USOO8883146 B2

(12) United States Patent (10) Patent No.: US 8,883,146 B2 Fraunhofer et al. (45) Date of Patent: *Nov. 11, 2014

(54) PROTEINFORMULATIONS AND METHODS (56) References Cited OF MAKING SAME U.S. PATENT DOCUMENTS (71) Applicant: AbbVie Inc., North Chicago, IL (US) 4,597,966 A 7/1986 Zolton et al. 4,877,608 A 10, 1989 Lee et al. (72) Inventors: Wolfgang Fraunhofer, Gurnee, IL (US); 5,237,054 A 8, 1993 Brinks et al. Annika Bartl, Ludwigshafen (DE); 5,608,038 A 3, 1997 Eiblet al. Hans-Juergen Krause, Biblis (DE); 5,702,699 A 12/1997 Hanish et al. Markus Tschoepe, Hessheim (DE); 6,090,382 A T/2000 Salfeld et al. Katharina Kaleta, Ludwigshafen (DE) 6,165.467 A 12/2000 Hagiwara et al. 6,171,586 B1 1/2001 Lam et al. 6,252,055 B1 6/2001 Relton (73) Assignee: AbbVie Inc., North Chicago, IL (US) 6,258,562 B1 7/2001 Salfeld et al. 6,281,336 B1 8/2001 Laursen et al. (*) Notice: Subject to any disclaimer, the term of this 6,436,397 B1 8/2002 Baker et al. patent is extended or adjusted under 35 6,485,932 B1 1 1/2002 McIntosh et al. U.S.C. 154(b) by 0 days. 6,509,015 B1 1/2003 Salfeld et al. 6,693,173 B2 2/2004 Mamidi et al. This patent is Subject to a terminal dis- 7,070,775 B2 7/2006 Le et al. claimer. 7,223,394 B2 5/2007 Salfeld et al. 7,250,165 B2 7/2007 Heavner et al. (21) Appl. No.: 13/774,735 7,276,239 B2 10/2007 Le et al. 7,541,031 B2 6/2009 Salfeld et al. (22) Filed: Feb. 22, 2013 7,588,761 B2 9/2009 Salfeld et al. 7,758,860 B2 7/2010 Warne et al. (65) Prior Publication Data 7,863,426 B2 1/2011 Wan et al. 7.919,264 B2 4/2011 Maksymowych et al. US 2013/0156760 A1 Jun. 20, 2013 8,034,906 B2 10/2011 Borhani et al. 8,092,998 B2 1/2012 Stuhlmuller et al. 8,093,045 B2 1 2012 Pla et al. Related U.S. Application Data 8, 187,836 B2 5, 2012 Hsieh (63) Continuation of application No. 12/325,049, filed on 8, 197,813 B2 6/2012 Salfeld et al. Nov. 28, 2008, now Pat. No. 8,420,081. (Continued) (60) Provisional application No. 61/004,992, filed on Nov. FOREIGN PATENT DOCUMENTS 30, 2007. EP 0486526 5, 1992 (51) Int. Cl. EP 419251 B 1, 1993 A 6LX39/395 (2006.01) (Continued) A6 IK39/00 (2006.01) A6 IK38/00 (2006.01) OTHER PUBLICATIONS C07K 16/00 (2006.01) C07K 6/24 (2006.01) Abbott “Humira Prescribing Info..”. Jan. 31, 2003.* CI2P 2/08 (2006.01) A6 IK9/00 (2006.01) (Continued) A6 IK9/08 (2006.01) 3. I 3.08: Primary Examiner — Robert Landsman A6 IK38/46 (2006.01) Assistant Examiner — Bruce D Hissong A6 IK38/48 (2006.01) (74) Attorney, Agent, or Firm — McCarter & English LLP: A6 IK38/50 (2006.01) Cristin H. Cowles; Deborah L. Nagle C07K L/34 (2006.01) (52) U.S. Cl. CPC ...... A61K.39/3955 (2013.01); A61 K9/0019 (57) ABSTRACT (2013.01); A61K 9/08 (2013.01); A61 K9/19 (2013.01); A61 K38/21 (2013.01); A61 K38/46 The invention provides an aqueous formulation comprising (2013.01); A61K 38/4893 (2013.01); A61 K water and a protein, and methods of making the same. The 38/50 (2013.01); A61K.39/395 (2013.01); aqueous formulation of the invention may be a high protein A61K.39/39591 (2013.01); C07K I/34 formulation and/or may have low levels of conductity result (2013.01) ing from the low levels of ionic excipients. Also included in USPC ... 424/130.1; 424/145.1: 514/1.1 : 530/387.1; the invention are formulations comprising water and proteins 530/388.23 having low osmolality. (58) Field of Classification Search None See application file for complete search history. 18 Claims, 36 Drawing Sheets US 8,883,146 B2 Page 2

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33,333 < Salie gigs 33 US 8,883,146 B2 1. 2 PROTEINFORMULATIONS AND METHODS tics. To date, biologic formulations require additional excipi OF MAKING SAME ents to maintain protein stability. Typically, liquid pharma ceutical formulations contain multiple additives for stability. RELATED APPLICATIONS For example, a liquid formulation for patient self-administra tion of human growth hormone, Norditropin Simplexx(R), This application is a continuation of U.S. patent applica contains the additives mannitol (a Sugar alcohol), histidine tion Ser. No. 12/325,049, filed on Nov. 28, 2008, which and poloxamer 188 (a surfactant) to stabilize the hormone. claims the benefit of priority to U.S. Provisional Application Pharmaceutical additives need to be soluble, non-toxic and No. 61/004,992, filed on Nov.30, 2007. The contents of the used at particular concentrations that provide stabilizing priority applications are hereby incorporated by reference 10 effects on the specific therapeutic protein. Since the stabiliz herein. ing effects of additives are protein- and concentration-depen dent, each additive being considered for use in a pharmaceu SEQUENCE LISTING tical formulation must be carefully tested to ensure that it does not cause instability or have other negative effects on the The instant application contains a Sequence Listing which 15 chemical or physical make-up of the formulation. Ingredients has been submitted in ASCII format via EFS-Web and is used to stabilize the protein may cause problems with protein hereby incorporated by reference in its entirety. Said ASCII stability over time or with protein stability in changing envi copy, created on Feb. 22, 2013, is named SEQLIST and is ronments during storage. 3,911 bytes in size. Typically, long shelf-life is achieved by storing the protein in frozen from (e.g., at -80°C.) or by subjecting the protein to BACKGROUND OF THE INVENTION a lyophilization process, i.e., by storing the protein in lyo philized form, necessitating a reconstitution step immedi A basic principle of pharmaceutical proteinformulations is ately before use and thus posing a significant disadvantage that certain instabilities must be overcome. Degradation path with regard to patient convenience. However, freezing a pro ways of proteins can be separated into two distinct classes, 25 tein formulation for storage may lead to localized high con involving chemical instability and physical instability. centrations of proteins and additives, which can create local Chemical instabilities lead to the modification of the protein extremes in pH. degradation and protein aggregation within through bond formation or cleavage. Examples of chemical the formulation. In addition, it is well known to those skilled instability problems include deamidation, racemization, in the art that freezing and thawing processes often impact hydrolysis, oxidation, beta elimination and disulfide 30 protein stability, meaning that even storage of the pharma exchange. Physical instabilities, on the other hand, do not ceutical protein in frozen form can be associated with the loss lead to covalent changes in proteins. Rather, they involve ofstability due to the freezing and thawing step. Also, the first changes in the higher order structure (secondary and above) process step of lyophilization involves freezing, which can of proteins. These include denaturation, adsorption to Sur negatively impact protein stability. In industry settings, a faces, aggregation and precipitation (Manning et al., Pharm. 35 pharmaceutical protein may be subjected to repeated freeze Res. 6,903 (1989)). thaw processing during Drug Substance manufacturing It is generally accepted that these instabilities, which can (holding steps, storage, re-freeze and re-thaw to increase have great effect on the commercial viability and efficacy of timing and batch size flexibility in Drug Product fill-finish pharmaceutical protein formulations, can be overcome by ing) and during Subsequent Drug Product fill-finishing (lyo including additional molecules in the formulation. Protein 40 philization). Since it is well known that the risk of encoun stability can be improved by including excipients that interact tering protein instability phenomena increases with with the protein in solution to keep the protein stable, soluble increasing the number of freeze-thaw cycles a pharmaceuti and unaggregated. For example, Salt compounds and other cal protein encounters, achieving formulation conditions that ionic species are very common additives to protein formula maintain protein stability over repeated freeze-thaw pro tions. They assistin fighting denaturation of proteins by bind 45 cesses is a challenging task. There is a need in the biophar ing to proteins in a non-specific fashion and increasing ther maceutical industry for formulations that can be frozen and mal stability. Salt compounds (e.g., NaCl, KCl) have been thawed without creating undesired properties in the formula used Successfully in commercial insulin preparations to fight tions, especially gradients of pH, osmolarity, density or pro aggregation and precipitation (ibid. at 911). Amino acids tein or excipient concentration. (e.g., histidine, arginine) have been shown to reduce alter 50 Often protein-based pharmaceutical products need to be ations in proteins secondary structures when used as formu formulated at high concentrations for therapeutic efficacy. lation additives (Tian et al., Int'l J. Pharm. 355, 20 (2007)). Highly concentrated protein formulations are desirable for Other examples of commonly used additives include polyal therapeutic uses since they allow for dosages with Smaller cohol materials such as glycerol and Sugars, and Surfactants Volumes, limiting patient discomfort, and are more economi Such as detergents, both nonionic (e.g., Tween, Pluronic) and 55 cally packaged and stored. The development of high protein anionic (sodium dodecyl sulfate). The near universal preva concentration formulations, however, presents many chal lence of additives in all liquid commercial protein formula lenges, including manufacturing, stability, analytical, and, tions indicates that protein solutions without Such compounds especially for therapeutic proteins, delivery challenges. For may encounter challenges with degradation due to instabili example, difficulties with the aggregation, insolubility and ties. 60 degradation of proteins generally increase as protein concen The primary goal of protein formulation is to maintain the trations in formulations are raised (for review, see Shire, S.J. stability of a given protein in its native, pharmaceutically et al. J. Pharm. Sci., 93, 1390 (2004)). Previously unseen active form over prolonged periods of time to guarantee negative effects may be caused by additives that, at lower acceptable shelf-life of the pharmaceutical protein drug. concentrations of the additives or the protein, provided ben Maintaining the stability and solubility of proteins in solu 65 eficial effects. The production of high concentration protein tion, however, is especially challenging in pharmaceutical formulations may lead to significant problems with opales formulations where the additives are included into therapeu cence, aggregation and precipitation. In addition to the poten US 8,883,146 B2 3 4 tial for normative protein aggregation and particulate forma teins in solution. As a result of the low level of ionic excipi tion, reversible self-association may occur, which may result ents, the aqueous formulation of the invention has low con in increased viscosity or other properties that complicate ductivity, e.g., less than 2 mS/cm. The methods and delivery by injection. High viscosity also may complicate compositions of the invention also provide aqueous protein manufacturing of high protein concentrations by filtration formulations having low osmolality, e.g., no greater than 30 approaches. mOsmol/kg. In addition, the formulations described herein Thus, pharmaceutical protein formulations typically care are preferred over standard formulations because they have fully balance ingredients and concentrations to enhance pro decreased immunogenicity due to the lack of additional tein stability and therapeutic requirements while limiting any agents needed for protein stabilization. negative side-effects. Biologic formulations should include 10 The methods and compositions of the invention may be stable protein, even at high concentrations, with specific used to provide anaqueous formulation comprising water and amounts of excipients reducing potential therapeutic compli any type of protein of interest. In one aspect, the methods and cations, storage issues and overall cost. compositions of the invention are used for large proteins, As proteins and other biomacromolecules gain increased including proteins which are larger than 47 kDa. Antibodies, interest as drug molecules, formulations for delivering Such 15 and fragments thereof, including those used for in vivo and in molecules are becoming an important issue. Despite the revo vitro purposes, are another example of proteins which may be lutionary progress in the large-scale manufacturing of pro used in the methods and compositions of the invention. teins for therapeutic use, effective and convenient delivery of Furthermore, the multiple step purification and concentra these agents in the body remains a major challenge due to tion processes that are necessary to prepare proteins and their intrinsic physicochemical and biological properties, peptide formulations often introduce variability in composi including poor permeation through biological membranes, tions, such that the precise composition of a formulation may large molecular size, short plasma half life, self association, vary from lot to lot. Federal regulations require that drug physical and chemical instability, aggregation, adsorption, compositions be highly consistent in their formulations and immunogenicity. regardless of the location of manufacture or lot number. 25 Methods of the invention can be used to create solutions of SUMMARY OF THE INVENTION proteins formulated in water to which buffers and excipients are added back in precise amounts, allowing for the creation The invention is directed towards the Surprising findings of proteinformulations with precise concentrations of buffers that proteins formulated in water maintain solubility, as well and/or excipients. as stability, even at high concentrations, during long-term 30 In one embodiment, the invention provides an aqueous liquid storage or other processing steps, such as freeze? thaw formulation comprising a protein and water, wherein the for ing and lyophilization. mulation has certain characteristics, such as, but not limited The present invention relates to methods and compositions to, low conductivity, e.g., a conductivity of less than about 2.5 for aqueous proteinformulations which comprise water and a mS/cm, a protein concentration of at least about 10 ug/mL, an protein, where the protein is stable without the need for addi 35 osmolality of no more than about 30 mOsmol/kg, and/or the tional agents. Specifically, the methods and compositions of protein has a molecular weight (M) greater than about 47 the invention are based on a diafiltration process wherein a kDa. In one embodiment, the formulation of the invention has first solution containing the protein of interest is diafiltered improved stability, such as, but not limited to, stability in a using water as a diafiltration medium. The process is per liquid form for an extended time (e.g., at least about 3 months formed such that there is at least a determined volume 40 or at least about 12 months) or stability through at least one exchange, e.g., a five fold Volume exchange, with the water. freeze/thaw cycle (if not more freeze/thaw cycles). In one By performing the methods of the invention, the resulting embodiment, the formulation is stable for at least about 3 aqueous formulation has a significant decrease in the overall months in a form selected from the group consisting of fro percentage of excipients in comparison to the initial protein Zen, lyophilized, or spray-dried. solution. For example, 95-99% less excipients are found in 45 In one embodiment, proteins included in the formulation of the aqueous formulation in comparison to the initial protein the invention may have a minimal size, including, for Solution. Despite the decrease in excipients, the protein example, a M. greater than about 47 kDa, a M. greater than remains soluble and retains its biological activity, even at high about 57 kDa, a M. greater than about 100 kDa, a M. greater concentrations. In one aspect, the methods of the invention than about 150 kDa, a Mgreater than about 200kDa, oraM, result in compositions comprising an increase in concentra 50 greater than about 250 kDa. tion of the protein while decreasing additional components, In one embodiment, the formulation of the invention has a Such as ionic excipients. As such, the hydrodynamic diameter low conductivity, including, for example, a conductivity of of the protein in the aqueous formulation is Smaller relative to less than about 2.5 mS/cm, a conductivity of less than about 2 the same protein in a standard buffering Solution, such as mS/cm, a conductivity of less than about 1.5 mS/cm, a con phosphate buffered saline (PBS). 55 ductivity of less than about 1 mS/cm, or a conductivity of less The formulation of the invention has many advantages over than about 0.5 mS/cm. standard buffered formulations. In one aspect, the aqueous In one embodiment, proteins included in the formulation of formulation comprises high protein concentrations, e.g., 50 to the invention have a given concentration, including, for 200 mg/mL or more. Proteins of all sizes may be included in example, a concentration of at least about 1 mg/mL, at least the formulations of the invention, even at increased concen 60 about 10 mg/mL, at least about 50 mg/mL, at least about 100 trations. Despite the high concentration of protein, the for mg/mL, at least about 150 mg/mL, at least about 200 mg/mL, mulation has minimal aggregation and can be stored using or greater than about 200 mg/mL. various methods and forms, e.g., freezing, without deleteri In one embodiment, the formulation of the invention has an ous effects that might be expected with high protein formu osmolality of no more than about 15 mCosmol/kg. lations. Formulations of the invention do not require excipi 65 In one embodiment, the invention provides an aqueous ents, such as, for example, Surfactants and buffering systems, formulation comprising water and a given concentration of a which are used in traditional formulations to stabilize pro protein, wherein the protein has a hydrodynamic diameter US 8,883,146 B2 5 6 (D) which is at least about 50% less than the D, of the protein The invention further provides a method of preparing an in a buffered solution at the given concentration. In one aqueous formulation of a protein, the method comprising embodiment, the D, of the protein is at least about 50% less providing the protein in a first solution; Subjecting the first than the D, of the protein in phosphate buffered saline (PBS) Solution to diafiltration using water as a diafiltration medium at the given concentration; the D, of the protein is at least until at least a five-fold volume exchange with the water has about 60% less than the D, of the protein in PBS at the given been achieved to thereby prepare a diafiltered protein solu concentration; the D of the protein is at least about 70% less tion; and concentrating the diafiltered protein solution to than the D of the protein in PBS at the given concentration. thereby prepare the aqueous formulation of the protein. In one In one embodiment, the invention provides an aqueous embodiment, the protein in the resulting formulation retains formulation comprising a protein, such as, but not limited to, 10 its biological activity. an antibody, or an antigen-binding fragment, wherein the In one embodiment, the concentration of the diafiltered protein has a hydrodynamic diameter (D) of less than about protein solution is achieved via centrifugation. 