration (mg/mL) ▼-H οό m OO m od <T) 00 00 ^_/ CN CN 00 <N o (N appearance, pH dish <eD HH II, BY7 6.35 •ifi3 dish HH time point! 1 i inch OO 鳑CN acid peak % main peak % alkaline peak % 15.5 66.9 17.6 15.3 66.2 18.5 15.3 68.9 15.8 v0 00 S 34997 Li Wei ro U # ω and Xfl cn % 窆1 hJ ο Ο 锲1 II S4 Ί 00 Ο (Ν <Ν <Ν (Ν 00 00 Ό 〇\ ΟΝ 00 ΟΝ 00 Ον 1 m 〇 with (4) 0.046 0.052 0.043 1 t h- 铡S 卜 c3) 卜 ^ ΓΟ m 00 cn 赍 00 00 Ο) ιη 戚Φ (Ν mw *&) »· »» ΗΗ ΗΗ ΗΗ ΪΕ轵密 CN inch ν〇132080.doc -126- 200909005 Deer ¥ Ίε ΓI Yangshuo Love 钵杷^ <^.«ls 9· IBFfo=l 衾:琛03驾^^莨:哒^莰佑«^

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Acid peak % Main peak % Basic peak % 14.9 70.7 14.4 14.9 69.8 15.4 15.3 69.7 15.0 inch On wo H τ-Η SEC total peak area ▲ 35903 35374 35697 34222 LMW% 〇Ο Ο Ο g Ο Aggregate % τ—Η Ο III 1 I 1 Ί »—Η σ\ ο (Ν monomer % 98.91 98.80 98.67 98.53 turbidity at 360 nm 0.106 0.104 0.108 0.123 concentration (mg/mL) 105.3 105.9 103.3 (Ν (Ν in ίη Appearance, pH value j 卜 jH III, BY6 6.33 03 y Time point 00 fS Acid peak % Main peak % Basic peak % ^ 00 〇〇 inch in — O ΓΟ iri vd Η ir! ^}* Ο —<N ^ \ύ 〇ό ^-Η τ—^ g 〇 〇 VO yn VO mmmm | gs CD ο 韬修 1 1 1 荽Ί >·^ <N r4 98.55 gN ON (N ON σί gm <N (N in 〇c5 1 —H d B "&0 cn o S, g deer S5 § o ΟΊ* 卜 inch rn 辋 Φ Φ 'w^ <N colorless ί 1 ____1 BY7 BY7 female • hH HH > Sh CN inch v〇132080 .doc -128- 200909005 f. Deer «1 day 6. £田钹浞|^刺杷^ -戚^1日0.寸阳岔0=1^ :琛B^^^M -.婼,『,举一绂茛龊迓赍学# "0i ooo-sd 10,韹«wws 9 , - Irfls/ss Ζ,.Ζ,ΓΝΙ, U3a)3uvqm/sm CNCN acid peak °/〇main peak % alkaline peak % O ir>r< η rn 4 “inch·f**"· τ-Η Ο ' Ον ^ Ο ^ (-«· τ—Η 15.3 70.5 14.2 SEC total peak area count 35322 35887 36694 35619 LMW% 0.09 0.06 0.06 0.07 Aggregate % II Ο |· < ί—Η ο II Gong Hao 0.58 0.54 0.54 0.69 Monomer% 99.32 99.28 99.28 99.24 Turbidity at 360 nm 0.047 0.042 0.064 0.045 Concentration (mg/mL) 37.6 Os cn 39.6 1 40.2 KF% of moisture ^s〇(Ν Ν/Α Ν/Α 1 Ν/Α Recombination Time (minutes) 00 4 νο <Ν Ο CN Appearance, pH III, colorless II, colorless II, ΒΥ7 6.34 II, colorless CQ ο I Wf 00 12 weeks 132080.doc -129- 200909005

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〇 acid peak % main peak % test peak % 15.5 66.3 18.2 15.3 64.2 20.6 15.0 66.7 18.3 V0 ^ Μ 〇s 00 00 00 〇〇cn mmgg 〇o 〇鲴1 1 Mil o 荽vn inch Ί <N <N \〇1 Η 97.35 00 ON ON | m 0.046 0.048 0.046 t η- Ο Os mm oo m 00 00 m ”, 奥奥£5琢r—H (N _ f order (N (N ffi <a) CL, Dish female HH H HH 5? 酹<N inch VO 132080.doc -130· 200909005 ^.«qm 9,lKf 0=1 衾.婼,['回H^f_!K?Iss:f drought# 08_sd 0 /0ΙΌ , 翘^^.sslrlI , 窭 pants Twss o.rnCN, S&31UY1日/Sm 〇〇1 ροο-ζ Acid peak % Main peak % Basic peak % ^ CN (N 00 00 1, gas On ^ r* ^ τ-Η ο Tt t*-»- ^·Η ^ \,〇ι/S 0's ^ V»〇jn <N vo m IT) l〇女Η擗00 ir> mmm C^i 1 gs 〇 〇osi 1 1 崃〇1 1 1 Ch Q\ 〇〇卜丨1荽o Monomer% (N <N m 00 oo Os 〇\ 00 〇\ od 〇\ od ON I Rebel (4) 0.120 0.109 0.134 0.20 1 "oi) 〇〇00 07.9 02.9 03.3 ·,, KF% of water, ι/Ί < z < Recombination time (minutes) Ο) CN Q\ X 〇. >H Disc PQ Female> HH >> ο 00 00 CN -131 - 132080.doc 200909005 \

CN Acid peak % Main peak % Alkaline peak % »—· Ον Ο OO (N Ο Bub. On t> ir! od vd »—< Ό t—( ^· od ί-Η ^s〇in in od H In in od rH r—H V0 jn ΓΛ ON 35334 mm OC 0 S goo 〇oo H-1 t> TH c\ r·*^ (N 00 CN do 〇o 蓉Ί Bu CN CN (N in ON (N On Cn CO monomer% 宕 CN CN CN vd ON 1 叛 Λ 0Λ (N (N Nd c> gt; g/m 卜 On T-H cn 00 〇 ui 辋 f let CN cn X < e3 l>>H Ph 41 ffl ffl^! ffl Appearance >>"^sd> HH S? ca 寸 \〇00 Acid Peak % Main Peak % Alkali Peak % ^T) 〇\ iri ON —tr> (N 145 55.0 30.5 卜5, r*^ t-*-1-Η CN v〇(N 00 ΙΛ) 衮犮 衮犮 S ΓΟ u iis m ω 03 〇τ—Η τ—Η S o Ο 〇mm Ό 〇\ 00 spider oo ο Ί as ΓΠ g vn S et g®(- in On rn Os 5: | ^ 0.132 § 0.133 0.139 /—SO) ^ *g 1—H ΓΛ金啦 g.,, 旦—哲 哲 in in in in in in in in in in in in in in in in in 1衾: Stepping on B benzene breaks ^莨:#>ϊ ooo-sd %so,^sfwss9 .岽,[,画H$cf!R^s5:舍学# 黎维quI/sm(N.6 , SJS31UV1S /3UI0 inch I (N acid peak % main peak % test peak % 00 inch OS uS inch - in ^-1 On Ο inch q 1 - H f**«. 〇\ , 丨丨 — ^ 's«C) Μ 〇v〇IT) (N 00 ITi 鸾犮〇m (N ro S r^) υ * 00 m | 〇〇O 鲴修1 1 1 os 00 T—^ '1荽〇单%% CN s tn 〇 \ Os 00 ON 00 ON 00 σ\ Ο JW 0.082 0.053 0.052 0.069 00 rn OO o vd m 00 m ON cn , KF% of moisture 〇ui z Recombination time (minutes) <N CM 00 <N \〇04 ON , pH value = i〇D ^63 Difficult and general appearance 1 ΗΗ KH -\ό hH HH Time point Ο Wl oo 鳑CN -133 - 132080.doc 200909005 〆