5um. In one embodiment, the protein has a D, of less than In one embodiment, the diafiltration medium consists of about 3 Lum. 15 Water. Any protein may be used in the methods and compositions In one embodiment, the first solution is subjected to diafil of the invention. In one embodiment, the formulation com tration with water until a Volume exchange greater than a prises a therapeutic protein. In one embodiment, the formu five-fold volume exchange is achieved. In one embodiment, lation comprises an antibody, or an antigen-binding fragment the first solution is subjected to diafiltration with water until at thereof. Types of antibodies, or antigen binding fragments, least about a six-fold Volume exchange is achieved. In one that may be included in the methods and compositions of the embodiment, the first solution is subjected to diafiltration invention include, but are not limited to, a chimeric antibody, with water until at least about a seven-fold volume exchange a human antibody, a humanized antibody, and a domain anti is achieved. body (dAb). In one embodiment, the antibody, or antigen In one embodiment, the aqueous formulation has a final binding fragment thereof, is an anti-TNFC, such as but not 25 concentration of excipients which is at least about 95% less limited to adalimumab or golimumab, or an anti-IL-12 anti than the first solution. body, such as but not limited to J695. In addition, the formu In one embodiment, the aqueous formulation has a final lation of the invention may also include at least two distinct concentration of excipients which is at least about 99% less types of proteins, e.g., adalimumab and Jó95. than the first solution. In yet another embodiment of the invention, the formula 30 In one embodiment, the first protein solution is obtained tion may further comprise a non-ionizable excipient. from a mammalian cell expression system and has been puri Examples of non-ionizable excipients include, but are not fied to remove host cell proteins (HCPs). limited to, a Sugar alcohol or polyol (e.g., mannitol or Sorbi In one embodiment, the method of the invention further tol), a non-ionic Surfactant (e.g., polysorbate 80, polysorbate comprises adding an excipient to the aqueous formulation. 20, polysorbate 40, polysorbate 60), and/or a Sugar (e.g., 35 Sucrose). Other non-limiting examples of non-ionizable BRIEF DESCRIPTION OF THE DRAWINGS excipients that may be further included in the formulation of the invention include, but are not limited to, non-trehalose, FIG. 1 shows the SEC chromatogram of Adalimumab ref raffinose, and maltose. erence standard AFP04C (bottom line), Adalimumab DS In one embodiment, the formulation does not comprise an 40 (drug substance before (middle line) and after DF/UF pro agent selected from the group consisting of a tonicity modi cessing (top line). fier, a stabilizing agent, a Surfactant, an anti-oxidant, a cryo FIG. 2 shows the impact of sorbitol (a non-ionizable protectant, a bulking agent, a lyroprotectant, a basic compo excipient) and NaCl (ionizable excipient) concentrations on nent, and an acidic component. the hydrodynamic diameter (Dh) of Adalimumab monomer The formulation of the invention may be suitable for any 45 upon addition of the excipient compound to DF/UF-pro use, including both in vitro and in vivo uses. In one embodi cessed Adalimumab monomer. ment, the formulation of the invention is suitable for admin FIG. 3 shows the IEC profile of Jó95 reference standard istration to a subject via a mode of administration, including, (bottom graph) and J695 DS, pH adjusted to pH 4.4 (top but not limited to, Subcutaneous, intravenous, inhalation, graph). intradermal, transdermal, intraperitoneal, and intramuscular 50 FIG. 4 shows the IEC profile of J695 after DF/UF with administration. The formulation of the invention may be used Milli-Q water, pH 4.7 (top graph), and J695 DS before in the treatment of a disorder in a subject. DF/UF. pH adjusted to pH 4.4 (bottom curve). Also included in the invention are devices that may be used FIG.5 graphically depicts the correlation of hydrodynamic to deliver the formulation of the invention. Examples of such diameter (Z-average) and concentration of Adalimumab (dis devices include, but are not limited to, a Syringe, a pen, an 55 solved in WFI). X: determined with an SOP using 1.1 mPasas implant, a needle-free injection device, an inhalation device, assumed sample viscosity. y: determined with an SOP using and a patch. 1.9 mPas as assumed sample viscosity. In one embodiment, the formulation of the invention is a FIG. 6 graphically depicts the correlation of hydrodynamic pharmaceutical formulation. diameter (peak monomer) and concentration of Adalimumab The invention also provides a method of preparing an aque 60 (dissolved in WFI). X: determined with an SOP using 1.1 ous formulation comprising a protein and water, the method mPas as assumed sample viscosity. y: determined with an comprising providing the protein in a first Solution, and Sub SOP using 1.9 mPas as assumed sample viscosity jecting the first solution to diafiltration using water as a dia FIG.7 graphically depicts the correlation of hydrodynamic filtration medium until at least a five fold volume exchange diameter (Z-average) and concentration of Jô95 (dissolved in with the water has been achieved to thereby prepare the aque 65 WFI). X: determined with an SOP using 1.1 mPas as assumed ous formulation. In one embodiment, the protein in the result sample viscosity: determined with an SOP using 1.9 mPas as ing formulation retains its biological activity. assumed sample viscosity US 8,883,146 B2 7 8 FIG.8 graphically depicts the correlation of hydrodynamic FIG.28 shows size distribution by intensity for 100 mg/mL diameter (peak monomer) and concentration of JG95 (dis Adalimumab in water at various pH levels. solved in WFI). X: determined with an SOP using 1.1 mPasas FIG. 29 also shows size distribution by intensity for 100 assumed sample viscosity. y: determined with an SOP using mg/mL Adalimumab in water at various pH levels. 1.9 mPas as assumed sample viscosity. FIG.30 shows monomer content (SEC) for Adalimumab in FIG. 9 shows the sum of lysine 0, 1 and 2 of Adalimumab Water. % in dependence on Adalimumab concentration in water for FIG.31 shows aggregate content (SEC) for Adalimumab in injection. Water. FIG. 10 shows the sum of peak 1 to 7 of Jó95 % in FIG. 32 shows the viscosity of two J695 solutions (WFI dependence on J695 concentration in water for injection. 10 formulations) as a function of Solution temperature. FIG. 11 shows the sum of acidic peaks of JG95% in FIG. 33 graphically depicts 1D4.7 antibody stability as dependence on J695 concentration in water for injection. measured by Subvisible particle (>1 um) during repeated FIG. 12 shows the sum of basic peaks of JG95 % in freeze/thaw (ft) cycles for a number of different formula dependence on J695 concentration in water for injection tions. (WFI). 15 FIG. 34 graphically depicts 13C5.5 antibody stability as FIG. 13 shows the efficiency of the dialysis performed in measured by subvisible particle (>10 um) during repeated Example 12, in terms of the reduction of components respon freeze/thaw (ft) cycles for a number of different formula sible for osmolality and conductivity of the formulation tions. (BDS, 74 mg/ml, 10 ml sample volume, SpectraPor7 FIG. 35 graphically depicts 13C5.5 antibody stability as MWCO10k). measured by Subvisible particle (>1 um) during repeated FIG. 14 shows the stability of pH levels in dialyzed Adali freeze/thaw (ft) cycles for a number of different formula mumab Bulk Solutions. pH levels before and after dialysis tions. against deionized water (1:1,000,000) are shown. (BDS, 74 FIG. 36 graphically depicts 7C6 antibody stability as mea mg/ml, 10 ml sample volume, SpectraPor7 MWCO10k) sured by subvisible particle (>1 um) during repeated freeze/ FIG. 15 shows bottle mapping density data for 250 mg/ml 25 thaw (ft) cycles for a number of different formulations. and 200 mg/ml low-ionic Adalimumab solutions after freeze thaw. DETAILED DESCRIPTION OF THE INVENTION FIG. 16 shows bottle mapping pH data for 250 mg/ml and 200 mg/ml low-ionic Adalimumab solutions after freeze I. Definitions thaw. 30 FIG. 17 shows bottle mapping concentration data for 250 In order that the present invention may be more readily mg/ml and 200 mg/ml low-ionic Adalimumabsolutions after understood, certain terms are first defined. freeze thaw. As used herein, the term “acidic component” refers to an FIG. 18 shows bottle mapping osmolality data for 250 agent, including a solution, having an acidic pH, i.e., less than mg/ml and 200 mg/ml low-ionic Adalimumab solutions after 35 7.0. Examples of acidic components include phosphoric acid, freeze thaw. hydrochloric acid, acetic acid, citric acid, oxalic acid, Suc FIG. 19 shows bottle mapping conductivity data for 250 cinic acid, tartaric acid, lactic acid, malic acid, glycolic acid mg/ml and 200 mg/ml low-ionic Adalimumab solutions after and fumaric acid. In one embodiment, the aqueous formula freeze thaw. tion of the invention does not include an acidic component. FIG. 20 shows SEC analysis of low-ionic Adalimumab 40 As used herein, the term “antioxidant’ is intended to mean (referred to as D2E7 in FIG. 20) solutions that were either an agent which inhibits oxidation and thus is used to prevent stored at 2-8°C. for 8.5 months after DF/UF (bottom curve) the deterioration of preparations by the oxidative process. or stored at -80° C. for 4.5 months after DF/UF (top curve). Such compounds include by way of example and without FIG. 21 shows the stability of the monoclonal antibody limitation, acetone, Sodium bisulfate, ascorbic acid, ascorbyl 1D4.7 formulated in various solutions and in water before 45 palmitate, citric acid, butylated hydroxyanisole, butylated freeze-thaw procedures (TO) and after each of four freeze hydroxytoluene, hydrophosphorous acid, monothioglycerol, thaws (T1, T2, T3 and T4). propyl gallate, methionine, sodium ascorbate, sodium citrate, FIG. 22 shows the stability of the monoclonal antibody Sodium Sulfide, sodium sulfite, Sodium bisulfite, Sodium 13C5.5 formulated in water and with various buffers before formaldehyde Sulfoxylate, thioglycolic acid, sodium met freeze-thaw procedures (TO) and after each of four freeze 50 abisulfite, EDTA (edetate), pentetate and others known to thaws (T1, T2, T3 and T4). Blank=WFI control sample. those of ordinary skill in the art. FIG. 23 shows the stability of the monoclonal antibody The term “aqueous formulation” refers to a solution in 13C5.5 formulated in water and with various excipients which the solvent is water. added, before freeze-thaw procedures (TO) and after each of As used herein, the term “basic component” refers to an four freeze-thaws (T1, T2, T3 and T4). Blank=WFI control 55 agent which is alkaline, i.e., pH greater than 7.0. Examples of sample. basic components include potassium hydroxide (KOH) and FIG. 24 shows the impact of the concentration of Adali sodium hydroxide (NaOH) mumab (WFI formulation) and solution pH on solution vis As used herein, the term “bulking agent' is intended to cosity. mean a compound used to add bulk to the reconstitutable solid FIG. 25 shows turbidity data for Adalimumab solutions 60 and/or assist in the control of the properties of the formulation (WFI formulations) of various concentrations and pH values. during preparation. Such compounds include, by way of FIG. 26 shows hydrodynamic diameter (Dh) data for example and without limitation, dextran, trehalose. Sucrose, Adalimumab solutions (WFI formulations) at various pH val polyvinylpyrrolidone, lactose, inositol, Sorbitol, dimethylsul ues and concentrations. foxide, glycerol, albumin, calcium lactobionate, and others FIG. 27 shows a size distribution by intensity graph (Dh 65 known to those of ordinary skill in the art. measurements) for Adalimumab in water Solutions, pH 5, at The term “conductivity, as used herein, refers to the ability various concentrations. of an aqueous solution to conduct an electric current between US 8,883,146 B2 10 two electrodes. Generally, electrical conductivity or specific Examples of non-ionic excipients include, but are not limited conductivity is a measure of a materials ability to conductan to, Sugars (e.g., Sucrose), Sugar alcohols (e.g., mannitol), and electric current. In solution, the current flows by ion transport. non-ionic Surfactants (e.g., polysorbate 80). Therefore, with an increasing amount of ions present in the The term “first protein solution” or “first solution” as used aqueous solution, the Solution will have a higher conductivity. herein, refers to the initial protein solution or starting material The unit of measurement for conductivity is mmhos (mS/cm), used in the methods of the invention, i.e., the initial protein and can be measured using a conductivity meter sold, e.g., by solution which is diafiltered into water. In one embodiment, Orion Research, Inc. (Beverly, Mass.). The conductivity of a the first protein Solution comprises ionic excipients, non Solution may be altered by changing the concentration of ions ionic excipients, and/or a buffering system. therein. For example, the concentration of ionic excipients in 10 The term “hydrodynamic diameter' or “D, of a particle the solution may be altered in order to achieve the desired conductivity. refers to the diameter of a sphere that has the density of water The term "cryoprotectants' as used herein generally and the same velocity as the particle. Thus the term “hydro includes agents, which provide stability to the protein from dynamic diameter of a protein’ as used herein refers to a size freezing-induced stresses. Examples of cryoprotectants 15 determination for proteins in Solution using dynamic light include polyols such as, for example, mannitol, and include scattering (DLS). A DLS-measuring instrument measures the saccharides Such as, for example, Sucrose, as well as includ time-dependent fluctuation in the intensity of light scattered ing Surfactants such as, for example, polysorbate, poloxamer from the proteins in Solution at a fixed scattering angle. Pro or polyethylene glycol, and the like. Cryoprotectants also tein Dhis determined from the intensity autocorrelation func contribute to the tonicity of the formulations. tion of the time-dependent fluctuation in intensity. Scattering As used herein, the terms “ultrafiltration' or “UF refers to intensity data are processed using DLS instrument Software any technique in which a solution or a suspension is subjected to determine the value for the hydrodynamic diameter and the to a semi-permeable membrane that retains macromolecules size distribution of the scattering molecules, i.e. the protein while allowing solvent and Small solute molecules to pass specimen. through. Ultrafiltration may be used to increase the concen 25 The term “lyoprotectant as used herein includes agents tration of macromolecules in a solution or Suspension. In a that provide Stability to a protein during water removal during preferred embodiment, ultrafiltration is used to increase the the drying or lyophilisation process, for example, by main concentration of a protein in water. taining the proper conformation of the protein. Examples of As used herein, the term “diafiltration' or “DF is used to lyoprotectants include saccharides, in particular di- or trisac mean a specialized class of filtration in which the retentate is 30 charides. Cryoprotectants may also provide lyoprotectant diluted with solvent and re-filtered, to reduce the concentra tion of soluble permeate components. Diafiltration may or effects. may not lead to an increase in the concentration of retained The term “pharmaceutical” as used herein with reference components, including, for example, proteins. For example, to a composition, e.g., an aqueous formulation, that it is useful in continuous diafiltration, a solvent is continuously added to 35 for treating a disease or disorder. the retentate at the same rate as the filtrate is generated. In this The term “protein' is meant to include a sequence of amino case, the retentate Volume and the concentration of retained acids for which the chain length is sufficient to produce the components does not change during the process. On the other higher levels of secondary and/or tertiary and/or quaternary hand, in discontinuous or sequential dilution diafiltration, an structure. This is to distinguish from "peptides' or other small ultrafiltration step is followed by the addition of solvent to the 40 molecular weight drugs that do not have such structure. In one retentate side; if the volume of solvent added to the retentate embodiment, the proteins used herein have a molecular side is not equal or greater to the Volume offiltrate generated, weight of at least about 47 kD. Examples of proteins encom then the retained components will have a high concentration. passed within the definition used herein include therapeutic Diafiltration may be used to alter the pH, ionic strength, salt proteins. A “therapeutically active protein’ or “therapeutic composition, buffer composition, or other properties of a 45 protein’ refers to a protein which may be used for therapeutic Solution or Suspension of macromolecules. purposes, i.e., for the treatment of a disorder in a Subject. It As used herein, the terms “diafiltration/ultrafiltration' or should be noted that while therapeutic proteins may be used “DF/UF refer to any process, technique or combination of for treatment purposes, the invention is not limited to Such techniques that accomplishes ultrafiltration and/or diafiltra use, as said proteins may also be used for in vitro studies. In tion, either sequentially or simultaneously. 50 a preferred embodiment, the therapeutic protein is a fusion As used herein, the term “diafiltration step’ refers to a total protein oran antibody, or antigen-binding portion thereof. In Volume exchange during the process of diafiltration. one embodiment, the methods and compositions of the inven The term “excipient” refers to an agent that may be added tion comprise at least two distinct proteins, which are defined to a formulation to provide a desired consistency, (e.g., alter as two proteins having distinct amino acid sequences. Addi ing the bulk properties), to improve stability, and/or to adjust 55 tional distinct proteins do not include degradation products of osmolality. Examples of commonly used excipients include, a protein. but are not limited to, Sugars, polyols, amino acids, Surfac The phrase “protein is dissolved in water as used herein tants, and polymers. The term "ionic excipient’ or “ionizable refers to a formulation of a protein wherein the protein is excipient as used interchangeably herein, refers to an agent dissolved in an aqueous solution in which the amount of small that has a net charge. In one embodiment, the ionic excipient 60 molecules (e.g., buffers, excipients, salts, Surfactants) has has a net charge under certain formulation conditions, such as been reduced by DF/UF processing. Even though the total pH. Examples of an ionic excipient include, but are not lim elimination of Small molecules cannot be achieved in an ited to, histidine, arginine, and sodium chloride. The term absolute sense by DF/UF processing, the theoretical reduc “non-ionic excipient’ or “non-ionizable excipient, as used tion of excipients achievable by applying DF/UF is suffi interchangeably herein, refers to an agent having no net 65 ciently large to create a formulation of the protein essentially charge. In one embodiment, the non-ionic excipient has no in water exclusively. For example, with 6 volume exchanges net charge under certain formulation conditions, such as pH. in a continuous mode DF/UF protocol, the theoretical reduc US 8,883,146 B2 11 12 tion of excipients is ~99.8% (cie, with ci being the initial reverse osmosis, also referred to herein as “pure water. In a excipient concentration, and X being the number of Volume preferred embodiment, water used in the methods and com exchanges). positions of the invention is excipient-free. In one embodi The term “pharmaceutical formulation” refers to prepara ment, water includes sterile water suitable for administration tions which are in Such a form as to permit the biological to a subject. In another embodiment, water is meant to include activity of the active ingredients to be effective, and, there water for injection (WFI). In one embodiment, water refers to fore. may be administered to a Subject for therapeutic use. distilled water or water which is appropriate for use in in vitro A “stable formulation is one in which the protein therein assays. In a preferred embodiment, diafiltration is performed essentially retains its physical stability and/or chemical sta in accordance with the methods of the invention using water bility and/or biological activity upon storage. Various analyti 10 cal techniques for measuring protein stability are available in alone as the diafiltration medium. the art and are reviewed in Peptide and Protein Drug Delivery, The term “antibody' as referred to herein includes whole 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, antibodies and any antigen binding fragment (i.e., “antigen N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: binding portion') or single chains thereof. An “antibody” 29-90 (1993), for example. In one embodiment, the stability 15 refers to a glycoprotein comprising at least two heavy (H) of the protein is determined according to the percentage of chains and two light (L) chains inter-connected by disulfide monomer protein in the solution, with a low percentage of bonds, or an antigen binding portion thereof. Each heavy degraded (e.g., fragmented) and/or aggregated protein. For chain is comprised of a heavy chain variable region (abbre example, an aqueous formulation comprising a stable protein viated herein as V) and a heavy chain constant region. The may include at least 95% monomer protein. Alternatively, an heavy chain constant region is comprised of three domains, aqueous formulation of the invention may include no more CH1, CH2 and CH3. Each light chain is comprised of a light than 5% aggregate and/or degraded protein. chain variable region (abbreviated herein as V) and a light The term “stabilizing agent” refers to an excipient that chain constant region. The light chain constant region is com improves or otherwise enhances stability. Stabilizing agents prised of one domain, CL. The V and V regions can be include, but are not limited to, C.-lipoic acid, C-tocopherol, 25 further subdivided into regions of hypervariability, termed ascorbyl palmitate, benzyl alcohol, biotin, bisulfites, boron, complementarity determining regions (CDR), interspersed butylated hydroxyanisole (BHA), butylated hydroxytoluene with regions that are more conserved, termed framework (BHT), ascorbic acid and its esters, carotenoids, calcium cit regions (FR). Each VandV, is composed of three CDRs and rate, acetyl-L-camitine, chelating agents, chondroitin, chro four FRS, arranged from amino-terminus to carboxy-termi mium, citric acid, coenzyme Q-10, cysteine, cysteine hydro 30 nus in the following order: FR1, CDR1, FR2, CDR2, FR3, chloride, 3-dehydroshikimic acid (DHS), EDTA CDR3, FR4. The variable regions of the heavy and light (ethylenediaminetetraacetic acid: edetate disodium), ferrous chains contain a binding domain that interacts with an anti Sulfate, folic acid, fumaric acid, alkyl gallates, garlic, glu gen. The constant regions of the antibodies may mediate the cosamine, grape seed extract, gugul, magnesium, malic acid, binding of the immunoglobulin to host tissues or factors, metabisulfite, N-acetyl cysteine, niacin, nicotinomide, nettle 35 including various cells of the immune system (e.g., effector root, ornithine, propyl gallate, pycnogenol, saw palmetto, cells) and the first component (C1q) of the classical comple Selenium, Sodium bisulfite, sodium metabisulfite, sodium ment system. Sulfite, potassium sulfite, tartaric acid, thiosulfates, thioglyc The term “antigen-binding portion of an antibody (or erol, thiosorbitol, tocopherol and their esters, e.g., tocopheral simply “antibody portion'), as used herein, refers to one or acetate, tocopherol Succinate, tocotrienal, d-C-tocopherol 40 more fragments of an antibody that retain the ability to spe acetate, Vitamin A and its esters, vitamin B and its esters, cifically bind to an antigen (e.g., TNFO, IL-12). It has been Vitamin C and its esters, vitamin D and its esters, vitamin E shown that the antigen-binding function of an antibody can be and its esters, e.g., vitamin Eacetate, Zinc, and combinations performed by fragments of a full-length antibody. Examples thereof. of binding fragments encompassed within the term “antigen The term "surfactants' generally includes those agents that 45 binding portion of an antibody include (i) a Fab fragment, a protect the protein from air/solution interface-induced monovalent fragment consisting of the V, V, CL and CH1 stresses and solution/surface induced-stresses. For example domains; (ii) a F(ab') fragment, a bivalent fragment compris Surfactants may protect the protein from aggregation. Suit ing two Fab fragments linked by a disulfide bridge at the hinge able Surfactants may include, e.g., polysorbates, polyoxyeth region; (iii) a Fd fragment consisting of the V, and C. ylene alkyl ethers such as Brij 35(R), or poloxamer such as 50 domains; (iv) a Fv fragment consisting of the V, and V. Tween 20, Tween 80, or poloxamer 188. Preferred detergents domains of a single arm of an antibody, (v) a dAb fragment are poloxamers, e.g., Poloxamer 188, Poloxamer 407: poly (Ward et al. (1989) Nature 341:544-546), which consists of a oxyethylene alkyl ethers, e.g., Brij 35(R), Cremophor A25, V or V, domain; and (vi) an isolated complementarity deter Sympatens ALM/230; and polysorbates/Tweens, e.g., mining region (CDR). Furthermore, although the two Polysorbate 20, Polysorbate 80, and Poloxamers, e.g., Polox 55 domains of the Fv fragment, V, and V, are coded for by amer 188, and Tweens, e.g., Tween 20 and Tween 80. separate genes, they can be joined, using recombinant meth As used herein, the term “tonicity modifier is intended to ods, by a synthetic linker that enables them to be made as a mean a compound or compounds that can be used to adjust the single protein chain in which the VL and VH regions pair to tonicity of a liquid formulation. Suitable tonicity modifiers form monovalent molecules (known as single chain FV include glycerin, lactose, mannitol, dextrose, sodium chlo 60 (scFV); see e.g., Bird et al. (1988) Science 242:423-426; and ride, magnesium sulfate, magnesium chloride, Sodium Sul Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 fate, Sorbitol, trehalose. Sucrose, raffinose, maltose and others 5883). Such single chain antibodies are also intended to be known to those or ordinary skill in the art. In one embodi encompassed within the term “antigen-binding portion of an ment, the tonicity of the liquid formulation approximates that antibody. These antibody fragments are obtained using con of the tonicity of blood or plasma. 65 ventional techniques known to those with skill in the art, and The term “water is intended to mean water that has been the fragments are screened for utility in the same manner as purified to remove contaminants, usually by distillation or are intact antibodies. In one embodiment of the invention, the US 8,883,146 B2 13 14 antibody fragment is selected from the group consisting of a antibody at one or more of the antibody's antigen binding Fab, an Fd, an Fd', a single chain Fv (scEv), an Schv, and a regions. In the context of the present invention, first and domain antibody (dAb). second "epitopes are understood to be epitopes which are Still further, an antibody orantigen-binding portion thereof not the same and are not bound by a single monospecific may be part of a larger immunoadhesion molecule, formed by 5 antibody, or antigen-binding portion thereof. covalent or noncovalent association of the antibody or anti The phrase “recombinant antibody” refers to antibodies body portion with one or more other proteins or peptides. that are prepared, expressed, created or isolated by recombi These other proteins or peptides can have functionalities that nant means, such as antibodies expressed using a recombi allow for the purification of antibodies or antigen-binding nant expression vector transfected into a host cell, antibodies portions thereofor allow for their association with each other 10 isolated from a recombinant, combinatorial antibody library, or other molecules. Thus examples of Such immunoadhesion antibodies isolated from an animal (e.g., a mouse) that is molecules include use of the Streptavidin core region to make transgenic for human immunoglobulin genes (see e.g., Taylor a tetrameric single chain variable fragment (ScPV) molecules et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies (Kipriyanov et al. (1995) Human Antibodies and Hybridomas prepared, expressed, created or isolated by any other means 6:93-101) and the use of a cysteine residue, a marker peptide 15 that involves splicing of particular immunoglobulin gene and a C-terminal polyhistidine tag to make bivalent and bioti sequences (such as human immunoglobulin gene sequences) nylated scFv molecules (Kipriyanov et al. (1994) Mol. Immu to other DNA sequences. Examples of recombinant antibod mol. 31:1047-1058). Antibody portions, such as Fab and ies include chimeric, CDR-grafted and humanized antibod F(ab')2 fragments, can be prepared from whole antibodies 1CS using conventional techniques, such as papain or pepsin The term “human antibody” refers to antibodies having digestion, respectively, of whole antibodies. Moreover, anti variable and constant regions corresponding to, or derived bodies, antibody portions and immunoadhesion molecules from, human germline immunoglobulin sequences as can be obtained using standard recombinant DNA tech described by, for example, Kabat et al. (See Kabat, et al. niques. (1991) Sequences of Proteins of Immunological Interest, Two antibody domains are “complementary' where they 25 Fifth Edition, U.S. Department of Health and Human Ser belong to families of structures which form cognate pairs or vices, NIH Publication No. 91-3242). The human antibodies groups or are derived from Such families and retain this fea of the invention, however, may include amino acid residues ture. For example, a VH domain and a VL domain of an not encoded by human germline immunoglobulin sequences antibody are complementary; two VH domains are not (e.g., mutations introduced by random or site-specific complementary, and two VL domains are not complementary. 30 mutagenesis in vitro or by Somatic mutation in vivo), for Complementary domains may be found in other members of example in the CDRs and in particular CDR3. the immunoglobulin superfamily, such as the Vol. and VB (or Recombinant human antibodies of the invention have vari gamma and delta) domains of the T-cell receptor. able regions, and may also include constant regions, derived The term “domain refers to a folded protein structure from human germline immunoglobulin sequences (See Kabat which retains its tertiary structure independently of the rest of 35 et al. (1991) Sequences of Proteins of Immunological Inter the protein. Generally, domains are responsible for discrete est, Fifth Edition, U.S. Department of Health and Human functional properties of proteins, and in many cases may be Services, NIH Publication No. 91-3242). In certain embodi added, removed or transferred to other proteins without loss ments, however, Such recombinant human antibodies are Sub of function of the remainder of the protein and/or of the jected to in vitro mutagenesis (or, when an animal transgenic domain. By single antibody variable domain is meanta folded 40 for human Ig sequences is used, in vivo Somatic mutagenesis) polypeptide domain comprising sequences characteristic of and thus the amino acid sequences of the VH and VL regions antibody variable domains. It therefore includes complete of the recombinant antibodies are sequences that, while antibody variable domains and modified variable domains, derived from and related to human germline VH and VL for example in which one or more loops have been replaced sequences, may not naturally exist within the human antibody by sequences which are not characteristic of antibody Vari 45 germline repertoire in vivo. In certain embodiments, how able domains, or antibody variable domains which have been ever, such recombinant antibodies are the result of selective truncated or comprise N- or C-terminal extensions, as well as mutagenesis or backmutation or both. folded fragments of variable domains which retain at least in The term “backmutation” refers to a process in which some part the binding activity and specificity of the full-length or all of the Somatically mutated amino acids of a human domain. 50 antibody are replaced with the corresponding germline resi Variable domains of the invention may be combined to dues from a homologous germline antibody sequence. The form a group of domains; for example, complementary heavy and light chain sequences of a human antibody of the domains may be combined, such as VL domains being com invention are aligned separately with the germline sequences bined with VH domains. Non-complementary domains may in the VBASE database to identify the sequences with the also be combined. Domains may be combined in a number of 55 highest homology. Differences in the human antibody of the ways, involving linkage of the domains by covalent or non invention are returned to the germline sequence by mutating covalent means. defined nucleotide positions encoding Such different amino A “dAb’ or “domain antibody” refers to a single antibody acid. The role of each amino acid thus identified as candidate variable domain (V or V.) polypeptide that specifically for backmutation should be investigated for a direct or indi binds antigen. 60 rect role in antigen binding and any amino acid found after As used herein, the term “antigenbinding region' or “anti mutation to affect any desirable characteristic of the human gen binding site' refers to the portion(s) of an antibody mol antibody should not be included in the final human antibody. ecule, or antigen binding portion thereof, which contains the To minimize the number of amino acids subject to backmu amino acid residues that interact with an antigen and confers tation those amino acid positions found to be different from on the antibody its specificity and affinity for the antigen. 65 the closest germline sequence but identical to the correspond The term "epitope' is meant to refer to that portion of any ing amino acid in a second germline sequence can remain, molecule capable of being recognized by and bound by an provided that the second germline sequence is identical and US 8,883,146 B2 15 16 colinear to the sequence of the human antibody of the inven obtained from a mammalian cell expression system and has tion for at least 10, preferably 12 amino acids, on both sides of been purified to remove host cell proteins (HCPs). Examples the amino acid in question. Backmutation may occur at any of methods of purification are described in U.S. application stage of antibody optimization. Ser. No. 1 1/732,918 (US 20070292442), incorporated by ref. The term "chimeric antibody” refers to antibodies which 5 erence herein. It should be noted that there is no special comprise heavy and light chain variable region sequences preparation of the first protein solution required in accor from one species and constant region sequences from another dance with the methods of the invention. species, such as antibodies having murine heavy and light Proteins which may be used in the compositions and meth chain variable regions linked to human constant regions. ods of the invention may be any size, i.e., molecular weight The term “CDR-grafted antibody” refers to antibodies 10 (M). For example, the protein may have a M equal to or which comprise heavy and light chain variable region greater than about 1 kDa, a M equal to or greater than about sequences from one species but in which the sequences of one 10 kDa, a M equal to or greater than about 47 kDa, a M. or more of the CDR regions of VH and/or VL are replaced equal to or greater than about 57 kDa, a M equal to or greater with CDR sequences of another species, such as antibodies than about 100 kDa, a M, equal to or greater than about 150 having murine heavy and light chain variable regions in 15 kDa, a M equal to or greater than about 200 kDa, or a M. which one or more of the murine CDRs (e.g., CDR3) has been equal to or greater than about 250kDa. Numbers intermediate replaced with human CDR sequences. to the above recited M, e.g., 47,48,49, 50,51,52,53,54,55, The term “humanized antibody' refers to antibodies which 56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71,72, comprise heavy and light chain variable region sequences 73, 74, 75,76, 77,78, 79,80, 81, 82,83, 84,85, 86, 87, 88,89, from a non-human species (e.g., amouse) but in which at least 90,91, 92,93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103,104, a portion of the VHand/or VL sequence has been altered to be 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, more "human-like', i.e., more similar to human germline 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, variable sequences. One type of humanized antibody is a 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, CDR-grafted antibody, in which human CDR sequences are 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,153, introduced into non-human VHand VL sequences to replace 25 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, the corresponding nonhuman CDR sequences. 165, 166, 167, 168, 169,170, and so forth, as well as all other Various aspects of the invention are described in further numbers recited herein, are also intended to be part of this detail in the following subsections. invention. Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended II. Methods of Invention 30 to be included in the scope of the invention. For example, proteins used in the invention may range in size from 57 kDa Generally, diafiltration is a technique that uses membranes to 250kDa, from 56 kDa to 242 kDa, from 60kDa to 270 kDa, to remove, replace, or lower the concentration of salts or and so forth. Solvents from Solutions containing proteins, peptides, nucleic The methods of the invention also include diafiltration of a acids, and other biomolecules. Protein production operations 35 first protein solution that comprises at least two distinct pro often involve final diafiltration of a protein solution into a teins. For example, the protein Solution may contain two or formulation buffer once the protein has been purified from more types of antibodies directed to different molecules or impurities resulting from its expression, e.g., host cell pro different epitopes of the same molecule. teins. The invention described herein provides a means for In one embodiment, the protein that is in solution is a obtaining an aqueous formulation by Subjecting a protein 40 therapeutic protein, including, but not limited to, fusion pro Solution to diafiltration using water alone as a diafiltration teins and enzymes. Examples of therapeutic proteins include, solution. Thus, the formulation of the invention is based on but are not limited to, Pulmozyme (Dornase alfa), Regranex using water as a formulation medium during the diafiltration (Becaplermin), Activase (Alteplase), Aldurazyme process and does not rely on traditional formulation mediums (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetin which include excipients, such as Surfactants, used to solubi 45 alfa), Becaplermin Concentrate, Betaseron (Interferon beta lize and/or stabilize the protein in the final formulation. The 1b). BOTOX (Botulinum Toxin Type A), Elitek (Rasbu invention provides a method for transferring a protein into ricase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel pure water for use in a stable formulation, wherein the protein (Etanercept), Fabrazyme (Agallsidase beta), Infergen (Inter remains in Solution and is able to be concentrated at high feron alfacon-1), Intron A (Interferon alfa-2a), Kineret levels without the use of other agents to maintain its stability. 