Cs| Acid peak % Main peak % Alkaline peak % O On »—· ος Inch ος t-η 〇s 〇^T) a\ in οό ^dr—H inch k »—1 Vs〇'―1 ir! ^sO 00 w^> in oo r-^ m 00 Os 〇\ tn (N mm C^) 00 00 〇ST-HO 〇〇oo system On 1—H as o (N 〇CN o (N 〇 inch ^4 00 As ΓΟ (N (N <N 00 ΓΊ (N s®l- ON VO 〇 〇嫂 四 (4) 0.046 0.053 0.063 0.049 t K ^ B IT) VO o as m 00 m 00 m as m ', 1 (N inch ΓΟ o rn CN , pH value < 〇 3 < 03 < fi3 dish « ° Appearance HH ^ 'O jH HH BWi» Se 酹南wf inch VO 00 -134- Acid peak % Main peak % Alkaline峰% cn ro cn 卜々卜oo <>ir; on in —<N ^ cc \〇—m (N ^ 00 ν〇—m ίΝ v0 \〇ιη 00 (N in m Bu mm ζΛ 卜. -1 < 〇T—^ ο 〇o ο 韬<N cn Ό 〇〇ο Ί as rn 卜 in ν〇(N in 00 in Bu 1-Η ti- Ch cn On (Ν ΟΝ 0.052 〇 ( (4) 0.047 0.055 /• V Agricultural ig/m IT) 00 C^i 00 00 fT^ Ο 00 β Recombination time (minutes) <N «3 <δ3 碡r« j—j HH ΗΗ £? CN 寸νο 132080.doc 200909005 8Tr&lt Deer Side Is 8dws.ss:^翠蚌埤钇

Bromine thorn 1 day 0.1 yang 赀 0 = 1 ^: SB benzene breaking 绂冢 SW. IK'®H$CMSISS: 赍单# ooo-sd %9S , 毽^ belongs to the art day inch (N, 窭槌Is/ox) day 8·9? s&luvls/s day 091 poo-fN acid peak % main peak % Basic peak % O in »〇0s; Inch O ^ t^* ON vd vd t> o ^ ("*** ^ ON i〇On ^ *S〇r~»4 33210 00 〇On 〇〇mv〇g U # 〇o 〇1 g 〇oo韬1 1 1 1 荽Ί C^· 〇s inch TH <N rH monomer % cn (N ON CN Os 00 ON 00 00 Os § 5 ^ 0.162 0.150 0.140 0.163 agricultural degree Lg/mL) 28.8 53.4 53.4 43.9 rH T-^ H Moisture KF% in < zz Recombination time (minutes) 〇OO o 00 inch X Oh <aD ^s3 卜>H PQ • Female>>>> 〇 OO wf C4 132080 .doc 135- 200909005 33⁄4 _ 15.2 69.0 15.8 σ\ rn 〇〇 · ^*^ m ^ 's^ 15.4 66.0 18.6 SEC total peak area count § S inch 40334 ITi o 40606 S 0.09 0.09 r~H ooo 韬崃0.18 1 (N o (N 〇# Ί 2.20 2.56 ON (N 3.44 黩s4- 97.53 97.15 96.67 96.16 turbidity at 360 nm 0.144 0.179 〇0.136 concentration (mg/mL) 153.1 156.6 131.7 ^Γ\ <〇卜^ Bu in OO appearance, PH value <63 discs > IV, ΒΥ7 IV, BY6 6.35 IV, BY7 Ss inch S 00 n ρο inch acid peak % main peak % alkaline % O OOCN iri ONvn in — inch wSo ON 寸 — in <n —>/Ί <N — mm 〇〇K〇璨女\〇νΒ 〇o 〇1 r· i< r* < »- H oo hJ 韬〇C^) m 〇\ Ο oo 荽Ί s CN 00 ί—H (N «ΤΊ ϊ> 韬O] ro ON Self-contained VO r—^ o 0.157 CN ί—H o Concentration (mg/mL ) oo w-> CN y—^ oar—H ^.Se§ 卜 ON 00 BY7 BY7 *>广>>> S5 (N inch. Ooo-sd %Γ0 ,^^^S6gl,t,flm/sule.69'sjg9-l->uvls/slu00l 132080.doc -136· 200909005

On ττ. Bromine «1UI Fuyang? 1葩赀荤14(Cup^*Deer 2nd 9.1昍哲0=1衾"^ m.岽,『,学I$CM Step s£f 举# 08-Sd./.10, 鍩崁s ss SI , 窭牦ιε/sxuΓη.69 , sa31UVTII/SUI 001 P 00 I fS Acid peak % Main peak % Alkaline peak % 04 Bu - (N α 0"\ Ο (N --H (N t***·· ^r! Os ^f inch·〆》ri r—^ ΟΛ UO r—4 '\Q v0 00 JO s (N oo 璨犮mm υ ί» ω ^ c/} rn ro cn 〇 ogg ο 〇o 韬φ 1 1 1 1 荽s τ τ " Ή m m Ί ί—H % 龉98.91 § vq 98.55 s吟00 〇\ 00 ON a 00 ogo § ik oor»H o T—H oos 'Sij rn iT) Sso ^-H recombination time (minutes) 〇CN p in ,pH\o >H ffl ^ 03 KH jH > l·-1 time point vo 00 cs •137- 132080.doc 200909005

Acid peak % main peak % basic peak % ^q r-; m O oo inch inch CN in v〇r^ O —Ό <N iri oo v〇V0 o inch m (N ro m ί g 〇〇韬1 1 1 1 (N CN et r«^ 〇 (N m CN <N OO m rn 00 Q\ σί σί t ^ oams 0.097 ooty z0"^ to inch cn dan g wS OO T-H -3⁄4 Se ^ r- Side f Φ (N (N CN , pH colorless BY7 colorless appearance hH Hj 1—| ft® £e South 恝 CN inch VO -138- 132080.doc 200909005 /%. Rhyme one: one day e_I yang s s 0 衅 衅 potassium 茫 * deer ¥ 1 9.1 昍 〇 〇 衾 衾 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩 踩SI , t^ls/ss S.SII , SJQO31UV1S/SUI 02

P8 — Z Acid peak % Main peak % Alkali peak % iri t> rH 00 0's 守 " W rH rH 'O »-h CN ^ d τι·- 1-H f*·"·» 15.6 70.6 13.8 SEC total peak Area count 36877 35319 34784 35667 LMW%; i 1 1 〇oo Aggregate % 1 1 o 1 1 1 1 Dimer % (N moo 00 VsO o fN 〇 monomer% 99.48 99.28 99.23 99.21 Turbidity at 360 nm 0.089 0.100 0.106 1_ 0.104 Concentration (mg/mL) ^sO (N 104.7 102.3 100.5 KF% of water Z < Z Recombination time (minutes) as CN inch rn 00 i〇 appearance, pH value -3⁄4) HH III, BY7 6.42 ___________ _ CQ HH Time point 00 00 compared to 132080.doc -139- 200909005 Acid peak % Main peak % Alkaline peak % 15.4 67.6 17.0 14.8 71.5 13.7 15.5 70.9 13.6 15.7 70.7 13.6 SEC total peak area count 35236 34035 33404 34370 LMW % 1 1 0.06 0.09 0.08 Aggregate % 1 1 0.11 1 1 1 1 Dimer % 0.46 0.46 0.58 0.58 Monomer % 99.54 99.37 99.33 99.34 Haze at 360 nm 0.090 0.085 0.096 0.092 Concentration (mg/mL) 82.0 100.6 97.3 100.5 KF% of moisture 〇CN N/AN/AN/A During reorganization (minutes) 卜rn Bu\ό οό Appearance, pH IV, colorless III, colorless III, ΒΥ7 6.44 III, ΒΥ7 Time point 〇f 00 12 weeks acid peak % Main peak % Alkaline peak % 15.3 71.2 13.5 15.3 70.6 14.1 00 1 -H (N 00 ^ CN 〆ci inch · lH {*-«· SEC total peak area count 34149 34039 33713 33713 LMW% 〇Ο oo Aggregate % 1 1 II 1 1 1 1 Dimer % Bu 〇Ο o cn 00 o monomer% 99.22 99.18 99.14 99.09 turbidity at 360 nm 0.096 0.088 0.097 0.100 concentration (mg/mL) 100.9 100.1 〇OS in rn inch cn O) ^J· Appearance, pH 1 III, colorless III, colorless III, BY6 6.37 III, BY7 Time point 00 12 weeks -140- 132080.doc 200909005

Acid peak % Main peak % Alkaline peak % 00^0 m (N m in t> 1 y-^ in <5 00 r-H i/t ί—H Ns〇in 〇m ^乐 m | g 1-1 〇〇韬1 1 1 荽00 ϊ—^ t"< (N o '1 韬(N (N 00 00 s®l- σ\ od oo 〇\ g(4) 粳〇 00 (N On 0.091 §昶ο 〇〇〇/—N 1 od 〇 s Recombination time (minutes) 00 inch, pH colorless BY7 colorless appearance 1-1 |—j HH Se South 1 inch Ό 132080.doc -141 200909005 Table 51 HMW% ABC 0 0.49 0.39 0.43 8 0.5 0.49 0.51 12 0.5 0.49 0.50 LMW% ABC 0 0.09 0 0.00 8 0.08 0.08 0.07 12 0.07 0.07 0.06 Monomer % ABC 0 99.42 99.61 99.57 8 99.43 99.43 99.42 12 99.43 99.44 99.43 Table 52 Kang Gan. C: Cold Dry 40 0 2 4 6 HMW% A1 A2 B3 0.50 0.51 0.45 1.48 1.47 0.77 2.10 2.16 1.08 2.62 2.59 1.27 B4 A5 A6 C7 0.44 0.51 0.58 0.79 0.78 1.45 1.45 4.23 1.11 2.13 2.14 6.43 1.25 2.61 2.56 7.98 C8 C9 A10 B11 0.76 0.77 0.52 0.46 4.31 4.32 1.46 0.78 6.32 6.47 2.10 1.11 7.74 8.14 2.57 1.25 LMW% A1 A2 0 0.00 0.00 2 0.00 0.00 4 0.09 0.08 6 0.10 0.09 B3 B4 A5 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.07 0.08 0.08 0.07 0.09 A6 C7 C8 0.09 0.00 0.00 0.11 0.00 0.17 0.08 0.10 0.10 0.09 0.11 0.10 C9 A10 B11 0.00 0.00 0.00 0.00 0.00 0.00 0.11 0.08 0.08 0.11 0.09 0.07 B3 B4 A5 99.55 99.56 99.49 99.23 99.22 98.55 98.84 98.82 97.79 98.65 98.68 97.29 A6 C7 C8 99.32 99.22 99.24 98.16 95.76 95.52 97.77 93.46 93.58 97.35 91.91 92.17 C9 A10 B11 99.23 99.48 99.54 95.67 98.54 99.22 93.42 97.82 98.81 91.75 97.33 98.68 Monomer % A1 A2 0 99.50 99.49 2 98.52 98.53 4 97.81 97.76 6 97.28 97.32 132080.doc -142- 200909005 Table 53 Cold and dry ο 4 6 812 5 8 2 4 7 9 β 5 9 1 2 3 ο ο 1 1 1- 5 12 2 4 9 ^5·5·0·1·2·3 π_3 11 11 11 It 4 2 5 ο 3 Μ 4 7 7 8 8 I ο ο ο ο ο 5 5 0 5 0 2 i34 7 7 8 8 γπ ♦ ♦ · · · 1 ο ο ο ο ο 2 12 2 3 8 ^^^-123 ο n 1- 1- 11 % V 1 ο 3 3 5 ο ^α1·5·°λ·2·4 11 Ί1 11 11

C 9 4 10 7 ·7·3·7·2·6 ο·2·2·3·Γ^ J 6 3 9 6 6 :87 3 6 1 6 C0·2·2·3·3· 16 15 6 3 1 4 777 8 B0·0·0·0·0· 0 2 2 4 4 7 1 5* Ό 9^ 3 nou 11 11 7 8 6 2 3 :97 3 7 2 7 C0·2·2·3· 3· LMW% Α1 Α2 Β3 Β4 Α5 0 0.00 0.00 0.00 0.00 0.00 4 0.07 0.08 0.07 0.06 0.07 6 0.08 0.07 0.07 0.06 0.07 S 0.11 0.09 0.09 0.08 0.10 12 0.08 0.08 0.08 0.07 0.08 Monomer % Α1 Α2 Β3 Β4 Α5 0 99.50 99.49 99.55 99.56 99.49 4 98.89 98.90 99.22 99.22 98.91 6 98.79 98.81 99.18 99.19 98.80 8 98.64 98.67 99.11 99.12 98.67 12 98.52 98.55 99.10 99.10 98.53 Α6 C7 C8 C9 Α10 Β11 0.09 0.00 0.00 0.00 0.00 0.00 0.07 0.09 0.08 0.09 0.07 0.06 0.07 0.09 0.09 0.09 0.07 0.06 0.08 0.09 0.11 0.11 0.09 0.10 0.07 0.10 0.10 0.10 0.08 0.07 Α6 C7 C8 C9 Α10 Β11 99.32 99.22 99.24 99.23 99.48 99.54 98.90 97.57 97.58 97.53 98.91 99.22 98.79 97.20 97.22 97.15 98.80 99.18 98.65 96.71 96.73 96.67 98.67 99.14 98.54 96.22 96.25 96.16 98.55 99.09 54

Freeze-drying 25〇C HMW% Α1 Α2 Β3 Β4 Α5 0 0.50 0.51 0.45 0.44 0.51 4 0.65 0.65 0.56 0.59 0.66 8 0.68 0.67 0.57 0.58 0.67 12 0.71 0.72 0.60 0.58 0.72 LMW% Α1 Α2 Β3 Β4 Α5 0 0.00 0.00 0.00 0.00 0.00 4 0.07 0.06 0.07 0.06 0.07 8 0.10 0.08 0.10 0.07 0.08 12 0.08 0.07 0.09 0.07 0.06 Α6 C7 C8 C9 Α10 Β11 0.58 0.79 0.76 0.77 0.52 0.46 0.65 0.99 1.01 1.02 0.66 0.57 0.67 1.10 1.09 1.14 0.68 0.58 0.69 1.17 1.18 1.21 0.72 0.58 Α6 C7 C8 C9 Α10 Β11 0.09 0.00 0.00 0.00 0.00 0.00 0.06 0.07 0.07 0.08 0.06 0.06 0.07 0.08 0.07 0.08 0.08 0.09 0.07 0.05 0.07 0.07 0.07 0.08 132080.doc -143 - 200909005 Monomer % A1 A2 B3 B4 A5 A6 C7 C8 C9 A10 0 99.50 99.49 99.55 99.56 99.49 99.48 4 99.25 99.23 99.48 99.35 99.28 99.37 99.35 99.27 99.28 98.94 98.92 98.90 99.28 8 99.22 99.25 99.34 99.34 99.24 99.25 98.83 98.84 98.79 99.23 12 99.21 99.21 99.32 99.35 99.22 99.24 98.78 98.75 98.72 99.21 Table 55 Pre-freeze-dried A A6 5 °C 5°C 25〇C Pre Freeze-dried B 40°C 5°C Pre-freeze-dried c 5°C C7 5°C 25〇C 40°C A1 5°C Stagnant in IV, becomes BY7 at 8 weeks C8 25〇C Stagnated in IV , becomes BY7 at 6 weeks, 5°C, 40°C, stagnates at IV, becomes BY7 at 4 weeks, 25°C, 40°C, A2, 5°C, stagnates at III, and becomes BY7 C9 at 8°C, 25 °C. Stagnated in III, changed to BY7 at 6 weeks, 5°C, 40°C, changed to IV at 6 weeks, changed to BY7 at 4 weeks, 25°C, 40°C, B3, 5°C, stopped at III, at 8 weeks. Change to BY7 A10 25 °C becomes IV at 12 weeks, becomes BY7 at 6 weeks 5°C 40°C Stagnates at III, becomes BY7 at 4 weeks 25°C 40°C B4 5°C No The main changes are all stagnant in II, colorless Bll 25 V 5 ° C 40 ° C 25 ° C 40 ° C A5 5 ° C 25 ° C 40 ° C 132080.doc -144 -