50 (Anakinra), MYOBLOC (Botulinum Toxin Type B), Neu Prior to diafiltration or DF/UF in accordance with the lasta (Pegfilgrastim), Neumega (Oprelvekin), Neupogen teachings herein, the method includes first providing a protein (Filgrastim), Ontak (Denileukin diftitox), PEGASYS in a first solution. The protein may be formulated in any first (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pul Solution, including formulations using techniques that are mozyme (Dornase alfa), Rebif (Interferon beta-1a), Regranex well established in the art, such as synthetic techniques (e.g., 55 (Becaplermin), Retavase (Reteplase), Roferon-A (Interferon recombinant techniques, peptide synthesis, or a combination alfa-2), TNKase (Tenecteplase), and Xigris (Drotrecogin thereof). Alternatively, the protein used in the methods and alfa), Arcalyst (Rilonacept), NPlate (Romiplostim), Mircera compositions of the invention is isolated from an endogenous (methoxypolyethylene glycol-epoetin beta), Cinry Ze (C1 source of the protein. The initial protein solution may be esterase inhibitor), Elaprase (idursulfase), Myozyme (alglu obtained using a purification process whereby the protein is 60 cosidase alfa), Orencia (abatacept), Naglazyme (galsulfase), purified from a heterogeneous mix of proteins. In one Kepivance (palifermin) and Actimmune (interferon gamma embodiment, the initial protein solution used in the invention 1b). is obtained from a purification method whereby proteins, The protein used in the invention may also be an antibody, including antibodies, expressed in a mammalian expression or antigen-binding fragment thereof. Examples of antibodies system are subjected to numerous chromatography steps 65 that may be used in the invention include chimericantibodies, which remove host cell proteins (HCPs) from the protein non-human antibodies, human antibodies, humanized anti Solution. In one embodiment, the first protein solution is bodies, and domain antibodies (dAbs). In one embodiment, US 8,883,146 B2 17 18 the antibody, or antigen-binding fragment thereof, is an anti referred to as a monoclonal antibody although it has antigenic TNFC. and/or an anti-IL-12 antibody (e.g., it may be a dual specificity for more than a single antigen. variable domain (DVD) antibody). Other examples of anti Monoclonal antibodies are obtained from a population of bodies, or antigen-binding fragments thereof, which may be Substantially homogeneous antibodies, i.e., the individual used in the methods and compositions of the invention antibodies comprising the population are identical except for include, but are not limited to, 1D4.7 (anti-IL-12/IL-23 anti possible naturally occurring mutations that may be present in body; Abbott Laboratories), 2.5(E)mg1 (anti-IL-18; Abbott minor amounts. Thus, the modifier "monoclonal indicates Laboratories), 13C5.5 (anti-IL-13 antibody; Abbott Labora the character of the antibody as not being a mixture of discrete tories), J695 (anti-IL-12; Abbott Laboratories), Afelimomab antibodies. (Fab 2 anti-TNF: Abbott Laboratories), Humira (adali 10 In a further embodiment, antibodies can be isolated from mumab) Abbott Laboratories), Campath (Alemtuzumab), antibody phage libraries generated using the techniques CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetux described in McCafferty et al., Nature, 348:552-554 (1990). imab), Herceptin (Trastuzumab), Myoscint (Imciromab Pen Clackson et al., Nature, 352:624-628 (1991) and Marks et al., tetate), ProstaScint (Capromab Pendetide), Remicade (Inflix 15 J. Mol. Biol., 222:581-597 (1991) describe the isolation of imab), ReoPro (Abciximab), Rituxan (Rituximab), Simulect murine and human antibodies, respectively, using phage (Basiliximab), Synagis (Palivizumab), Verluma (Nofetumo libraries. Subsequent publications describe the production of mab). Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin high affinity (nM range) human antibodies by chain shuffling (Ibritumomab Tiuxetan), Orthoclone OKT3 (Muromonab (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as CD3), Panorex (Edrecolomab), Mylotarg (Gemtuzumab ozo combinatorial infection and in vivo recombination as a strat gamicin), golimumab (Centocor), Cimzia (Certolizumab egy for constructing very large phage libraries (Waterhouse et pegol), Soliris (Eculizumab), CNTO 1275 (ustekinumab), al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these Vectibix (panitumumab), Bexxar (to situmomab and I' tosi techniques are viable alternatives to traditional monoclonal tumomab), an anti-IL-17 antibody Antibody 7 as described in antibody hybridoma techniques for isolation of monoclonal International Application WO 2007/149032 (Cambridge 25 antibodies. Antibody Technology), the entire contents of which are incor Antibodies and antibody fragments may also be isolated porated by reference herein, the anti-IL-13 antibody CAT-354 from yeast and other eukaryotic cells with the use of expres (Cambridge Antibody Technology), the anti-human CD4 sion libraries, as described in U.S. Pat. Nos. 6,423,538; 6,696, antibody CE9y4PE (IDEC-151, clenoliximab) (Biogen 251; 6,699,658; 6,300,065; 6,399,763; and 6,114,147. IDEC/Glaxo Smith Kline), the anti-human CD4 antibody 30 Eukaryotic cells may be engineered to express library pro IDEC CE9.1/SB-210396 (keliximab) (Biogen IDEC), the anti-human CD80 antibody IDEC-114 (galiximab) (Biogen teins, including from combinatorial antibody libraries, for IDEC), the anti-Rabies Virus Protein antibody CR4098 display on the cell Surface, allowing for selection of particular (foravirumab), and the anti-human TNF-related apoptosis cells containing library clones for antibodies with affinity to inducing ligand receptor 2 (TRAIL-2) antibody HGS-ETR2 35 select target molecules. After recovery from an isolated cell, (lexatumumab) (Human Genome Sciences, Inc.), and Avastin the library clone coding for the antibody of interest can be (bevacizumab). expressed at high levels from a suitable mammalian cell line. Techniques for the production of antibodies are provided Additional methods for developing antibodies of interest below. include cell-free screening using nucleic acid display tech Polyclonal Antibodies 40 nology, as described in U.S. Pat. Nos. 7,195,880; 6,951,725: Polyclonal antibodies generally refer to a mixture of anti 7,078, 197; 7,022,479, 6,518,018; 7,125,669; 6,846,655; bodies that are specific to a certain antigen, but bind to dif 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804: ferent epitopes on said antigen. Polyclonal antibodies are 6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; generally raised in animals by multiple subcutaneous (sc) or 6,623,926; 6,416,950; 6,660,473; 6,312,927; 5,922,545; and intraperitoneal (ip) injections of the relevant antigen and an 45 6,348,315. These methods can be used to transcribe a protein adjuvant. It may be useful to conjugate the relevantantigento in vitro from a nucleic acid in Such a way that the protein is a protein that is immunogenic in the species to be immunized, physically associated or bound to the nucleic acid from which e.g., keyhole limpet hemocyanin, serum albumin, bovine thy it originated. By selecting for an expressed protein with a roglobulin, or soybean trypsin inhibitor using a bifunctional target molecule, the nucleic acid that codes for the protein is or derivatizing agent, for example, maleimidobenzoyl Sulfo 50 also selected. In one variation on cell-free screening tech Succinimide ester (conjugation through cysteine residues), niques, antibody sequences isolated from immune system N-hydroxysuccinimide (through lysine residues), glutaralde cells can be isolated and partially randomized polymerase hyde, succinic anhydride, SOCl, or RNCNR, where R and chain reaction mutagenesis techniques to increase antibody Rare different alkyl groups. Methods for making polyclonal diversity. These partially randomized antibody genes are then antibodies are known in the art, and are described, for 55 expressed in a cell-free system, with concurrent physical example, in Antibodies. A Laboratory Manual, Lane and association created between the nucleic acid and antibody. Harlow (1988), incorporated by reference herein. The DNA also may be modified, for example, by substi Monoclonal Antibodies tuting the coding sequence for human heavy- and light-chain A "monoclonal antibody” as used herein is intended to constant domains in place of the homologous murine refer to a hybridoma-derived antibody (e.g., an antibody 60 sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. secreted by a hybridoma prepared by hybridoma technology, Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently such as the standard Kohler and Milstein hybridoma method joining to the immunoglobulin coding sequence all or part of ology). For example, the monoclonal antibodies may be made the coding sequence for a non-immunoglobulin polypeptide. using the hybridoma method first described by Kohler et al., Typically such non-immunoglobulin polypeptides are Sub Nature, 256:495 (1975), or may be made by recombinant 65 stituted for the constant domains of an antibody, or they are DNA methods (U.S. Pat. No. 4,816,567). Thus, a hybridoma Substituted for the variable domains of one antigen-combin derived dual-specificity antibody of the invention is still ing site of an antibody to create a chimeric bivalent antibody US 8,883,146 B2 19 20 comprising one antigen-combining site having specificity for sequences of the antibodies, are described in U.S. Pat. No. an antigen and another antigen-combining site having speci 6,090,382 (referred to as D2E7), incorporated by reference ficity for a different antigen. herein.\ Chimeric or hybrid antibodies also may be prepared in In one embodiment, the human neutralizing, antibody, or vitro using known methods in synthetic protein chemistry, 5 an antigen-binding portion thereof, having a high affinity for including those involving crosslinking agents. For example, human TNFC. is an isolated human antibody, or an antigen immunotoxins may be constructed using a disulfide-ex binding portion thereof, with a light chain variable region change reaction or by forming a thioether bond. Examples of (LCVR) having a CDR3 domain comprising the amino acid Suitable reagents for this purpose include iminothiolate and sequence of SEQID NO:3, or modified from SEQID NO:3 methyl-4-mercaptobutyrimidate. 10 by a single alanine Substitution at position 1, 4, 5, 7 or 8, and Humanized Antibodies with a heavy chain variable region (HCVR) having a CDR3 Methods for humanizing non-human antibodies are well domain comprising the amino acid sequence of SEQ ID known in the art. Generally, a humanized antibody has one or NO:4, or modified from SEQ ID NO:4 by a single alanine more amino acid residues introduced into it from a source 15 substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, which is non-human. These non-human amino acid residues the LCVR further has a CDR2 domain comprising the amino are often referred to as “import residues, which are typically acid sequence of SEQID NO:5 (i.e., the D2E7 VLCDR2)and taken from an “import' variable domain. Humanization can the HCVR further has a CDR2 domain comprising the amino be essentially performed following the method of Winter and acid sequence of SEQID NO: 6 (i.e., the D2E7 VHCDR2). co-workers (Jones et al., Nature, 321:522-525 (1986); Riech Even more preferably, the LCVR further has CDR1 domain mann et al., Nature, 332:323-327 (1988); Verhoeyen et al., comprising the amino acid sequence of SEQ ID NO:7 (i.e., Science, 239:1534-1536 (1988)), by substituting non-human the D2E7 VL CDR1) and the HCVR has a CDR1 domain (e.g., rodent) CDRS or CDR sequences for the corresponding comprising the amino acid sequence of SEQ ID NO:8 (i.e., sequences of a human antibody. Accordingly, such "human the D2E7 VHCDR1). ized antibodies are chimericantibodies (U.S. Pat. No. 4,816, 25 In another embodiment, the human neutralizing, antibody, 567), wherein substantially less than an intact human variable or an antigen-binding portion thereof, having a high affinity domain has been Substituted by the corresponding sequence for human TNFC. is an isolated human antibody, or an anti from a non-human species. In practice, humanized antibodies gen-binding portion thereof, with a light chain variable region are typically human antibodies in which some CDR residues (LCVR) comprising the amino acid sequence of SEQ ID and possibly some framework (FR) residues are substituted 30 NO:1 (i.e., the D2E7 VL) and a heavy chain variable region by residues from analogous sites in rodent antibodies. Addi (HCVR) comprising the amino acid sequence of SEQ ID tional references which describe the humanization process NO:2 (i.e., the D2E7 VH). In certain embodiments, the anti include Sims et al., J. Immunol. 151:2296 (1993); Chothia et body comprises a heavy chain constant region, such as an al., J. Mol. Biol., 196:901 (1987); Carter et al., Proc. Natl. IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or Ig) constant Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 35 region. Preferably, the heavy chain constant region is an IgG1 151:2623 (1993), each of which is incorporated by reference heavy chain constant region or an IgG4 heavy chain constant herein. region. Furthermore, the antibody can comprise a light chain Human Antibodies constant region, either a kappa light chain constant region or Alternatively, it is now possible to produce transgenic ani a lambda light chain constant region. Preferably, the antibody mals (e.g., mice) that are capable, upon immunization, of 40 comprises a kappa light chain constant region. producing a full repertoire of human antibodies in the absence In one embodiment, the formulation of the invention com of endogenous immunoglobulin production. For example, it prises an antibody, or antigen-binding portion thereof, which has been described that the homozygous deletion of the anti binds human IL-12, including, for example, the antibody body heavy-chain joining region (J) gene in chimeric and J695 (Abbott Laboratories; also referred to as ABT-874) germ-line mutant mice results in complete inhibition of 45 (U.S. Pat. No. 6,914,128). J695 is a fully human monoclonal endogenous antibody production. Transfer of the human antibody designed to target and neutralize interleukin-12 and germ-line immunoglobulin gene array in Such germ-line interleukin-23. In one embodiment, the antibody, or antigen mutant mice will result in the production of human antibodies binding fragment thereof, has the following characteristics: it upon antigen challenge. See, e.g., Jakobovits et al., Proc. dissociates from human IL-1C. with a K, of 3x107M or less; Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., 50 dissociates from human IL-1B with a K, of 5x10 Morless; Nature, 362:255-258 (1993); Bruggermann et al., Year in and does not bind mouse IL-1C. or mouse IL-1B. Examples Immuno. 7:33 (1993). Human antibodies can also be derived and methods for making human, neutralizing antibodies from phage-display libraries (Hoogenboom et al., J. Mol. which have a high affinity for human IL-12, including Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 sequences of the antibody, are described in U.S. Pat. No. 597 (1991)). 55 6,914,128, incorporated by reference herein. In one embodiment, the formulation of the invention com In one embodiment, the formulation of the invention com prises an antibody, or antigen-binding portion thereof, which prises an antibody, or antigen-binding portion thereof, which binds human TNFC., including, for example, adalimumab binds human IL-18, including, for example, the antibody (also referred to as Humira, adalimumab, or D2E7; Abbott 2.5(E)mg1 (Abbott Bioresearch; also referred to as ABT-325) Laboratories). In one embodiment, the antibody, or antigen 60 (see U.S. Patent Application No. 2005/0147610, incorporated binding fragment thereof, dissociates from human TNFC. by reference herein). with a K of 1x10 M or less and a K rate constant of In one embodiment, the formulation of the invention com 1x10s' or less, both determined by surface plasmon reso prises an anti-IL-12/anti-IL-23 antibody, or antigen-binding nance, and neutralizes human TNFC. cytotoxicity in a stan portion thereof, which is the antibody 1D4.7 (Abbott Labo dard in vitro L929 assay with an ICso of 1x107M or less. 65 ratories; also referred to as ABT-147) (see WO 2007/005608 Examples and methods for making human, neutralizing anti A2, published Jan. 11, 2007, incorporated by reference bodies which have a high affinity for human TNFO, including herein). US 8,883,146 B2 21 22 In one embodiment, the formulation of the invention com Such antibodies have, for example, been proposed to target prises an anti-IL-13 antibody, or antigen-binding portion immune system cells to unwanted cells (U.S. Pat. No. 4,676, thereof, which is the antibody f C5.5 (Abbott Laboratories: 980), and for treatment of HIV infection (WO 91/00360, WO also referred to as ABT-308) (see. PCT/US2007/19660 (WO 92/200373, and EP 03089). Heteroconjugate antibodies may 08/127,271), incorporated by reference herein). be made using any convenient cross-linking methods. Suit In one embodiment, the formulation of the invention com able cross-linking agents are well known in the art, and are prises an antibody, or antigen-binding portion thereof, which disclosed in U.S. Pat. No. 4,676.980, along with a number of is the antibody 7C6, an anti-amyloid f antibody (Abbott cross-linking techniques. Laboratories; see PCT publication WO 07/062,852, incorpo Techniques for generating bispecific antibodies from anti rated by reference herein). 10 body fragments have also been described in the literature. The Bispecific Antibodies following techniques can also be used for the production of Bispecific antibodies (BSAbs) are antibodies that have bivalent antibody fragments which are not necessarily bispe binding specificities for at least two different epitopes. Such cific. For example, Fab' fragments recovered from E. coli can antibodies can be derived from full length antibodies or anti be chemically coupled in vitro to form bivalent antibodies. body fragments (e.g., F(ab')2 bispecific antibodies). 15 See, Shalaby et al., J. Exp. Med., 175:217-225 (1992). Methods for making bispecific antibodies are known in the Various techniques for making and isolating bivalent anti art. Traditional production of full length bispecificantibodies body fragments directly from recombinant cell culture have is based on the coexpression of two immunoglobulin heavy also been described. For example, bivalent heterodimers have chain-light chain pairs, where the two chains have different been produced using leucine Zippers. Kostelny et al., J. specificities (Millstein et al., Nature, 305:537-539 (1983)). Immunol., 148(5): 1547-1553 (1992). The leucine zipper Because of the random assortment of immunoglobulin heavy peptides from the Fos and Junproteins were linked to the Fab' and light chains, these hybridomas (quadromas) produce a portions of two different antibodies by gene fusion. The anti potential mixture of 10 different antibody molecules, of body homodimers were reduced at the hinge region to form which only one has the correct bispecific structure. Purifica monomers and then re-oxidized to form the antibody het tion of the correct molecule, which is usually done by affinity 25 erodimers. The “diabody’ technology described by Hollinger chromatography steps, is rather cumbersome, and the product et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has yields are low. Similar procedures are disclosed in WO provided an alternative mechanism for making bispecific/ 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 bivalent antibody fragments. The fragments comprise a (1991). heavy-chain variable domain (VH) connected to a light-chain According to a different approach, antibody variable 30 variable domain (VL) by a linker which is too short to allow domains with the desired binding specificities (antibody-an pairing between the two domains on the same chain. Accord tigen combining sites) arefused to immunoglobulin constant ingly, the VH and VL domains of one fragment are forced to domain sequences. pair with the complementary VL and VH domains of another The fusion preferably is with an immunoglobulin heavy fragment, thereby forming two antigen-binding sites. chain constant domain, comprising at least part of the hinge, 35 Another strategy for making bispecific/bivalent antibody CH2, and CH3 regions. It is preferred to have the first heavy fragments by the use of single-chain FV (SFV) dimers has also chain constant region (CH1) containing the site necessary for been reported. See Gruber et al., J. Immunol. 