Bll 99.54 99.37 99.33 99.34 200909005 Table 56 There is no major change in turbidity at any temperature.

40°G A1 A2 B3 B4 A5 A6 C7 C8 C9 A10 B11 0 0.126 0.095 0.112 0.054 0.100 0.047 0.109 0.082 0.162 0.089 0.090 2 0.124 0.106 0.107 0.046 0.112 0.046 0.133 0.052 0.163 0.102 0.089 4 0.113 0.110 0.102 0.052 0.123 0.048 0.132 0.047 0.157 0.097 0.092 6 0.113 0.111 0.114 0.043 0.115 0.046 0.139 0.055 0.152 0.100 0.091 25〇C A1 A2 B3 B4 A5 A6 C7 C8 C9 A10 B11 0 0.126 0.095 0.112 0.054 0.100 0.047 0.109 0.082 0.162 0.089 0.090 4 0.111 0.101 0.093 0.040 0.106 0.040 0.129 0.046 0.144 0.098 0.096 6 0.132 0.106 0.109 0.044 0.104 0.046 0.148 0.053 0.179 0.100 0.088 8 0.116 0.101 0.099 0.053 0.108 0.054 0.124 0.063 0.155 0.108 0.097 12 0.119 0.105 0.118 0.052 0.123 0.046 0.122 0.049 0.136 0.101 0.100 5°C A1 A2 B3 B4 A5 A6 C7 C8 C9 A10 Bll 0 0.126 0.095 0.112 0.054 0.100 0.047 0.109 0.082 0.162 0.089 0.090 4 0.101 0.111 0.124 0.041 0.112 0.042 0.120 0.053 0.140 0.100 0.085 8 0.110 0.107 0.105 0.046 0.107 0.064 0.134 0.052 0.150 0.106 0.096 12 0.108 0.107 0.116 0.051 0.105 0.045 0.201 0. 069 0.163 0.104 0.092 Pre-freeze drying 5°C A B C 0 0.044 0.041 0.041 8 0.053 0.040 0.041 12 0.041 0.044 0.043 See also Examples 25A, B, C and D. 132080.doc -145 200909005 Table 57 Recombination stability at 40 ° C for 4 weeks at 5 ° C for 4 weeks --- 4089-1L monomer % 4089-1L monomer % —--- '**" - Μ. 5°C 0 96.5 5°C υ 97.78 0 96.56 0 ~~97J7^ 1 96.57 1 1 — 1 96.61 "1 1 ~97J9 ~~---- 25 °C 0 96.55 25 °C 0 — 0 96.52 0 -----—. 1 96.72 1 -:— 1 96.7 1 ----

96.06^6!〇7~ Example 5 The formulation described herein is a freeze-dried cake containing natalizumab, sucrose, histidine, and polysorbate 80. The pre-lyophilized bulk drug substance contained 40 mg/mL antibody, 41 mg/mL sucrose, 0.04% polysorbate 8 〇, and 6 mM histidine hydrochloride (pH 6.0). This material was filled in 4 mL per vial 'freeze-dried'! • 〇 mL water was reconstituted to 12 〇 mg/mL, ι 23 mg/mL sucrose, 〇. 12% polysorbate 8 〇 and 18 mM histidine hydrochloride (pH 6.0). The pre-V, East Dry natalizumab composition was supplied by B^gen Idec. 132080.doc * 146 - 200909005 It is formulated in phosphate buffered saline and treated in ammonium sulfate solution. This material was then diafiltered into the formulation buffer (no polysorbate) and concentrated to 40 mg/mL. An appropriate amount of polysorbate 80 was then added, and the material was sterile filtered into a polypropylene bottle and stored at 2-8 ° C for 4 weeks, followed by filling and lyophilization. Filling and freeze drying were performed in non-GMP groups using written batch records. Prior to filling, the set was sterilized and all filling operations were performed under a laminar flow hood. Use Virtis Gensis 25 EL cold; East dryer to perform cold drying. The stability study consists of three parts, one part of which tests the pre-freeze-dried natalizumab composition for up to one year at the recommended storage temperature (2-8 °C) and over the accelerated temperature of 25 °C. Stability for up to 6 months. The lyophilized composition vials were stored at the recommended storage temperature (2-8 ° C) for 12 months, at 25 ° C for 6 months, and at 40 ° C for 3 months. The vials from the 2-8 °C fraction were reconstituted at various time points and stored at 2-8 ° C (recommended temperature) and 25 ° C (acceleration) for 1 week. Regarding appearance, A280, pH, non-reducing and reducing SDS-PAGE, cation exchange chromatography and size exclusion chromatography, quality control stability tests were performed. Materials, methods and test time points The following samples will be tested. Table 58. Temple | Product Configuration Lot No. / Item No. Description Biogenldec Pre-cooled Bead Drying Item No.: LYO121504 2 mL Cold-dried vial filled with 160 mg active substance/vial in a polypropylene vial No. K65001 5 mL vial containing freeze-dried Cake and butyl rubber stopper and easy-open lid. The vials were reconstituted with 1 mL of WFI or other pure water. 132080.doc •147· 200909005 Table 59. Method of study Method number Title required volume EOP 000-01291 Determination of the appearance of freeze-dried vials 2 vials * EOP 000-01291 Moisture 2 vials EOP 000-01292 Determination of reconstitution time 2 vials * AAM 001-00141 Determination of the appearance of Antegren and matching placebo final containers** 2 vials * AAM 001-00143 Determination of pH of Antegren drug substance, final container and placebo for Antegren 1 mL* AAM 001-00380 Non-reducing SDS-Page and Reducing SDS-PAGE** 50 pL EOP 001-01198 Determination of the charge distribution of Antegren drug substance and final container by weak cation exchange chromatography** 50 μί EOP 001-01205 Size Exclusion Chromatography** 50 [iL AAM 001-00025 Concentration of drug substance according to A280, final container and placebo for Antegren** 200 μί * No damage, use vials for subsequent testing. **The method needs to be corrected, see section 3.3 below. Table 60, Study Method Method No. Title Volume required 160a.213 VCAM lysate 1 vial β 22d.530 Reducing gel wafer CE 1 vial** 22d.596 Non-reducing gel wafer CE 1 vial# 22d.544b and 22d.552a 曱 thioacetate oxidation 1 vial β 22d.84 small amount of parenteral microparticles 1 vial β * * All 5 assays use a vial. 132080.doc 148- 200909005 Table 61. Stability test and time point test method for pre-freeze-dried cakes Initial 1 month 2 months 3 months 6 months 12 months or EOS 2-8 25 2-8 25 2-8 25 2-8 25 2-8 Appearance (AAM 001-00141) XXXXXXXXXX Protein concentration according to A280 XXXXXXXXXX Size Exclusion Chromatography (SEC) XXXXXXXXXX Cation Exchange Chromatography (CEX) XXXXXXXXXX SDS-PAGE : Reducing XXXXXXXXXX SDS-PAGE: non-reducing XXXXXXXXXX pH XXXXXXXXXX VCAM lysate* XXXX Reducing gel wafer* XXXX Non-reducing gel wafer CE* XXXX Methionine oxidation* XXXX Under Elan, every condition at each time Number of samples taken 1 1 1 1 1 1 1 1 1 1 132080.doc 149- 200909005 Step on LBE 哲 ^绂荩趄矽灌W瘅, f' W forged life. 2 < 12 months or EOS 00 AXXXXXXXXXXXXXXX <N 6 months 1/5 XXXXXXXXXXXXXXX (N op AXXXXXXXXXXXXXXX <N 3 months ο XXXXXXXXXXXXXXX CN in η XXXXXXXXXXXXXX CN op fN XXXXXXXXX CN 2 months o XXXXXXXXX (N f fS XXXXXXXXX (N op fS XXXXXXXXX CN 1 month o XXXXXXXXX (N in <s XXXXXXXXX (N op fS XXXXXXXXX <N initial XXXXXXXXXXXXXXX CN test method 1 Appearance of cake Recombination time Appearance of water recombination solution According to A280 protein concentration size exclusion chromatography (SEC) Cation exchange chromatography (CEX) SDS-PAGE: Reductive SDS-PAGE: non-reducing pH VCAM solution Cellular* Reducing Gel Wafer* Non-reducing Gel Wafer* Methionine Oxidation* SVP Particles* 63⁄4 ΠΠ #七-VK Ή §接ω幸132080.doc -150- 200909005 Recombination stability at the following times Point, 4 vials were removed from 2-8 ° C, reconstituted and then placed at 2-8 ° C and 25 ° C for one week to verify the stability of the recombinant material. To facilitate the use of analytical equipment and time, samples are taken one week prior to the scheduled time point, reconstituted, placed at the desired temperature and then assayed with other samples. Table 63. Stability test and time point test method for recombinant vials Initial 3 geese month 6 months month 12 months or EOS 2-8 25 2-8 25 2-8 Appearance (AAM 001-00141) XXXXXX Protein concentration according to A280 XXXXXX Size Exclusion Chromatography (SEC) XXXXXX Cation Exchange Chromatography (CEX) XXXXXX SDS-PAGE: Reductive XXXXXX SDS-PAGE: Non-reducing XXXXXX pH XXXXXX VCAM lysate* XXX Reducing gel wafer* XXX Non Reductive gel wafer CE* XXX 曱 thioacetate oxidation * XXX Under Elan, the number of samples taken at each time point per condition 2 2 2 2 2 2 Note: Remove the vial from 2-8 ° C at the listed time points Reconstituted and then stored at 2-8 ° C or 25 ° C for one week and assayed as above. Information Appendix A: Total number of vials placed under each condition. Note: The 2-8 ° number includes the initial time point test as needed. 132080.doc -151 - 200909005 Table 64 Pre-freeze-dried block samples require temperature Elan test required number of vials too many vials required total 5 ° C 6 5 11 vials 25〇C/60%RH 4 4 8 vials total 5°C and 25°C/60% RH study 19 vials Table 65 Freeze-dried sample requirements (including for reconstitution) Temperature Elan test required The number of vials is too large. The total number of vials required is 5 ° C. 24 12 3 6 vials 25 〇 C / 60% RH 8 5 13 vials 40 〇 C / 75% RH 6 4 10 vials total 5 ° C and 25 ° C / 60 % RH Study 59 vials of materials, methods and test time points Table 66. Method of study Method number Title required volume EOP 000-01291 Determination of the appearance of freeze-dried vials 2 vials * EOP 000-01290+ or EOP 03201299** Moisture 2 vials E0P 000-01292 Determination of reconstitution time 2 vials * AAM 001-00141 Determination of the appearance of Antegren and matching placebo final containers** 2 vials * AAM 001-00143 Antegren drug substance, final container and for Determination of pH of Antegren's placebo 1 mL* AA M 001-00380 Non-reducing SDS-PAGE and Reducing SDS-PAGE** 50 pL EOP 001-01198 Determination of the charge distribution of Antegren drug substance and final container by weak cation exchange chromatography** 50 pL EOP 001-01205 Size Exclusion Chromatography** 50 μί AAM 001-00025 Concentration of drug substance according to A280, final container and placebo for Antegren* * _ 200 μί * No damage, use vials for subsequent testing. * * The method needs to be corrected, see sections 3 and 3 below. + Determine the appropriate SOP, see the text. 132080.doc -152- 200909005 Table 67. Method of Study Method No. Title Volume required 22d.394+ Karl Fischer 2 vials 160a.213 VCAM lysate 1 vial # 22d.530 Reducing gel wafer CE 1 vial β 22d.596 Non-reducing gel wafer CE 1 vial # 22d.544b and 22d.552a Methionine oxidation 1 vial # 22d.84 A small amount of parenteral particles 1 vial** A vial is used for all 5 tests. + Determine the appropriate SOP, see the text. Required Corrections for Calibration The size exclusion chromatography was modified to include data collection at A280 nm wavelength. Note: The increased mass of approximately 40 pg to 400 pg on the column requires the use of smaller sensitivity wavelengths. Section 3.5, Table 5: This table was modified to show that a total of 4 vials were required for the initial time point test at 3 months and at 12 months or at the end of the study. Note: This table ignores the other 2 vials required to consider the moisture test at these points in time. This test is destructive and therefore the contents of the vial cannot be recycled for other uses. 132080.doc -153 - 200909005

/V Record 葩啻«^铖茛趄祝灌w^,f' W 锻佑务^·89< 12 months or EOS 00 rs X x XXXXXXXXXXXXX inch 6 months\n <NXXXXXXXXXXXXXXX inch 00 <s XXXXXXXXXXXXXXX Inch 3 months ο XXXXXXXXXXXXXX (N ITi fS XXXXXXXXXXXXXX <N 00 fS XXXXXXXXXXXXX CN 2 months o XXXXXXXXX (N ΙΛ fS XXXXXXXXX CN op <s XXXXXXXXX (N 1 month o XXXXXXXXX (N IT) n XX: XXXXXXX CN Op AXXXXXXX! XX (N initial XXXXXXXXXXXXXXX inch test method cake appearance reconstitution time moisture reconstitution solution appearance according to A280 protein concentration size exclusion chromatography (SEC) cation exchange chromatography (CEX) SDS-PAGE: reducing SDS -PAGE : Non-reducing pH VCAM lysate * Reducing gel wafer * Non-reducing gel wafer * Methionine oxidation * SVP particles * S® ππ - Seven self-deficient S-connected t Φ 132080.doc 154. 200909005 uorsisDV so^s inch 0SIOA1 face 驷 Gong 荽 old W锄铋¥§f 一 *龊佑餸令ύΐ·#— - S 69< Τ1:-ρετρς " $$ :琛01锄铋