152:5368 light chain binding, present in at least one of the fusions. (1994). DNAS encoding the immunoglobulin heavy chain fusions In one embodiment, the formulation of the invention com and, if desired, the immunoglobulin light chain, are inserted 40 prises an antibody which is bispecific for IL-1 (including into separate expression vectors, and are co-transfected into a IL-1C. and IL-1B). Examples and methods for making bispe suitable host organism. This provides for great flexibility in cific IL-1 antibodies can be found in U.S. Provisional Applin. adjusting the mutual proportions of the three polypeptide No. 60/878,165, filed Dec. 29, 2006. fragments in embodiments when unequal ratios of the three Diafiltration/Ultrafiltration (also generally referred to polypeptide chains used in the construction provide the opti 45 herein as DF/UF) selectively utilizes permeable (porous) mum yields. It is, however, possible to insert the coding membrane filters to separate the components of solutions and sequences for two or all three polypeptide chains in one Suspensions based on their molecular size. A membrane expression vector when the expression of at least two retains molecules that are larger than the pores of the mem polypeptide chains in equal ratios results in high yields or brane while Smaller molecules such as salts, solvents and when the ratios are of no particular significance. 50 water, which are permeable, freely pass through the mem In a preferred embodiment of this approach, the bispecific brane. The solution retained by the membrane is known as the antibodies are composed of a hybrid immunoglobulin heavy concentrate or retentate. The solution that passes through the chain with a first binding specificity in one arm, and a hybrid membrane is known as the filtrate or permeate. One param immunoglobulin heavy chain-light chain pair (providing a eter for selecting a membrane for concentration is its reten second binding specificity) in the other arm. It was found that 55 tion characteristics for the sample to be concentrated. As a this asymmetric structure facilitates the separation of the general rule, the molecular weight cut-off (MWCO) of the desired bispecific compound from unwanted immunoglobu membrane should be /3rd to /6th the molecular weight of the lin chain combinations, as the presence of an immunoglobu molecule to be retained. This is to assure complete retention. lin light chain in only one half of the bispecific molecule The closer the MWCO is to that of the sample, the greater the provides for a facile way of separation. This approach is 60 risk for some Small product loss during concentration. disclosed in WO94/04690 published Mar. 3, 1994. For fur Examples of membranes that can be used with methods of the ther details of generating bispecific antibodies see, for invention include OmegaTMPES membrane (30kDaMWCO, example, Suresh et al., Methods in Enzymology, 121:210 i.e. molecules larger than 30 kDa are retained by the mem (1986). brane and molecules less than 30 kDa are allowed to pass to Bispecific antibodies include cross-linked or "heterocon 65 the filtrate side of the membrane) (Pall Corp., Port Washing jugate' antibodies. For example, one of the antibodies in the ton, N.Y.); Millex(R-GV Syringe Driven Filter Unit, PVDF heteroconjugate can be coupled to avidin, the other to biotin. 0.22 um (Millipore Corp., Billerica, Mass.); Millex(R)-GP US 8,883,146 B2 23 24 Syringe Driven Filter Unit, PES 0.22 um; SteriveXR0.22 um and water Volumes, number of process steps, and other Filter Unit (Millipore Corp., Billerica, Mass.); and Vivaspin parameters of particular embodiments of the invention, see concentrators (MWCO 10 kDa, PES: MWCO3 kDa, PES) the Examples section below. (Sartorius Corp., Edgewood, N.Y.). In order to prepare a Alternative methods to diafiltration for buffer exchange low-ionic protein formulation of the invention, the protein where a protein is re-formulated into water in accordance solution (which may be solubilized in a buffered formulation) with the invention include dialysis and gel filtration, both of is subjected to a DF/UF process, whereby water is used as a which are techniques known to those in the art. Dialysis DF/UF medium. In a preferred embodiment, the DF/UF requires filling a dialysis bag (membrane casing of defined medium consists of water and does not include any other porosity), tying off the bag, and placing the bag in a bath of excipients. 10 water. Through diffusion, the concentration of salt in the bag will equilibrate with that in the bath, whereinlarge molecules, Any water can be used in the DF/UF process of the inven e.g., proteins that cannot diffuse through the bag remain in the tion, although a preferred water is purified or deionized water. bag. The greater the volume of the bath relative to the sample Types of water known in the art that may be used in the Volume in the bags, the lower the equilibration concentration practice of the invention include water for injection (WFI) 15 that can be reached. Generally, replacements of the bath water (e.g., HyPure WFI Quality Water (HyClone), AQUA are required to completely remove all of the salt. Gel filtration NOVAR WFI (Aqua Nova)), UltraPureTM Water (Invitrogen), is a non-adsorptive chromatography technique that separates and distilled water (Invitrogen; Sigma-Aldrich). molecules on the basis of molecular size. In gel filtration, There are two forms of DF/UF, including DF/UF in dis large molecules, e.g., proteins, may be separated from Smaller continuous mode and DF/UF in continuous mode. The meth molecules, e.g., salts, by size exclusion. ods of the invention may be performed according to either In a preferred embodiment of the invention, the first protein mode. Solution is subjected to a repeated Volume exchange with the Continuous DF/UF (also referred to as constant volume water, Such that an aqueous formulation, which is essentially DF/UF) involves washing out the original buffer salts (or water and protein, is achieved. The diafiltration step may be other low molecular weight species) in the retentate (sample 25 performed any number of times, depending on the protein in or first protein solution) by adding water or a new buffer to the Solution, wherein one diafiltration step equals one total Vol retentate at the same rate as filtrate is being generated. As a ume exchange. In one embodiment, the diafiltration process result, the retentate Volume and product concentration does is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to as many times are not change during the DF/UF process. The amount of salt deemed necessary to remove excipients, e.g., salts, from the 30 first protein solution, Such that the protein is dissolved essen removed is related to the filtrate volume generated, relative to tially in water. A single round or step of diafiltration is the retentate volume. The filtrate volume generated is usually achieved when a volume of water has been added to the referred to in terms of “diafiltration volumes”. A single dia retentate side that is equal to the starting Volume of the protein filtration volume (DV) is the volume of retentate when dia Solution. filtration is started. For continuous diafiltration, liquid is 35 In one embodiment, the protein solution is Subjected to at added at the same rate as filtrate is generated. When the least 2 diafiltration steps. In one embodiment, the diafiltration volume of filtrate collected equals the starting retentate vol step or Volume exchange with water may be repeated at least ume, 1 DV has been processed. four times, and preferably at least five times. In one embodi Discontinuous DF/UF (examples of which are provided ment, the first protein solution is subjected to diafiltration below in the Examples section) includes two different meth 40 with water until at least a six-fold Volume exchange is ods, discontinuous sequential DF/UF and volume reduction achieved. In another embodiment, the first protein solution is discontinuous DF/UF. Discontinuous DF/UF by sequential subjected to diafiltration with water until at least a seven-fold dilution involves first diluting the sample (or first protein Volume exchange is achieved. Ranges intermediate to the solution) with water to a predetermined volume. The diluted above recited numbers, e.g., 4 to 6 or 5 to 7, are also intended sample is then concentrated back to its original volume by UF. 45 to be part of this invention. For example, ranges of values Discontinuous DF/UF by volume reduction involves first using a combination of any of the above recited values as concentrating the sample to a predetermined Volume, then upper and/or lower limits are intended to be included. diluting the sample back to its original Volume with water or In a preferred embodiment, loss of protein to the filtrate replacement buffer. As with continuous DF/UF, the process is side of an ultrafiltration membrane should be minimized. The repeated until the level of unwanted solutes, e.g., ionic excipi 50 risk of protein loss to the filtrate side of a particular membrane ents, are removed. varies in relation to the size of the protein relative to the DF/UF may be performed in accordance with conventional membrane's pore size, and the protein's concentration. With techniques known in the art using water, e.g., WFI, as the increases in protein concentration, risk of protein loss to the DF/UF medium (e.g., Industrial Ultrafiltration Design and filtrate increases. For a particular membrane pore size, risk of Application of Diafiltration Processes, Beaton & Klinkowski, 55 protein loss is greater for a smaller protein that is close in size J. Separ. Proc. Technol. 4(2) 1-10 (1983)). Examples of to the membrane’s MWCO than it is for a larger protein. Thus, commercially available equipment for performing DF/UF when performing DF/UF on a smaller protein, it may not be include Millipore LabscaleTM TFF System (Millipore), LV possible to achieve the same reduction in Volume, as com CentramateTM Lab Tangential Flow System (Pall Corpora pared to performing DF/UF on a larger protein using the same tion), and the UniFlux System (GE Healthcare). 60 membrane, without incurring unacceptable protein losses. In For example, in a preferred embodiment, the Millipore other words, as compared to the ultrafiltration of a solution of LabscaleTM Tangential Flow Filtration (TFF) system with a a smaller protein using the same equipment and membrane, a 500 mL reservoir is used to perform a method of the invention solution of a larger protein could be ultrafiltered to a smaller to produce a diafiltered antibody solution. The DF/UF proce Volume, with a concurrent higher concentration of protein in dure is performed in a discontinuous manner, with 14 process 65 the solution. DF/UF procedures using a particular pore size steps used to produce a high concentration antibody formu membrane may require more process steps for a smaller pro lation in water. For additional exemplary equipment, Solution tein than for a larger protein; a greater Volume reduction and US 8,883,146 B2 25 26 concentration for a larger protein permits larger Volumes of tivity of less than 2 mS/cm. In yet another embodiment, the water to be added back, leading to a larger dilution of the aqueous formulation has a conductivity of less than 1 mS/cm. remaining buffer or excipient ingredients in the protein Solu In one aspect of the invention, the aqueous formulation has a tion for that individual process step. Fewer process steps may conductivity of less than 0.5 mS/cm. Ranges intermediate to therefore be needed to achieve a certain reduction in solutes the above recited numbers, e.g., 1 to 3 mS/cm, are also for a larger protein than for a smaller one. A person with skill intended to be encompassed by the invention. For example, in the art would be able to calculate the amount of concentra ranges of values using a combination of any of the above tion possible with each process step and the number of overall recited values as upper and/or lower limits are intended to be process steps required to achieve a certain reduction in Sol included. In addition, values that fall within the recited num utes, given the protein size and the pore size of the ultrafil 10 bers are also included in the invention, e.g., 0.5,0.6,0.7, 0.8. tration device to be used in the procedure. 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, As a result of the diafiltration methods of the invention, the 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 and so forth. concentration of solutes in the first protein solution is signifi An important aspect of the invention is that the diafiltered cantly reduced in the final aqueous formulation comprising protein solution (solution obtained following the diafiltration essentially water and protein. For example, the aqueous for 15 process of the first protein solution) can be concentrated. By mulation may have a final concentration of excipients which following this process, it has been discovered that high con is at least 95% less than the first protein solution, and prefer centrations of protein are stable in water. Concentration fol ably at least 99% less than the first protein solution. For lowing diafiltration results in an aqueous formulation con example, in one embodiment, to dissolve a protein in WFI is taining water and an increased protein concentration relative a process that creates a theoretical final excipient concentra to the first protein solution. Thus, the invention also includes tion, reached by constant volume diafiltration with five dia diafiltering a protein solution using water as a diafiltration filtration Volumes, that is equal or approximate to Ci medium and Subsequently concentrating the resulting aque e=0.00674, i.e., an approximate 99.3% maximum excipient ous solution. Concentration of the diafiltered protein solution reduction. In one embodiment, a person with skill in the art may be achieved through means known in the art, including may perform 6 volume exchanges during the last step of a 25 centrifugation. For example, following diafiltration, the commercial DF/UF with constant volume diafiltration, i.e., water-based diafiltrated protein solution is subjected to a cen Ci would be C, e=0.0025. This would provide an approxi trifugation process which serves to concentrate the protein mate 99.75% maximum theoretical excipient reduction. In via ultrafiltration into a high concentration formulation while another embodiment, a person with skill in the art may use 8 maintaining the water-based solution. Means for concentrat diafiltration volume exchanges to obtain a theoretical ~99.9% 30 ing a solution via centrifugation with ultrafiltration mem maximum excipient reduction. branes and/or devices are known in the art, e.g., with Vivaspin The term "excipient-free” or “free of excipients” indicates centrifugal concentrators (Sartorius Corp. Edgewood, N.Y.). that the formulation is essentially free of excipients. In one The methods of the invention provide a means of concen embodiment, excipient-free indicates buffer-free, salt-free, trating a protein at very high levels in water without the need Sugar-free, amino acid-free, Surfactant-free, and/or polyol 35 for additional stabilizing agents. The concentration of the free. In one embodiment, the term “essentially free of excipi protein in the aqueous formulation obtained using the meth ents' indicates that the solution or formulation is at least 99% ods of the invention can be any amountinaccordance with the free of excipients. It should be noted, however, that in certain desired concentration. For example, the concentration of pro embodiments, a formulation may comprise a certain specified tein in an aqueous solution made according to the methods non-ionic excipient, e.g., Sucrose or mannitol, and yet the 40 herein is at least about 10 g/mL, at least about 1 mg/mL, at formulation is otherwise excipient free. For example, a for least about 10 mg/mL, at least about 20 mg/mL, at least about mulation may comprise water, a protein, and mannitol, 50 mg/mL, at least about 75 mg/mL, at least about 100 wherein the formulation is otherwise excipient free. In mg/mL, at least about 125 mg/mL, at least about 150 mg/mL, another example, a formulation may comprise water, a pro at least about 175 mg/mL; at least about 200 mg/mL, at least tein, and polysorbate 80, wherein the formulation is other 45 about 220 mg/mL, at least about 250 mg/mL, at least about wise excipient free. In yet another example, the formulation 300 mg/mL, or greater than about 300 mg/mL. Ranges inter may comprise water, a protein, a Sorbitol, and polysorbate 80, mediate to the above recited concentrations, e.g., at least wherein the formulation is otherwise excipient free. about 113 mg/mL, at least about 214 mg/mL, and at least When water is used for diafiltering a first protein solution in about 300 mg/mL, are also intended to be encompassed by the accordance with the methods described herein, ionic excipi 50 invention. In addition, ranges of values using a combination ents will be washed out, and, as a result, the conductivity of of any of the above recited values (or values between the the diafiltered aqueous formulation is lower than the first ranges described above) as upper and/or lower limits are protein solution. If an aqueous solution conducts electricity, intended to be included, e.g., 100 to 125 mg/mL, 113 to 125 then it must contain ions, as found with ionic excipients. A mg/mL, and 126 to 200 mg/mL or more. low conductivity measurement is therefore indicative that the 55 The methods of the invention provide the advantage that aqueous formulation of the invention has significantly the resulting formulation has a low percentage of protein reduced excipients, including ionic excipients. aggregates, despite the high concentration of the aqueous Conductivity of a solution is measured according to meth protein formulation. In one embodiment, the aqueous formu ods known in the art. Conductivity meters and cells may be lations comprising water and a high concentration of a pro used to determine the conductivity of the aqueous formula 60 tein, e.g., antibodies, contains less than about 5% protein tion, and should be calibrated to a standard solution before aggregates, even in the absence of a surfactant or other type of use. Examples of conductivity meters available in the art excipient. In one embodiment, the formulation comprises no include MYRON L Digital (Cole Parmer R), Conductometer more than about 7.3% aggregate protein; the formulation (Metrohm AG), and Series 3105/31 15 Integrated Conductiv comprises no more than about 5% aggregate protein; the ity Analyzers (Kemotron). In one embodiment, the aqueous 65 formulation comprises no more than about 4% aggregate formulation has a conductivity of less than 3 mS/cm. In protein; the formulation comprises no more than about 3% another embodiment, the aqueous formulation has a conduc aggregate protein; the formulation comprises no more than US 8,883,146 B2 27 28 about 2% aggregate protein; or the formulation comprising In accordance with the methods and compositions of the no more than about 1% aggregate protein. In one embodi invention, buffer composition changes during DF/UF opera ment, the