®M·^娜日一 S wa-^H'ltve^^ : «B 苯 趄 趄 耱 耱 Twsso 锖 锖 s 糇 : 糇 糇 糇 尺寸 尺寸 尺寸 尺寸 尺寸 尺寸 尺寸 L L L L L L L L L L L L L L L L L L L L L L L -^ (Ν Ο (N 〇 purity % 99.7 99.7 SDS-PAGE reported non-reducing 韬〇Τ-Η On consistent with the reference standard MW consistent with reducing IgG% in cK Os 99.2 cation exchange chromatography (CEX) Reported value 4th peak % 69.6 69.4 Higher isoforms % rn 00 — Lower isoforms % or—< 00 in 1—^ pH value reported o Concentration reported value (mg/mL) Appearance reported observations «V · _赛id趦^*rb»iv *s O *^3^3 Carbon Φ ^ «V · · 'Wf ·- 〇<ώ3 Time point (month) o 1-H 132080.doc -155- 200909005

The test size exclusion chromatography performed Η τ Η (N 〇 (Ν m cn m ο 99.7 99.7 99.7 99.7 SDS-PAGE 00 cn od 卜 Os coincides with the coincidence 99.5 99.4^ 99.4 100.0 cation exchange chromatography ( CEX) 70.8 69.4 68.9 i 70.2 CN* 12.4 14.2 〇\ <'i »-Η 17.2 18.2 16.9 17.9 pH CN 'Ο 2 Concentration r·'< Ο »—H 1—( Appearance is colorless, slightly milky white. : 0 «V 1 •so 哉*ffi 〇Colorless, slightly milky white. Granules: 0: colorless, slightly milky white. Granules: 0 time point (month) CN VO 132080.doc -156- 200909005 uo(N:si>v : ^典#VOJTf/ v/N^^.wfF^/<«09 玑佑馎女蜱#乱赊0卜< :琛B?F鹓寸 os^—ιΟΛΙ 1-ΜΜη日sw-B -^, f, 肊铋 揉 苯 苯 苯 苯 ^ 黎 黎 黎 黎 黎 黎 黎 : : : ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ L L L L L L L L L L L L L Τ-Η i 1 荽(N Ο m ο m ο mo 寸ο 〇 〇 On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On On Mm 纥nn -«r 柒m -Hr street mr Authentic IgG% 1 IT) σ; On f"»H Os ON cK 〇\ ϊ—H 〇< ON 00 od O m 4 1 Disadvantages 麻 OS cK VO od <N cK <N ^dv〇〇 n?荽寸寸·inch o (N 〇rn lower with i function variant % 〇ϊ> ir> v〇(N 卜姆Μ α 磔Ο vd ί—Η ^d ^dv〇rH ^d reported value (mg /mL) inch τ-^ inch t^H inch inch Oi 襄®^· 璘φ睽^°r 蕤〇趄 蕤〇趄 ΙΝ / /.: difficult to light <fi3襄IIUd feed carbon light O is i®Un pier φ踏^〇c? 魂寒·轻〇<N mv〇132080.doc -157- 200909005 uoCN^cyv Ι00ς9β inch osioahj

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Size Exclusion Chromatography Reported Value Impurity % LMW2% r—( ι~Η oligo % m ο m ο Formula 1 α; <η ο SDS-PAGE Reported value non-reducing ο 鲽S ^s ·φ® Consistently compatible reducing IgG% 99.52 Ον cK as cation exchange chromatography (CEX) reported value 4th peak % inch 〇 < inch cK higher isoforms % inch cn Ι"·Η in — lower isoforms % &lt ;Ν r*^ Recombination time recombination time i 1 llm29s 7m38s IZJ ΙΛ OO TH — inch BS Bu pH value ο 2 Moisture 1 1 1_ Reported value _ 0.3% i.._— Pd z Concentration reported value (mg/mL ί____ m rN Appearance 1 reported observations 1 solution -. β寒<s3 Zhuo* g. <fi] _ <s3 single disc invitation φ i white 1 cake <fi3 荽•ffi ~ 銮蓉 time Point (month) 〇:t«H 132080.doc -158- 200909005 Test size exclusion chromatography performed 1 1 rn iH 〇' cn rn 寸 o 〇 α m α; 〇\ ΓΛ α; as Ch Os 1〇 〇; SDS-PAGE ο 00 <η 〇〇απ 女纥m street mr ΠΠ -Wr ir>σ< ON Bu Os Bu On Os 〇〇 cation exchange Analysis (CEX) Ο in cK 00 00 Ό in ο CN TH 卜 (N CN »n VO (Ν <N —·_·Η oo 卜: 〇\ in Ον 'Ο rH Recombination time 6m47s 7m08s 5m22s 4m 16s 卜m57s 4m40s 4ml5s 6m36s 8ml7s Κ & 2 CN <Ν \ό 〇< Pi !z <N 〇οο ο ITi ro TH »—H (N (Ν rH colorless, _ lightly milky white. granules. - product. < E3 ^ ^ 〇 invite φ i 5 ^ \ltV ° , e 寒 ^ H H 碡邈 · ® i丄ό • g. Ο寒 <e3 〇 〇 Invitation Φ趁ό White 1 Piece<n ^ ^ 5 White 1 Piece White 1 Pie Time Point (Month) (N m (Ν ο££0-ιη0-ΙΟ#Ή&¥#·£ D^fJKw^^K-?wls£♦Β衾铖柃盘-驷驷靼ΗβΝ. Οοο-ιηο #ΉΙΊ^4Ν#ΞΙ触岭^)甽蹀恻遂-驷硪^茛&#;#100絮苍^. ^寒筚令%4*-^竦-1教¥衾款二132080.doc -159- 200909005 uo(n:sdv 1001093⁄4 .0 V/N#^_W?F 铋¥[«筑091一广^ :琛B锄铋.崩崩 itchy seven Zhaoye tan leather 嘁 嘁 EM qm 一阳.谰4fe屮蝴蝶鲶录^h^xl#^ rate W龊佑餸佥杷命HH'qmln inch 0SICNIOA1 :辗驷钿铋: «B stray plow race qm / SUI0 (NI ? F 雄 ^ ΐ * S : Fortunate %ιη - Η δ 0 / 009 / p (n-hps (n 漤砟獾 size exclusion chromatography reported value Μ LMW2% 1—Η m ο ο Purity % v〇cK m ο SDS-PAGE Reported value non-reducing ◎ l> o T~H ξ Y 4 field απ match 2 match! Reducing IgG% 99.522 00 cK ο\ cation exchange chromatography (CEX) Reported value 4th peak % inch Os VO CN Higher isoforms % inch cn t—^ Lower isoforms % CN Τ-Η ^-Η Recombination time recombination time llm29s 7m38s ΧΑ ζΠ 00 Τ-Η Η Inch β ε 卜 〇 〇 pH value reported o Moisture report value 0.3% Ρί ζ Concentration reported value (mg / mL) 1 m T-H Ό CN Appearance observation results solution colorless, slightly milky white. Particle 1 'colorless, light milk i white. granules · 1 cake white 1 1 white 1 cake time point (month) 〇132080.doc •160· 200909005 1—Η r-^ 〇 00 Ο Ο CN cK —_Η σν ΟΝ 00 00 Os m ο τ-Η (Ν οό inch 00 coincidence 1 1 coincidence coincidence cK ON 卜^sO cK C\ o ι〇cK m 00 VO CN (Ν y—io 〇l> vd W VI (N On mo ssv〇v〇6ml7s 5m41s 4m37s 4m45s 6m27s3 (Ν Pi 2 o o 卜^―H iT) m ,^._ · β _ <a3 light - the dish is colorless, slightly milky white. Particles 0 colorless, light milk _ white. Particles · 0,0, 1 > ο3 White 1 Pie _ ί White 1 Pie 1 White 1 Pie (Ν m οΓηεο-(τιο-ΙΟ^ΉΙα^^.ε♦Β衾Β衾柃讅驷柃讅驷^=ΉΝ. (卜00;0 #£Ί^4#ΞΙ舾冷¥)蜊蹀恻#驷嫦祝茛<Ying 100 Driving Cang_<Ν 132080.doc -161 - 200909005 uoCN^av5059:3⁄4 inchΟΠΠΟΛΊ /~ \ "0 捃,『,/-^«09 鲅V/M 龚劣赛W拉铋佑餸佥*运衅#华听qul/sm§ $$ KB喇鹖

.崩瘰瘰七^^衮莩钵嘁EM Ί 祀 1祀.渊4fe屮蚋鲶趁fh^^^^ rate>玑佑«佥杷^婼,『,18 5 智裸τ?:ΓΗΉΟ/ος -HS o/oszycu-Hpo inch size exclusion chromatography reported impurity % LMW2% F—1 Τ—Η 荽m· CN Purity % 99.6 98.7 SDS-PAGE Reported value non-reducing half antibody % 10.7 〇\ «l· t <β® on match 2 match 1 1 Reduced IgG% 99.5 ^ 99.6 Cation exchange chromatography (CEX) Reported value 4th peak % 69.4 68.8 Higher isoforms % 13.4 15.0 1 Lower isoforms % 17.2 16.2 Recombination time Recombination time llm29s 7m38s 5m30s 1 4m40s pH report o Water report Value 0.3% NR Concentration reported (mg/mL) ^O y ·Ή in (N Appearance observations The solution is colorless, slightly milky white. Particles Γ Colorless, light milk: white. Particles: 1 Piece white 1 Pie White 1 Piece BWi /—n S£ Πί: 〇T^H 132080.doc -162· 200909005 (N 〇v〇〇ΟΪ 98.3 97.7 〇〇σ< (N 00 coincidence 99.5 99.4 69.5 68.6 13.2 12.9 17.2 1 "18.4 5m40s 5m38s 4m38s 6m30s 2 CN NR NR m as (N none Color, slightly milky white. Granules = 1 colorless, slightly milky white. Granules 0 white 1 cake white 1 cake 1 CN cn 132080.doc 。 箪 令 % ^ ^ ^ ^ ^ 令 令 令 令 令 徙 徙 . (Bu 00;0#^11 舾琮驷4舾琮驷岭1〇甽杂蜊螽驷嫦^莨<垅1〇〇笨苍^^163 - 200909005 uoCNisov so^s inch srslloAl

:糇I 1S/3UI § , /§\ "S tr-^ Cui, f, deer side 婼7/-^#091 载佑 «4^运鲚#^ Apart one: - aeTPlrl, 荠 bracelet drama I &lt Is ς fF Zen "$$ : «B Lag size exclusion chromatography reported impurity % LMW2% 1—( inch 〇荽 〇荽 rn rn 〇 〇 purity % 1 99.6 1 m cK cK ON SDS-PAGE reported value Non-reducing half-antibody % rH 〇 & 6 < 〇 B απ conforms to coincidence reduction | IgG% 99.5 99.7 99.7 cation exchange chromatography (CEX) reported value 4th peak % 69.4 1 69.5 69.0 higher isoforms % rb Inch (N > 1 < lower isoforms % \〇00 Ον recombination time recombination time i 1 Pi 5ml7s 4m27s C/3 C/3 ι»Η 〇SB inch Ό pH report value 〇 'O 2 concentration Reported value (mg/mL) CN mt-H mm Appearance observation The solution is colorless, slightly milky white. Particles Γ Colorless, slightly milky white. Granules: 0 colorless, cake white 1 cake white 1 cake white 83⁄4 5a Nc: 〇m 132080.doc -164- 200909005 in 〇99.5 10.5 1 match 100.0 70.5 12.5 17.1 5m 59s 6ml8s (N VO 137 is slightly milky white. Granules · 0, 1 colorless, slightly milky white. Granules 0, 1 1 cake white 1 cake 1 CN 132080.doc . ^ 苯令令%^*,^锶4敦¥衾_1 -165- 200909005 uofNisbv 100593⁄4 inch οςιποΛΊ

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0/OS $$ SB 嘲 l lTf, 镩镩 1 1 - Is dM - s mi c / 009 / p (NTPln (N size exclusion chromatography reported impurity % LMW2% τ-Η inch ο rn ο purity % 99.6 99.2 SDS-PAGE j Reported value non-reducing Ο Ο 与 与 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 还原 阳离子 阳离子 6 6 6 6 6 6 cK higher isoforms % rn Os r~H r~H lower isoforms % O vd Γ 重组 recombination time recombination time Pd Z 1 7m 16s 5m05s pH value reported 〇»—H concentration reported value (mg/mL 1 CN T-^ Inch appearance report observation solution 1 1 1 Colorless, slightly milky white. Particle r colorless, light milk 1 white. Granules. 0 Pie 1 Pci White 1 Pie 93⁄4 ^ Sa Ο r〇132080.doc •166- 200909005 w-' ΙΌ Ό Ό<ν·66 6·α Street Essentials·66 S.69 6·α Inch./,1 s Bu ςιπς si Γ9 s I , 0 :#碟οβφ Ί5Ι* Fantai I β-ίπ 绂莨W哋铋K-?fs£啻农贼柃龠ik鳔皞=ΉΝ.智单絮令%^*-^珲-1軚^葙葙! 132080.doc -167 200909005 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the formation of a high molecular weight material at 30 °C. Figure 2 shows the formation of high molecular weight materials under hunger. Figure 3 shows the formation of low molecular weight materials in a pre-lyophilized solution at 30 C. Figure 4 shows the formation of a low molecular weight material in a solution that is dried under pre-cooling. Formation of Forms of Low Molecular Weight Substances and Low Molecular Weight Substances Figure 5 shows the total monomer loss caused by high molecular weight species at 5 °C. Figure 6 shows the total monomer loss caused by high molecular weight formation at 30 °C. Figure 7 shows the total monomer loss caused by the polymer at 40 °C. Figure 8 shows the shape of the substance and the low molecular weight substance. It does not cool down at 5 C. The mouth of the east dry mouth + n depletes the monomer loss over time in your ligand. Figure 9 shows the cold and dry at 30 °C. The mouth lost from the monomer in the % < formulation over time. Figure 10 shows monomer loss over time in a 4 (TCf cold-bundled formulation. Figure 11 shows the formation of low molecular weight species for each formulation at 30 C. Figure 12 shows the formulation at 40 C. Formation of Low Molecular Weight Substances Figure 13 shows the formation of high molecular weight species in a pre-freeze dried formulation at 40 C. Figure 14 shows the monomer loss caused by the formation of aggregates at 5 C. Figure 15 shows at 30 C Monomer loss caused by the formation of aggregates. 132080.doc -168· 200909005 Figure 16 shows the monomer loss caused by the formation of aggregates at 40 ° C. Figure 17 shows the pre-freeze drying at 40 ° C Formation of high molecular weight material in the sample. Figure 18 shows the formation of low molecular weight substances in the pre-freeze-dried sample at 4 (TC). Figure 19 shows the total number of high molecular weight substances and low molecular weight substances formed by tf at 40 C. Volume loss Figure 20 shows the recombination time at 40 ° C. Figure 21 shows the formation of high molecular weight material in a freeze-dried sample at 40 ° C. Figure 22 shows the sample in lyophilization at 40 ° C Formation of low molecular weight substances. 23 shows the total monomer loss caused by the formation of high molecular weight substances and low molecular weight substances at 40 ° C. / Figure 24A shows the formation of high molecular weight substance I in a pre-lyophilized sample at 5 ° C. Figure 24B shows The formation of low molecular weight material in the pre-lyophilized sample at 5 ° C. Figure 24C is shown in 5. The monomer loss in the pre-freeze-dried sample. Figure 25 shows the sample freeze-dried at 40 ° C. Formation of medium to high molecular weight materials. Figure 26A shows the formation of high molecular weight species in recombinant samples at 40 ° C. Figure 26B shows the formation of low molecular weight species in recombinant samples at 40 ° C. Figure 26C shows recombination at 40 ° C Loss of monomer in the sample. Figure 26D shows the time of recombination. 132080.doc •169-