formulation comprises at least about 92%, at least tions can be circumvented by using pure water as diafiltration about 93%, at least about 94%, at least about 95%, at least medium. By concentrating the protein ~20% more than the about 96%, at least about 97%, at least about 98%, or at least concentration desired in the final Bulk DS, excipients could about 99% monomer protein. Ranges intermediate to the Subsequently be added, for instance, via highly concentrated above recited concentrations, e.g., at least about 98.6%, no excipient stock solutions. Excipient concentrations and solu more than about 4.2%, are also intended to be part of this tion pH could then be guaranteed to be identical as labeled. invention. In addition, ranges of values using a combination The aqueous formulation of the invention provides an of any of the above recited values as upper and/or lower limits 10 advantage as a starting material, as it essentially contains no are intended to be included. excipient. Any excipient(s) which is added to the formulation Many protein-based pharmaceutical products need to be following the diafiltration in water can be accurately calcu formulated at high concentrations. For example, antibody lated, i.e., pre-existing concentrations of excipient(s) do not based products increasingly tend to exceed 100 mg/mL in interfere with the calculation. Examples of pharmaceutically their Drug Product (DP) formulation to achieve appropriate 15 acceptable excipients are described in Remington’s Pharma efficacy and meet a typical patient usability requirement of a ceutical Sciences 16th edition, Osol, A. Ed. (1980), incorpo maximal ~ 1 mL injection Volume. Accordingly, downstream rated by reference herein. Thus, another aspect of the inven processing steps. Such as diafiltration into the final formula tion includes using the aqueous formulation obtained through tion buffer or ultrafiltration to increase the protein concentra the methods described herein, for the preparation of a formu tion, are also conducted at higher concentrations. lation, particularly a pharmaceutical formulation, having Classic thermodynamics predicts that intermolecularinter known concentrations of excipient(s), including non-ionic actions can affect the partitioning of Small solutes across a excipient(s) or ionic excipient(s). One aspect of the invention dialysis membrane, especially at higher protein concentra includes an additional step where an excipient(s) is added to tions, and models describing non-ideal dialysis equilibrium the aqueous formulation comprising water and protein. Thus, and the effects of intermolecular interactions are available 25 the methods of the invention provide an aqueous formulation (Tanford Physical chemistry or macromolecules. New York, which is essentially free of excipients and may be used as a John Wiley and Sons, Inc., p. 182, 1961; Tester and Modell starting material for preparing formulations comprising Thermodynamics and its applications, 3" ed. Upper Saddle water, proteins, and specific concentrations of excipients. River, NL, Prentice-Hall, 1997). In the absence of the avail In one embodiment, the methods of the invention may be ability of detailed thermodynamic data in the process devel 30 used to add non-ionic excipients, e.g., Sugars or non-ionic opment environment, which is necessary to apply these type Surfactants, such as polysorbates and poloxamers, to the for of models, intermolecular interactions rarely are taken into mulation without changing the characteristics, e.g., protein account during the design of commercial DF/UF operations. concentration, hydrodynamic diameter of the protein, con Consequently, DP excipient concentrations may differ sig ductivity, etc. nificantly from the concentration labeled. Several examples 35 Additional characteristics and advantages of aqueous for of this discrepancy in commercial and development products mulations obtained using the above methods are described are published, e.g., chloride being up to 30% lower than below in section III. Exemplary protocols for performing the labeled in an IL-1 receptor antagonist, histidine being 40% methods of the invention are also described below in the lower than labeled in a PEG-STNF receptor, and acetate being Examples. up to 200% higher than labeled in a fusion conjugate protein 40 (Stoner et al., J. Pharm. Sci., 93, 2332-2342 (2004)). There III. Formulations of Invention are several reasons why the actual DP may be different from the composition of the buffer the protein is diafiltered into, The invention provide an aqueous formulation comprising including the Donnan effect (Tombs and Peacocke (1974) a protein and water which has a number of advantages over Oxford; Clarendon Press), non-specific interactions (Ar 45 conventional formulations in the art, including stability of the akawa and Timasheff, Arch. Biochem. Biophys., 224, 169-77 protein in water without the requirement for additional (1983); Timasheff, Annu. Rev. Biophys. Biomol. Struct., 22. excipients, increased concentrations of protein without the 67-97 (1993)), and volume exclusion effects. Volume exclu need for additional excipients to maintain solubility of the sion includes most protein partial specific Volumes are protein, and low osmolality. The formulations of the inven between 0.7 and 0.8 mL/g. Thus, for a globular protein at 100 50 tion also have advantageous storage properties, as the pro mg/mL, protein molecules occupy approx. 7.5% of the total teins in the formulation remain stable during storage, e.g., Solution Volume. No significant intermolecular interactions stored as a liquid form for more than 3 months at 7° C. or assumed, this would translate to a solute molar concentration freeze? thaw conditions, even at high protein concentrations on the retentate side of the membrane that is 92.5% of the and repeated freeze? thaw processing steps. In one embodi molar concentration on the permeate side of the membrane. 55 ment, formulations of the invention include high concentra This explains why basically all protein solution compositions tions of proteins such that the aqueous formulation does not necessarily change during ultrafiltration processing. For show significant opalescence, aggregation, or precipitation. instance, at 40 mg/mL, the protein molecules occupy approx. The aqueous formulation of the invention does not rely on 3% of the total solution volume, and an ultrafiltration step standard excipients, e.g., a tonicity modifier, a stabilizing increasing the concentration to 150 mg/mL will necessarily 60 agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulk induce molar excipient concentrations to change by more ing agent, a lyroprotectant, a basic component, and an acidic than 8% (as protein at 150 mg/mL accounts for more than component. In other embodiments of the invention, the for 11% of total solution volume). Ranges intermediate to the mulation contains water, one or more proteins, and no ionic above recited percentages are also intended to be part of this excipients (e.g., salts, free amino acids). invention. In addition, ranges of values using a combination 65 In certain embodiments, the aqueous formulation of the of any of the above recited values as upper and/or lower limits invention comprises a protein concentration of at least 50 are intended to be included. mg/mL and water, wherein the formulation has an osmolality US 8,883,146 B2 29 30 of no more than 30 mOsmol/kg. Lower limits of osmolality of Protein aggregation is a common problem in protein solu the aqueous formulation are also encompassed by the inven tions, and often results from increased concentration of the tion. In one embodiment the osmolality of the aqueous for protein. The instant invention provides a means for achieving mulation is no more than 15 mOsmol/kg. The aqueous for a high concentration, low protein aggregation formulation. mulation of the invention may have an osmolality of less than Formulations of the invention do not rely on a buffering 30 mOsmol/kg, and also have a high protein concentration, system and excipients, including Surfactants, to keep proteins e.g., the concentration of the protein is at least 100 mg/mL, in the formulation soluble and from aggregating. Formula and may be as much as 200 mg/mL or greater. Ranges inter tions of the invention can be advantageous for therapeutic mediate to the above recited concentrations and osmolality purposes, as they are high in protein concentration and water 10 based, not relying on other agents to achieve high, stable units are also intended to be part of this invention. In addition, concentrations of proteins in solution. ranges of values using a combination of any of the above The majority of biologic products (including antibodies) recited values as upper and/or lower limits are intended to be are subject to numerous degradative processes which fre included. quently arise from non-enzymatic reactions in solution. The concentration of the aqueous formulation of the inven 15 These reactions may have a long-term impact on product tion is not limited by the protein size and the formulation may stability, safety and efficacy. These instabilities can be include any size range of proteins. Included within the scope retarded, if not eliminated, by storage of product at Subzero of the invention is an aqueous formulation comprising at least temperatures, thus gaining a tremendous advantage for the 50 mg/mL and as much as 200 mg/mL or more of a protein, manufacturer in terms of flexibility and availability of Sup which may range in size from 5 kDa to 150 kDa or more. In plies over the product life-cycle. Although freezing is often one embodiment, the protein in the formulation of the inven the safest and most reliable method of biologics product tion is at least about 15 kD in size, at least about 20 kD in size; storage, it has inherent risks. Freezing can induce stress in at least about 47 kD in size; at least about 60 kD in size; at proteins through cold denaturation, by introducing ice-liquid least about 80 kD in size; at least about 100 kD in size; at least interfaces, and by freeze-concentration (cryoconcentration) about 120 kD in size; at least about 140 kD in size; at least 25 of solutes when the water crystallizes. about 160 kD in size; or greater than about 160 kD in size. Cryoconcentration is a process in which a flat, uncontrolled Ranges intermediate to the above recited sizes are also moving ice front is formed during freezing that excludes intended to be part of this invention. In addition, ranges of Solute molecules (Small molecules Such as Sucrose, salts, and values using a combination of any of the above recited values other excipients typically used in protein formulation, or as upper and/or lower limits are intended to be included. 30 macromolecules such as proteins), leading to Zones in which The aqueous formulation of the invention may be charac proteins may be found at relatively high concentration in the terized by the hydrodynamic diameter (D) of the proteins in presence of other solutes at concentrations which may poten solution. The hydrodynamic diameter of the protein in solu tially lead to local pH or ionic concentration extremes. For tion may be measured using dynamic light scattering (DLS), most proteins, these conditions can lead to denaturation and which is an established analytical method for determining the 35 in some cases, protein and Solute precipitation. Since buffer D, of proteins. Typical values for monoclonal antibodies, salts and other Solutes are also concentrated under Such con e.g., IgG, are about 10 nm. Low-ionic formulations, like those ditions, these components may reach concentrations high described herein, may be characterized in that the D, of the enough to lead to pH and/or redox changes in Zones within the proteins are notably lower than proteinformulations compris frozen mass. The pH shifts observed as a consequence of ing ionic excipients. It has been discovered that the D values 40 buffer salt crystallization (e.g., phosphates) in the solutions of antibodies in aqueous formulations made using the DF/UF during freezing can span several pH units, which may impact process using pure water as an exchange medium, are notably protein stability. lower than the D, of antibodies in conventional formulations Concentrated Solutes may also lead to a depression of the independent of protein concentration. In one embodiment, freezing point to an extent where the solutes may not be antibodies in the aqueous formulation of the invention have a 45 frozen at all, and proteins will exist within a solution under D, of less than 4 nm, or less than 3 nm. these adverse conditions. Often, rapid cooling may be applied In one embodiment, the D, of the protein in the aqueous to reduce the time period the protein is exposed to these formulation is smaller relative to the D of the same protein in undesired conditions. However, rapid freezing can induce a a buffered solution, irrespective of protein concentration. large-area ice-water interface, whereas slow cooling induces Thus, in certain embodiments, protein in an aqueous formu 50 Smaller interface areas. For instance, rapid cooling of six lation made inaccordance with the methods described herein, model proteins during one freeze? thaw step was shown to will have a D, which is at least 25% less than the D, of the reveal a denaturation effect greater than 10 cycles of slow protein in a buffered solution at the same given concentration. cooling, demonstrating the great destabilization potential of Examples of buffered solutions include, but are not limited to hydrophobic ice surface-induced denaturation. phosphate buffered saline (PBS). In certain embodiments, 55 The aqueous formulation of the invention has advanta proteins in the aqueous formulation of the invention have a D, geous stability and storage properties. Stability of the aque that is at least 50% less than the D, of the protein in PBS in at ous formulation is not dependent on the form of storage, and the given concentration; at least 60% less than the D, of the includes, but is not limited to, formulations which are frozen, protein in PBS at the given concentration; at least 70% less lyophilized, or spray-dried. Stability can be measured at a than the D of the protein in PBS at the given concentration; 60 selected temperature for a selected time period. In one aspect or more than 70% less than the D, of the protein in PBS at the of the invention, the protein in the aqueous formulations is given concentration. Ranges intermediate to the above recited stable in a liquid form for at least 3 months; at least 4 months, percentages are also intended to be part of this invention, e.g., at least 5 months; at least 6 months; at least 12 months. 55%, 56%, 57%, 64%. 68%, and so forth. In addition, ranges Ranges intermediate to the above recited time periods are also of values using a combination of any of the above recited 65 intended to be part of this invention, e.g., 9 months, and so values as upper and/or lower limits are intended to be forth. In addition, ranges of values using a combination of any included, e.g., 50% to 80%. of the above recited values as upper and/or lower limits are US 8,883,146 B2 31 32 intended to be included. Preferably, the formulation is stable complementary activities that do not adversely affect the at room temperature (about 30°C.) or at 40°C. for at least 1 other protein. For example, it may be desirable to provide two month and/or stable at about 2-8°C. for at least 1 year, or or more antibodies which bind to TNF or IL-12 in a single more preferably stable at about 2-8°C. for at least 2 years. formulation. Furthermore, anti-TNF or anti-IL12 antibodies Furthermore, the formulation is preferably stable following may be combined in the one formulation. Such proteins are freezing (to, e.g., -80° C.) and thawing of the formulation, Suitably present in combination in amounts that are effective hereinafter referred to as a “freeze/thaw cycle.” for the purpose intended. Stability of a protein can be also be defined as the ability to Examples of proteins that may be included in the aqueous remain biologically active. A protein “retains its biological formulation include antibodies, orantigen-binding fragments activity in a pharmaceutical formulation, if the protein in a 10 thereof. Examples of different types of antibodies, or antigen pharmaceutical formulation is biologically active upon binding fragments thereof, that may be used in the invention administration to a subject. For example, biological activity include, but are not limited to, a chimeric antibody, a human of an antibody is retained if the biological activity of the antibody, a humanized antibody, and a domain antibody antibody in the pharmaceutical formulation is within about (dAb). In one embodiment, the antibody used in the methods 30%, about 20%, or about 10% (within the errors of the assay) 15 and compositions of the invention is an anti-TNFC. antibody, of the biological activity exhibited at the time the pharmaceu orantigen-binding portion thereof, oran anti-IL-12 antibody, tical formulation was prepared (e.g., as determined in an orantigenbinding portion thereof. Additional examples of an antigen binding assay). antibody, or antigen-binding fragment thereof, that may be Stability of a protein inanaqueous formulation may also be used in the invention includes, but is not limited to, 1D4.7 defined as the percentage of monomer, aggregate, or frag (anti-IL-12/anti-IL-23; Abbott Laboratories), 2.5(E)mg 1 ment, or combinations thereof, of the protein in the formula (anti-IL-18; Abbott Laboratories), 13C5.5 (anti-1'-13: Abbott tion. A protein “retains its physical stability” in a formulation Laboratories), J695 (anti-IL-12; Abbott Laboratories), Afeli if it shows Substantially no signs of aggregation, precipitation momab (Fab 2 anti-TNF: Abbott Laboratories), Humira (ad and/or denaturation upon visual examination of color and/or alimumab (D2E7); Abbott Laboratories), Campath (Alemtu clarity, or as measured by UV light scattering or by size 25 Zumab), CEA-Scan Arcitumomab (fab fragment), Erbitux exclusion chromatography. In one aspect of the invention, a (Cetuximab), Herceptin (Trastuzumab), Myoscint (Imciro stable aqueous formulation is a formulation having less than mab Pentetate), ProstaScint (Capromab Pendetide), Remi about 10%, and preferably less than about 5% of the protein cade (Infliximab), ReoPro (Abciximab), Rituxan (Ritux being present as aggregate in the formulation. imab), Simulect (Basiliximab), Synagis (Palivizumab), Another characteristic of the aqueous formulation of the 30 Verluma (Nofetumomab), Xolair (Omalizumab), Zenapax invention is that, in some instances, diafiltering a protein (Daclizumab), Zevalin (Ibritumomab Tiuxetan), Orthoclone using water results in an aqueous formulation having OKT3 (Muromonab-CD3), Panorex (Edrecolomab), and improved viscosity features in comparison to the first protein Mylotarg (Gemtuzumab ozogamicin) golimumab (Cento solution (i.e., the viscosity of the diafiltered protein solution is cor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), reduced in comparison to the first protein Solution.) A person 35 CNTO 1275 (ustekinumab), Vectibix (panitumumab), with skill in the art will recognize that multiple methods for Bexxar (to situmomab and I'' to situmomab)and Avastin (be measuring viscosity can be used in the preparation of formu vacizumab). lations in various embodiments of the invention. For example, In one alternative, the protein is a therapeutic protein, kinematic viscosity data (cSt) may be generated using capil including, but not limited to, Pulmozyme (Dornase alfa), laries. In other embodiments, dynamic viscosity data is 40 Regranex (Becaplermin), Activase (Alteplase), Aldurazyme stated, either alone or with other viscosity data. The dynamic (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetin Viscosity data may be generated by multiplying the kinematic alfa), Becaplermin Concentrate, Betaseron (Interferon beta Viscosity data by the density. 