Claims (1)

200909005 X. Patent Application Range: 1 · A stable freeze-dried formulation prepared by freeze-drying an aqueous formulation, wherein the aqueous formulation comprises: (a) from about 20 mg/ml to about 80 mg/ml Natalizumab; (b) a buffer having a pH of from about 5.5 to about 6.5; (c) from about 20 mg/ml to about 80 mg/ml of sucrose; and (d) from about 0.02% to about 0.08% polysorbate. 2. The stable freeze-dried formulation of claim 1, wherein the aqueous formulation comprises from about 30 mg/ml to about 80 mg/ml of natalizumab. 3. A stable freeze-dried formulation according to claim 2, wherein the aqueous formulation comprises about 40 mg/ml natalizumab. 4. The stable cold-dried formulation of claim 1, wherein the buffer has a pH of about 6.0. 5. The stable freeze-dried formulation of claim 1, wherein the buffer is histamine. 6 'A stable freeze-dried formulation as claimed in claim 5, wherein the histamine is present in the aqueous formulation at a concentration of from about 1 ηι to about 12 mM. 7. The stable freeze-dried formulation of claim 6, wherein the histamine is present in the aqueous formulation at a level of 6 mM. 8. A stable freeze-dried formulation according to claim 1, wherein the sucrose is present in the aqueous formulation at a concentration of from 20 mg/mi to about 80 mg/ml. A stable freeze-dried formulation of Item 8 wherein the sucrose is present in the aqueous formulation at a concentration of the spoon. 132080.doc 200909005 A stable freeze-dried formulation of Kiss 1 wherein the polysorbate is polysorbate 80. A stable freeze-dried formulation according to claim 10, wherein the polysorbate @曰80 is present in the aqueous formulation at a concentration of from about 2% to about 8%. 12. The stable freeze-dried formulation of the invention of claim 11, wherein the polysorbate is present in the aqueous formulation at a concentration of about 0.1%. The stable cold-dried formulation of Ge Xiang 1 wherein the weight ratio of natalizumab to sucrose in the aqueous formulation is from about 〇 5 : 丨 to about 2: 丄. 1. A stable freeze-dried formulation such as Kiss 13, wherein the weight ratio of natalizumab to sucrose in the aqueous formulation is about 1:1. 15. The stable freeze-dried formulation of claim ,, wherein the molar ratio of sucrose to natalizumab in the aqueous formulation is about 3 〇〇: 丨 to about 500:1 〇 16. The stable freeze-dried formulation of item 15 wherein the molar ratio of sucrose to natalizumab in the aqueous formulation is about 4 〇〇: 丨 to about 50,000: 〇 17. as in claim 16 A stable, reduced dry formulation wherein the molar ratio of sucrose to natalizumab in the aqueous formulation is about 4 5 〇: i. 18. A stable freeze-dried formulation according to claim i, wherein the aqueous formulation further comprises a bulking agent. 19. A stable cold, east dry formulation of the present invention, wherein the aqueous formulation further comprises a tonicity modifier. 2〇·如凊求! A stable freeze-dried formulation wherein the aqueous formulation 132080.doc 200909005 comprises: (a) about 40 mg/ml natalizumab; (b) about 6 mM histidine, having a pH of about 6.0; ) about 41 mg/ml sucrose; and (d) about 0.04% polysorbate 80. 2 1. A stable recombinant formulation comprising: (i) from about 80 mg/ml to about 160 mg/ml of natalizumab; (Π) a Η value of about 6.0 to about 18 mM histidine; (iii) about 123 mg/ml sucrose; and (iv) about 0.12% polysorbate 80; wherein: the recombinant formulation is a stable cold/east dry formulation prepared by freeze-drying an aqueous formulation To prepare, the aqueous formulation comprises: (a) about 40 mg/ml natalizumab; (b) about 6 mM histidine, pH about 6.0; (c) about 41 mg/ml sucrose; (d) about 0. 4% polysorbate 80. 22. The stable recombinant formulation of claim 21, wherein the stable recombinant formulation comprises about 120 mg/ml natalizumab. A method of preparing a stable recombinant formulation comprising recombining a lyophilized formulation prepared by lyophilizing an aqueous formulation comprising: (a) from about 30 mg/ml to about 60 mg /ml of natalizumab; (b) a buffer having a pH of from about 5.5 to about 6_5; (c) sucrose from about 20 mg/ml to about 50 mg/ml; and 132080.doc 200909005 (d) From about 0.02% to about 0.08% polysorbate. 24. The method of claim 24 wherein the aqueous formulation comprises from about 4 mg/ml to about 50 mg/ml of natalizumab. 25. The method of claim 25 wherein the aqueous formulation comprises about 4 mg/ml natalizumab. 26_ The method of claim 23, wherein the buffer has a pH of about 6. 27. The method of claim 23 wherein the buffer is histamine. [28. The method of claim 28, wherein the histamine acid is present in the aqueous formulation at a concentration of from about 1 mM to about 12 'mM. The method of claim 29, wherein the histamine acid is present in the aqueous formulation at a concentration of about 6 mM. 30. The method of claim 23, wherein the sucrose is present in the aqueous formulation at a concentration of from about 2 mg/ml to about 5 mg/ml. 3. A stable freeze-dried formulation according to claim 31, wherein the sucrose is present in the aqueous formulation at a concentration of about 40 mg/ml. (32) The method of claim 23, wherein the polysorbate is polysorbate 80 = 33. The method of claim 32, wherein the polysorbate 8 is about 2% to about 0.08 The concentration of 5% is present in the aqueous formulation. The method of claim 33, wherein the polysorbate (tetra) is present in the aqueous formulation at a concentration of about omega. The method wherein the weight ratio of natalizumab to sucrose in the aqueous formulation is from about :5:1 to about 2:1. 36. The method of claim 35, wherein the aqueous formulation is The ratio of the ratio of the sucrose to the sucrose is about 丨:j. The sucrose to the sucrose of the sucrose to him. 37. The method of claim 23, wherein the aqueous preparation of the benzumab The ear ratio is about 300: 丨 to about 5 〇〇: 1. 3 8. The method of claim 37, wherein the molar ratio of the benzumab in the aqueous formulation is about 4 〇〇: 1 to about 5 〇〇. 39. The method of claim 38, wherein the molar ratio of the recommended sugar to natalizumab in the aqueous formulation is about 450:1. The method of claim 23, wherein the aqueous formulation further comprises a swelling agent. The method of claim 23, wherein the aqueous formulation further comprises a tonicity adjusting agent. 42. The method of claim 23, wherein the aqueous formulation Contains: (a) about 40 mg/ml natalizumab; (b) about 6 mM histidine, pH about 6.0; (c) about 41 mg/ml sucrose; and (d) about 0.04% poly sorbitol Alcohol ester 80. 4 3. A method of treating an individual comprising administering to the individual a therapeutically effective amount of a stable recombinant formulation, such as a clearing item 23, wherein the individual has a Anti-treatment of the disease. 132080.doc