1b). BOTOX (Botulinum Toxin Type A), Elitek (Rasbu In one embodiment, the invention also provides a method ricase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel for adjusting a certain characteristic, such as the osmolality 45 (Etanercept), Fabrazyme (Agallsidase beta), Infergen (Inter and/or viscosity, as desired in high protein concentration feron alfacon-1), Intron A (Interferon alfa-2a), Kineret water Solutions, by adding non-ionic excipients, such as man (Anakinra), MYOBLOC (Botulinum Toxin Type B), Neu nitol, without changing other desired features, such as non lasta (Pegfilgrastim), Neumega (Oprelvekin), Neupogen opalescence. As such, it is within the scope of the invention to (Filgrastim), Ontak (Denileukin diftitox), PEGASYS include formulations which are water-based and have high 50 (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pul concentrations of protein, where, either during or following mozyme (Dornase alfa), Rebif (Interferon beta-1a), Regranex the transfer of the protein to water or during the course of the (Becaplermin), Retavase (Reteplase), Roferon-A (Interferon diafiltration, excipients are added which improve, for alfa-2), TNKase (Tenecteplase), and Xigris (Drotrecogin example, the osmolality or viscosity features of the formula alfa), Arcalyst (Rilonacept), NPlate (Romiplostim), Mircera tion. Thus, it is also within the scope of the invention that such 55 (methoxypolyethylene glycol-epoetin beta), Cinry Ze (C1 non-ionic excipients could be added during the process of the esterase inhibitor), Elaprase (idursulfase), Myozyme (alglu transfer of the protein into the final low ionic formulation. cosidase alfa), Orencia (abatacept), Naglazyme (galsulfase), Examples of non-ionizable excipients which may be added to Kepivance (palifermin) and Actimmune (interferon gamma the aqueous formulation of the invention for altering desired 1b). characteristics of the formulation include, but are not limited 60 Other examples of proteins which may be included in the to, mannitol, Sorbitol, a non-ionic Surfactant (e.g., polysor methods and compositions described herein, include mam bate 20, polysorbate 40, polysorbate 60 or polysorbate 80), malian proteins, including recombinant proteins thereof, Sucrose, trehalose, raffinose, and maltose. Such as, e.g., growth hormone, including human growth hor The formulation herein may also contain more than one mone and bovine growth hormone; growth hormone releas protein. With respect to pharmaceutical formulations, an 65 ing factor, parathyroid hormone; thyroid stimulating hor additional, distinct protein may be added as necessary for the mone; lipoproteins; C-1-antitrypsin; insulin A-chain; insulin particular indication being treated, preferably those with B-chain; proinsulin, follicle stimulating hormone; calcitonin; US 8,883,146 B2 33 34 luteinizing hormone:glucagon; clotting factors such as factor include mammals, e.g., humans, dogs, cows, horses, pigs, VIIIC, factor IX, tissue factor, and von Willebrands factor; sheep, goats, cats, mice, rabbits, rats, and transgenic non anti-clotting factors such as Protein C. atrial natriuretic fac human animals. In specific embodiments of the invention, the tor; lung Surfactant; a plasminogen activator, Such as uroki Subject is a human. nase or tissue-type plasminogen activator (t-PA); bombazine; The term “treatment” refers to both therapeutic treatment thrombin; tumor necrosis factor-C. and -3 enkephalinase; and prophylactic or preventative measures. Those in need of RANTES (regulated on activation normally T-cell expressed treatment include those already with the disorder, as well as and secreted); human macrophage inflammatory protein those in which the disorder is to be prevented. (MIP-1-C); serum albumin such as human serum albumin; The aqueous formulation may be administered to a mam mullerian-inhibiting Substance; relaxin A-chain; relaxin 10 mal, including a human, in need of treatment in accordance B-chain; prorelaxin, mouse gonadotropin-associated pep with known methods of administration. Examples of methods tide; DNase; inhibin; activin; vascular endothelial growth of administration include intravenous administration, such as factor (VEGF); receptors for hormones or growth factors; an a bolus or by continuous infusion over a period of time, integrin: protein A or D, rheumatoid factors; a neurotrophic intramuscular, intraperitoneal, intracerobrospinal, Subcuta factor such as bone-derived neurotrophic factor (BDNF), 15 neous, intra-articular, intrasynovial, intrathecal, intradermal, neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a transdermal, oral, topical, or inhalation administration. nerve growth factor such as NGF-?3; platelet-derived growth In one embodiment, the aqueous formulation is adminis factor (PDGF): fibroblast growth factor such as aFGF and tered to the mammal by Subcutaneous administration. For bFGF: epidermal growth factor (EGF); transforming growth Such purposes, the formulation may be injected using a factor (TGF) such as TGFC. and TGF-B, including TGF-B 1. Syringe, as well as other devices including injection devices TGF-B 2, TGF-B3, TGF-B4, or TGF-35; insulin-like growth (e.g., the Inject-ease and Genject devices); injector pens (such factor-I and -II (IGF-I and IGF-II): des(1-3)-IGF-I (brain as the GenPen); needleless devices (e.g., MediJector and IGF-I); insulin-like growth factor binding proteins; CD pro Biojectorr 2000); and subcutaneous patch delivery systems. teins such as CD3, CD4, CD8, CD19 and CD20; erythropoi In one embodiment, the device, e.g., a Syringe, autoinjector etin (EPO); thrombopoietin (TPO); osteoinductive factors: 25 pen, contains a needle with a gauge ranging in size from 25 G immunotoxins; a bone morphogenetic protein (BMP); an or Smaller in diameter. In one embodiment, the needle gauge interferon Such as interferon-C, -3., and -Y., colony Stimulat ranges in size from 25G to 33 G (including ranges interme ing factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; inter diate thereto, e.g., 25sG,26,26sG,27G,28G,29G,30G,31G, leukins (ILS), e.g., IL-1 to IL-10; Superoxide dismutase; 32G, and 33G). In a preferred embodiment, the smallest T-cell receptors; Surface membrane proteins; decay acceler 30 needle diameter and appropriate length is chosen in accor ating factor (DAF); a viral antigen Such as, for example, a dance with the viscosity characteristics of the formulation portion of the AIDS envelope; transport proteins; homing and the device used to deliver the formulation of the inven receptors; addressins; regulatory proteins; immunoadhesins; tion. antibodies; and biologically active fragments or variants of One advantage of the methods/compositions of the inven any of the above-listed polypeptides. 35 tion is that they provide large concentrations of a protein in a Solution which may be ideal for administering the protein to IV. Uses of Invention a subject using a needleless device. Such a device allows for dispersion of the protein throughout the tissue of a subject The formulations of the invention may be used both thera without the need for an injection by a needle. Examples of peutically, i.e., in Vivo, or as reagents for in vitro or in situ 40 needleless devices include, but are not limited to, Bioectorr purposes. 2000 (Bioject Medical Technologies), Cool. Click (Bioject Therapeutic Uses Medical Technologies), Iject (Bioject Medical Technolo The methods of the invention may also be used to make a gies), Vitajet 3, (Bioject Medical Technologies), Mhi500 water-based formulation having characteristics which are (The Medical House PLC), Injex30 (INJEX Equidyne Sys advantageous for therapeutic use. The aqueous formulation 45 tems), InjeX 50 (INJEX Equidyne Systems), Injex 100 (IN may be used as a pharmaceutical formulation to treat a dis JEX-Equidyne Systems), Jet Syringe (INJEX Equidyne order in a Subject. Systems), Jetinjector (Becton-Dickinson), J-Tip (National The formulation of the invention may be used to treat any Medical Devices, Inc.), Medi-Jector VISION (Antares disorder for which the therapeutic protein is appropriate for Pharma), MED-JET (MIT Canada, Inc.), Dermo Jet (Akra treating. A “disorder is any condition that would benefit 50 Dermojet), Sonoprep (Sontra Medical Corp.), PenJet (PenJet from treatment with the protein. This includes chronic and Corp.), MicroPor (Altea Therapeutics), Zeneo (Crossject acute disorders or diseases including those pathological con Medical Technology), Mini-Ject (Valeritas Inc.), ImplaJect ditions which predispose the mammal to the disorder in ques (Caretek Medical LTD), Intraject (Aradigm), and Serojet tion. In the case of an anti-TNFC. antibody, a therapeutically (Bioject Medical Technologies). effective amount of the antibody may be administered to treat 55 Also included in the invention are delivery devices that an autoimmune disease. Such as rheumatoid arthritis, an house the aqueous formulation. Examples of Such devices intestinal disorder, such as Crohn's disease, a spondyloarthr include, but are not limited to, a Syringe, a pen (such as an opathy, Such as ankylosing spondylitis, or a skin disorder, autoinjector pen), an implant, an inhalation device, a needle Such as psoriasis. In the case of an anti-IL-12 antibody, a less device, and a patch. An example of an autoinjection pen therapeutically effective amount of the antibody may be 60 is described in U.S. application Ser. No. 1 1/824,516, filed administered to treat a neurological disorder, such as multiple Jun. 29, 2007. Sclerosis, or a skin disorder, Such as psoriasis. Other examples The invention also includes methods of delivering the for of disorders in which the formulation of the invention may be mulations of the invention by inhalation and inhalation used to treat include cancer, including breast cancer, leuke devices containing said formulation for Such delivery. In one mia, lymphoma, and colon cancer. 65 embodiment, the aqueous formulation is administered to a The term "subject' is intended to include living organisms, Subject via inhalation using a nebulizer or liquid inhaler. e.g., prokaryotes and eukaryotes. Examples of Subjects Generally, nebulizers use compressed air to deliver medicine US 8,883,146 B2 35 36 as wet aerosol or mist for inhalation, and, therefore, require (Boehringer). Respimat(R) is a multi-dose reservoir system that the drug be soluble in water. Types of nebulizers include that is primed by twisting the device base, which is com jet nebulizers (air-jet nebulizers and liquid-jet nebulizers) and pressed a spring and transfers a metered Volume of formula ultrasonic nebulizers. tion from the drug cartridge to the dosing chamber. When the Examples of nebulizers include AkitaTM (Activaero device is actuated, the spring is released, which forces a GmbH) (see US2001037806, EP1258264). AkitaTM is a table micro-piston into the dosing chamber and pushes the Solution top nebulizer inhalation system (Wt: 7.5 kg. BxWxH: 260x through a uniblock; the uniblock consists of a filter structure 170x270) based on Paris LC Star that provides full control with two fine outlet nozzle channels. The MMAD generated over patient’s breathing pattern. The device can deliver as by the Respimat(R) is 2 um, and the device is suitable for low much as 500 mg drug in solution in less than 10 min with a 10 dose drugs traditionally employed to treat respiratory disor very high delivery rates to the lung and the lung periphery. ders. 65% of the nebulized particles are below 5 microns and the Compositions of the invention may also be delivered using mass median aerodynamic diameter (MMAD) is 3.8 microns the Collegium NebulizerTM (Collegium Pharma), which is a at 1.8 bar. The minimum fill volume is 2 mL and the maxi nebulizer system comprised of drug deposited on membrane. mum volume is 8 mL. The inspiratory flow (200 mL/sec) and 15 The dosage form is administered to a patient through oral or nebulizer pressure (0.3-1.8 bar) are set by the Smart card. The nasal inhalation using the Collegium Nebulizer after recon device can be individually adjusted for each patient on the stitution with a reconstituting solvent. basis of a lung function test. Another example of a nebulizer device which may also be Another example of a nebulizer which may be used with used with compositions of the invention includes the Inspi compositions of the invention includes the Aeroneb.R. ration(R) 626 (Respironics). The 626 is a compressor based Go/Pro/Lab nebulizers (AeroGen). The AeronebR nebulizer nebulizer for home care. The 626 delivers a particle size is based on OnOTM technology, i.e., an electronic micropump between 0.5 to 5 microns. (3/8 inch in diameter and wafer-thin) comprised of a unique Nebulizers which can be used with compositions of the dome-shaped aperture plate that contains over 1,000 preci invention may include Adaptive Aerosol Delivery(R) technol sion-formed tapered holes, surrounded by a vibrational ele 25 ogy (Respironics), which delivers precise and reproducible ment. AeronebR. Go is a portable unit for home use, whereas inhaled drug doses to patients regardless of the age, size or Aeroneb(R) Pro is a reusable and autoclavable device for use in variability in breathing patterns of such patients. AADR) sys hospital and ambulatory clinic, and Aeroneb.R. Lab is a device tems incorporate electronics and sensors within the hand for use in pre-clinical aerosol research and inhalation studies. piece to monitor the patient's breathing pattern by detecting The features of the systems include optimization and cus 30 pressure changes during inspiration and expiration. The sen tomization of aerosol droplet size; low-velocity aerosol deliv sors determine when to pulse the aerosol delivery of medica ery with a precisely controlled droplet size, aiding targeted tion during the first part of inspiration. Throughout the treat drug delivery within the respiratory system; flexibility of ment, the sensors monitor the preceding three breaths and dosing; accommodation of a custom single dose ampoule adapt to the patients inspiratory and expiratory pattern. containing a fixed Volume of drug in solution or Suspension, 35 Because AADR systems only deliver medication when the or commercially available solutions for use in general pur patient is breathing through the mouthpiece, these devices pose nebulizers; continuous, breath-activated or program allow the patient to take breaks in therapy without medication mable; and adaptable to the needs of a broad range of patients, waste. Examples of AADR) system nebulizers include the including children and the elderly; single or multi-patient use. HaloLite(R). AADR), ProDose(RAADR), and I-NebRAADR). AerocurrentTM (AerovertRX corp) may also be used with 40 The HaloLite(R) Adaptive Aerosol Delivery (AAD)(R) compositions of the invention (see WO2006006963). This (Respironics) is a pneumatic aerosolisation system powered nebulizer is a portable, vibrating mesh nebulizer that features by a portable compressor. The AADR) technology monitors a disposable, pre-filled or user filled drug cartridge. the patient’s breathing pattern (typically every ten millisec Staccato TM (Alexza Pharma) may also be used with com onds) and, depending upon the system being used, either positions of the invention (see WO03095.012). The key to 45 releases pulses of aerosolized drug into specific parts of the Staccato TM technology is vaporization of a drug without ther inhalation, or calculates the dose drawn during inhalation mal degradation, which is achieved by rapidly heating a thin from a “standing aerosol cloud' (see EP 09 10421, incorpo film of the drug. In less than half a second, the drug is heated rated by reference herein). to a temperature sufficient to convert the solid drug film into The ProDos AADR (Respironics) is a nebulizing system a vapor. The inhaler consists of three core components: a 50 controlled by “ProDose DiscTM” system. (Respironics). Pro heating Substrate, a thin film of drug coated on the Substrate, Dos AADR) is a pneumatic aerosol system powered by a and an airway through which the patient inhales. The inhaler portable compressor, in which the dose to be delivered is is breath-actuated with maximum dose delivered to be 20-25 controlled by a microchip-containing disc inserted in the mg and MMAD in the 1-2 micron range. system that, among other things, instructs the system as to the AERX(R) (Aradigm) may also be used with compositions of 55 dose to deliver. The ProDose DiscTM is a plastic disc contain the invention (see WO9848873, U.S. Pat. No. 5,469,750, U.S. ing a microchip, which is inserted into the ProDose AADR) Pat. No. 5,509,404, U.S. Pat. No. 5,522,385, U.S. Pat. No. System and instructs it as to what dose to deliver, the number 5,694,919, U.S. Pat. No. 5,735,263, U.S. Pat. No. 5,855,564). of doses, which may be delivered together with various con AERX(R) is a hand held battery operated device which utilizes trol data including drug batch code and expiry date (see a piston mechanism to expel formulation from the AERX(R) 60 EP1245244, incorporated by reference herein). Promixin R Strip. The device monitors patients inspiratory air flow and can be delivered via Prodose AADR) for management of fires only when optimal breathing pattern is achieved. The pseudomonas aeruginosa lung infections, particularly in cys device can deliver about 60% of the dose as emitted dose and tic fibrosis. Promixin R is supplied as a powder for nebuliza 50-70% of the emitted dose into deep lung with <25% inter tion that is reconstituted prior to use. subject variability. 65 The I-neb AADR) is a handheld AADR) system that delivers Another example of a nebulizer device which may also be precise and reproducible drug doses into patients’ breathing used with compositions of the invention includes Respimat(R) patterns without the need for a separate compressor (“I- US 8,883,146 B2 37 38 Neb”). The I-neb AADR) is a miniaturized AADR inhaler The MysticTM device is breath activated, and has been used based upon a combination of electronic mesh-based aerosoli with Corus 1030TM (lidocaine HCl), Resmycin R) (doxorubi sation technology (Omron) and AADR technology to control cin hydrochloride), Acuair (fluticasone propionate), NCE dosing into patients’ breathing patterns. The system is with ViroPharm, and NCE with Pfizer. Additional details approximately the size of a mobile telephone and weighs less 5 regarding the MysticTM nebulizer may be found in U.S. Pat. than 8 ounces. I-neb AADR) has been used for delivery of No. 6,397,838, incorporated by reference herein. Ventavis(R) (iloprost) (CoTherix/Schering AG). Additional methods for pulmonary delivery of the formu Another example of a nebulizer which may be used with lation of the invention are provided in U.S. application Ser. compositions of the invention is AriaTM (Chrysalis). Aria is No. 12/217.972, incorporated by reference herein. based on a capillary aerosol generation system. The aerosol is 10 formed by pumping the drug formulation through a small, The appropriate dosage (“therapeutically effective electrically heated capillary. Upon exiting the capillary, the amount”) of the protein will depend, for example, on the formulation rapidly cooled by ambient air to produce an condition to be treated, the severity and course of the condi aerosol with MMAD ranging from 0.5-2.0 um. tion, whether the protein is administered for preventive or In addition the TouchSprayTM nebulizer (Odem) may be 15 therapeutic purposes, previous therapy, the patient’s clinical used to deliver a composition of the invention. The Touch history and response to the protein, the type of protein used, SprayTM nebulizer is a hand-held device which uses a perfo and the discretion of the attending physician. The protein is rate membrane, which vibrates at ultrasonic frequencies, in Suitably administered to the patientatone time or over a series contact with the reservoir fluid, to generate the aerosol cloud. of treatments and may be administered to the patient at any The vibration action draws jets of fluid though the holes in the time from diagnosis onwards. The protein may be adminis membrane, breaking the jets into drug cloud. The size of the tered as the sole treatment or in conjunction with other drugs droplets is controlled by the shape/size of the holes as well as or therapies useful in treating the condition in question. the Surface chemistry and composition of the drug solution. The formulations of the invention overcome the common This device has been reported to deliver 83% of the metered problem of protein aggregation often associated with high dose to the deep lung. Details of the TouchSprayTM nebulizer 25 concentrations of protein, and, therefore, provide a new are described in U.S. Pat. No. 6,659.364, incorporated by means by which high levels of a therapeutic protein may be reference herein. administered to a patient. The high concentration formulation Additional nebulizers which may be used with composi of the invention provides an advantage in dosing where a tions of the invention include nebulizers which are portable higher dose may be administered to a Subject using a volume units which maximize aerosol output when the patient inhales 30 which is equal to or less than the formulation for standard and minimize aerosol output when the patient exhales using treatment. Standard treatment for a therapeutic protein is two one-way valves (see PARI nebulizers (PARI GmbH). described on the label provided by the manufacturer of the Baffles allow particles of optimum size to leave the nebulizer. protein. For example, in accordance with the label provided The result is a high percentage of particles in the respirable by the manufacturer, infliximab is administered for the treat range that leads to improved drug delivery to the lungs. Such 35 ment of rheumatoid arthritis by reconstituting lyophilized nebulizers may be designed for specific patient populations, protein to a concentration of 10 mg/mL. The formulation of such a patients less than three years of age (PARI BABYTM) the invention may comprise a high concentration of inflix and nebulizers for older patients (PARILCPLUSR) and PARI imab, where a high concentration would include a concentra LC STARR). tion higher than the standard 10 mg/mL. In another example, An additional nebulizer which may be used with compo 40 in accordance with the label provided by the manufacturer, sitions of the invention is the e-Flow(R) nebulizer (PARI Xolair (omalizumab) is administered for the treatment of GmbH) which uses vibrating membrane technology to aero asthma by reconstituting lyophilized protein to a concentra Solize the drug Solution, as well as the Suspensions or colloi tion of 125 mg/mL. In this instance, the high concentration dal dispersions (, TouchSprayTM: ODEM (United Kingdom)). formulation of the invention would include a concentration of An e-Flow(R) nebulizer is capable of handling fluid volumes 45 the antibody omalizumab which is greater than the standard from 0.5 ml to 5 ml, and can produce aerosols with a very high 125 mg/mL. density of active drug, a precisely defined droplet size, and a Thus, in one embodiment, the formulation of the invention high proportion of respirable droplets delivered in the shortest comprises a high concentration which is at least about 10%, at possible amount of time. Drugs which have been delivered least about 20%, at least about 30%, at least about 40%, at using the e-Flow(R) nebulizer include aztreonam and 50 least about 50%, at least about 60%, at least about 70%, at lidocaine. Additional details regarding the e-Flow(R) nebulizer least about 80%, at least about 90%, at least about 100%, at are described in U.S. Pat. No. 6,962,151, incorporated by least about 110%, at least about 120%, at least about 130%, at reference herein. least about 140%, at least about 150%, at least about 175%, at Additional nebulizers which may be used with composi least about 200%, at least about 225%, at least about 250%, at tions of the invention include a Microair R electronic nebu 55 least about 275%, at least about 300%, at least about 325%, at lizer (Omron) and a MysticTM nebulizer (Ventaira). The least about 350%, at least about 375%, at least about 400%, MicroairR) nebulizer is extremely small and uses Vibrating and so forth, greater than the concentration of a therapeutic Mesh Technology to efficiently deliver solution medications. protein in a known, standard formulation. The Microair device has 7 mL capacity and produces drug In another embodiment, the formulation of the invention particle MMAD size around 5 microns. For additional details 60 comprises a high concentration which is at least about 2 times regarding the Microair R nebulizer see US patent publication greater than, at least about 3 times greater than, at least about no. 2004.045547, incorporated by reference herein. The Mys 4 times greater than, at least about 5 times greater than, at least ticTM nebulizer uses strong electric field to break liquid into a about 6 times greater than, at least about 7 times greater than, spray of nearly monodispersed, charged particles. The MyS at least about 8 times greater than, at least about 9 times ticTM System includes a containment unit, a dose metering 65 greater than, at least about 10 times greater than and so forth, system, aerosol generation noZZles, and Voltage converters the concentration of a therapeutic protein in a known, stan which together offer multi-dose or unit-dose delivery options. dard formulation. US 8,883,146 B2 39 40 Characteristics of the aqueous formulation may be tility to foods as these proteins have an ability to mimic fats improved fortherapeutic use. For example, the viscosity of an mouthfeel and texture. As such, whey- and Soy-derived pro antibody formulation may be improved by Subjecting an anti teins may be added to foods to decrease the overall fat con body protein solution to diafiltration using water without tent, without sacrificing satisfaction. Thus, an aqueous for excipients as the diafiltration medium. As described above in mulation comprising Suitable amounts of whey- and Soy Section II, excipients, such as those which improve viscosity, derived proteins may be formulated and used to Supplement may be added back to the aqueous formulation Such that the food products. final concentration of excipient is known and the specific Articles of Manufacture characteristic of the formulation is improved for the specified In another embodiment of the invention, an article of use. For example, one of skill in the art will recognize that the 10 manufacture is provided which contains the aqueous formu desired viscosity of a pharmaceutical formulation is depen lation of the present invention and provides instructions for its dent on the mode by which the formulation is being delivered, use. The article of manufacture comprises a container. Suit e.g., injected, inhaled, dermal absorption, and so forth. Often able containers include, for example, bottles, vials (e.g., dual the desired viscosity balances the comfort of the subject in chamber vials), Syringes (such as dual chamber Syringes), receiving the formulation and the dose of the protein in the 15 autoinjector pen containing a syringe, and test tubes. The formulation needed to have atherapeutic effect. For example, container may be formed from a variety of materials such as generally acceptable levels of viscosity for formulations glass, plastic or polycarbonate. The container holds the aque being injected are viscosity levels of less than about 100 ous formulation and the label on, or associated with, the mPas, preferentially less than 75 mPas, even more preferen container may indicate directions for use. For example, the tially less than 50 mPas. As such, viscosity of the aqueous label may indicate that the formulation is useful or intended formulation may be acceptable for therapeutic use, or may for Subcutaneous administration. The container holding the require addition of an excipient(s) to improve the desired formulation may be a multi-use vial, which allows for repeat characteristic. administrations (e.g., from 2-6 administrations) of the aque In one embodiment, the invention provides an aqueous ous formulation. The article of manufacture may further com formulation comprising water and a human TNFC. antibody, 25 prise a second container. The article of manufacture may or antigen-binding portion thereof, wherein the formulation further include other materials desirable from a commercial is excipient-free, wherein the formulation has viscosity which and user standpoint, including other buffers, diluents, filters, makes it advantageous for use as a therapeutic, e.g., low needles, syringes, and package inserts with instructions for viscosity of less than 40 cp at 8°C., and less than 25cPat 25° SC. C. when the protein concentration is about 175 mg/mL. In one 30 The contents of all references, patents and published patent embodiment, the concentration of the antibody, or antigen applications cited throughout this application are incorpo binding portion thereof, in a formulation having improved rated herein by reference viscosity is at least about 50 mg/mL. In one embodiment, the This invention is further illustrated by the following formulation of the invention has a viscosity ranging between examples which should not be construed as limiting. about 1 and about 2 mPas. 35 Non-Therapeutic Uses EXAMPLES The aqueous formulation of the invention may also be used for non-therapeutic uses, i.e., in vitro purposes. The following examples describe experiments relating to Aqueous formulations described herein may be used for an aqueous formulation comprising water as the Solution diagnostic or experimental methods in medicine and biotech 40 medium. It should be noted that in Some instances, decimal nology, including, but not limited to, use in genomics, pro places are indicated using European decimal notation. For teomics, bioinformatics, cell culture, plant biology, and cell example, in Table 31 the number"0.296' is synonymous with biology. For example, aqueous formulations described herein “O.296. may be used to provide a protein needed as a molecular probe in a labeling and detecting methods. An additional use for the 45 Example 1 formulations described herein is to provide supplements for cell culture reagents, including cell growth and protein pro Diafiltration/Ultrafiltration with Adalimumab and duction for manufacturing purposes. J695 Aqueous formulations described herein could be used in protocols with reduced concern regarding how an excipient in 50 Materials and Methods the formulation may react with the experimental environ Adalimumab and J695 were diafiltered using pure water. ment, e.g., interfere with another reagent being used in the After an at least 5-fold volume exchange with pure water, the protocol. In another example, aqueous formulations contain protein solutions were ultrafiltered to a final target concentra ing high concentrations of proteins may be used as a reagent tion of at least 150 mg/mL. Osmolality, visual inspection and for laboratory use. Such highly concentrated forms of a pro 55 protein concentration measurements (OD280) were per tein would expand the current limits of laboratory experi formed to monitor the status of the proteins during DF/UF mentS. processing. Another alternative use for the formulation of the invention Size exclusion chromatography and ion exchange chroma is to provide additives to food products. Because the aqueous tography were used to characterize protein stability in each formulation of the invention consists essentially of water and 60 final DF/UF product as compared to the starting formulation, protein, the formulation may be used to deliver high concen e.g., drug Substance (DS) starting material and protein stan trations of a desired protein, such as a nutritional Supplement, dard. Drug substance or “DS represents the active pharma to a food item. The aqueous formulation of the invention ceutical ingredient and generally refers to a therapeutic pro provides a high concentration of the protein in water, without tein in a common bulk solution. the concern for excipients needed for stability/solubility 65 Adalimumab Drug Substance, (Adalimumab extinction which may not be suitable for human consumption. For coefficient 280 nm: 1.39 mL/mg cm). Drug Substance example, whey- and Soy-derived proteins are lending versa did not contain polysorbate 80. DS composition: 5.57 US 8,883,146 B2 41 42 mM sodium phosphate monobasic, 8.69 mM sodium The stock Solution was used to prepare 1 mg/mL Adali phosphate dibasic, 106.69 mM sodium chloride, 1.07 mumab solution in pure water with various concentra mM sodium citrate, 6.45 mM citric acid, 66.68 mM tions of sorbitol (10, 20, 30, and 40 mg/mL). mannitol. HPLC Methods Adalimumab Solution used for dynamic light scattering 5 Adalimumab, SEC analysis: Sephadex 200 column (Phar (DLS) measurements: Adalimumab solution that was macia Cat. No. 175175-01, S/N 0504057). Mobile phase diafiltered using pure water as exchange medium was 20 mM sodium phosphate, 150 mM sodium chloride, pH adjusted to 1 mg/mL concentration by diluting the 7.5, 0.5 mL/min flow rate, ambient temperature, detec Adalimumab solution with Milli-Q water and excipient tion UV 214 nm and 280 nm. Each sample was diluted to stock solutions (excipients dissolved in Milli-Q water), 10 1.0 mg/mL with Milli-Q water, sample injection load 50 respectively. ug (duplicate injection). J695 Drug Substance, (J695 extinction coefficient 280 nm: 1.42 mL/mg cm). DS composition: Histidine, Methion Adalimumab, IEC analysis: Dionex, Propac WCX-10 col ine, Mannitol, pH 5.8, and polysorbate 80. umn (p/n 054993) along with a corresponding guard Millipore LabscaleTM Tangential Flow Filtration (TFF) 15 column (p/n 054994). Separation conditions: mobile system, equipped with a 500 mL reservoir. The Labscale phase A: 10 mM sodium phosphate, pH 7.5; mobile TFF system was operated in discontinuous mode at phase B 10 mM Sodium phosphate, 500 mM Sodium ambient temperature according to Millipore Operating chloride, pH 5.5. 1.0 mL/min flow rate, ambient tem Instructions. Stirrer speed was set to approx. 1.5, and the perature. Each sample was diluted to 1.0 mg/mL with pump speed was set to approximately 3. The target inlet Milli-Q water, sample injection load 100 ug, duplicate and outlet pressures were 15 mm psig approximately 50 injection. mm psig, respectively. J695, SEC analysis: Tosoh Bioscience G3000swxl, 7.8 MinimateTM Tangential Flow Filtration capsule, equipped mmx30 cm, 5um (Cat. No. 08541). Mobile phase 211 with an OmegaTM PES membrane, 30 kDa cut-off. The mM NaSO/92 mM NaHPO, pH 7.0. 0.3 mL/min capsule was rinsed for 30 min with 0.1 N NaOH and for 25 flow rate, ambient temperature, detection UV 214 nm another 30 min with Milli-Q water. and 280 nm. Each sample was diluted to 2.5 mg/mL with 780 pH meter, Metrohm, equipped with pH probe Pt1000, Milli-Q water, sample injection load 50 lug (duplicate No. 6.0258.010, calibrated with buffer calibration solu injection). tions VWR, pH4.00 buffer solution red, Cat. No.34.170 J695, IEC analysis: Dionex, Propac WCX-10 column (p/n 127, and pH 7.00 buffer solution yellow, Cat. No.34.170 30 054993) along with a corresponding guard column (p/n 130. 054994). Separation conditions: mobile phase A: 10 Varian 50 Bio UV visible spectrophotometer, AI 9655, mM NaHPO, pH 6.0; mobile phase B 10 mM with a fixed Cary 50 cell was used for protein concen NaHPO 500 mMNaCl, pH 6.0.1.0 mL/min flow rate, tration measurements (280 nm wavelength). A 100 uL 35° C. temperature. Each sample was diluted to 1.0 protein sample was diluted with water (Milli-Q water for 35 mg/mL with Milli-Q water, sample injection load 100 HPLC) to a final volume of 50.00 mL for protein con ug. J695 Reference standard 29001 BF was run in tripli centration measurements of all J695 samples and the cate as a comparison and was diluted to 1 mg/ml in Adalimumab solution after DF/UF. Concentration of all Milli-Q water based on the concentration from the Cer other Adalimumab samples was monitored by diluting tificate of Analysis. Calculation of the Protein Concentration 40 uL sample solution with 1960 uL Milli-Q water. 40 Disposable UV cuvettes, 1.5 mL, semi-micro, Poly(m- Calculation Formula: ethyl methacrylate) (PMMA), were used for concentra tion measurements, Milli-Q water was used as OD 280 blank. Milli-Q water for HPLC grade was used as DF/UF 45 medium. A Malvern Zetasizer Nano ZS, Instrument No. AI 94.94 e—absorption coefficient was used for DLS measurements. c—concentration Hellma precision cells, suprasil, Type No. 105.251-QS, d—length of cuvette that the light has to pass light path 3 mm, center 8.5, were used for DLS measure 50 E—absorbance ments (filled with 75 uL sample, Malvern Mastersizer Io initial light intensity Nano ZS, Item No. AI9494). I—light intensity after passing through sample Knauer Osmometer Automatic, Instr. No. 83963, Berlin, Germany, was used for osmolality measurement (cali 55 brated with 400 mOsmol/kg NaCl calibration solution, &Adalimumab = 1.39 Art. No. Y 1241, Herbert Knauer GmbH, Berlin, Ger mgXcm many). 250 mL Corning cell culture flasks, 75 cm, polystyrene, &695 F 'mgxem sterile, Corning, N.Y., USA, were used for storage of the protein solutions after the DF/UF operation. 60 &HSA = 1.042 Sodium chloride: J. T. Baker was used for preparing a 2M mgXcm NaCl stock solution. The stock solution was used to prepare 1 mg/mL Adalimumab Solution in pure water 1.1: DF/UF Processing of Adalimumab with various concentrations of NaCl (10, 20, 30, and 50 DF/UF experiments are carried out following the standard mM) 65 operating procedures of the DF/UF equipment manufactur D-sorbitol, Sigma Chemical Co., St. Louis, Mo. 63178 was ers. For example, the Millipore LabscaleTMTFF system was used for preparing a 200 mg/mL Sorbitol stock solution. equipped with a 500 mL reservoir and the system operated in US 8,883,146 B2 43 44 discontinuous mode at ambient temperature, in accord with TABLE 2 Millipore operating instructions. Stirrer speed was set to approximately 1.5, and the pump speed was set to approxi Impact of DFIUF processing on Adalimumab Solution mately 3. The target inlet and outlet pressures were 15 mm solution Solution psig and approximately 50 mm psig, respectively, and the parameter before DFUF after DFAUF target pressures were monitored to ensure that they were not pH 5.19 S.22 exceeded. concentration (mg/mL) 54.66 175.05 oSmolality (mCSmol/kg) 305 24 A MinimateTM Tangential Flow Filtration capsule *SEC data (% aggregate, O.26 OO.SO equipped with an OmegaTM PES membrane (Pall Corp., Port 10 monomer, 99.74 99.50 Washington, N.Y.), 30 kDa MWCO, was used. The capsule fragment) O.OO O.OO was rinsed for 30 min with 0.1 N NaOH and for another 30 * IEC data (acidic regions, 13.89 14.07 lys 0, 62.OS 61.97 min with Milli-Q water before use. lys 1, 1914 18.51 Approximately 500 mL of Adalimumab solution were lys 2,%) 4.83 4.73 15 placed into the TFF reservoir and DF/UF processing was *samples were subjected to one freezethaw step (-80° C.25°C.) before analysis via SEC started in discontinuous mode. Table 1 provides details on the and IEC In-Process-Control (IPC) data characterizing the DF/UF pro In the course of DF/UF processing, Adalimumab concen CCSS, tration exceeded 210 mg/mL. Throughout the experiment, the protein solution remained clear, and no solution haziness or TABLE 1. protein precipitation, which would have indicated Adali mumab solubility limitations, was observed. Compared to the Overview on Adalimumab DF/UF Processing original Adalimumab DS solution (-55 mg/mL), Adali Volume mumab solution diafiltered by using pure water as DF/UF of Approx. 25 exchange medium revealed lower opalescence, despite a Milli-Q volume of Adalimumab Adalimumab more than 3-fold increase in protein concentration (~175 Proc- water Adalimumab concentration OSmolality concentration mg/mL). ess added Solution in of retentate (mCSmol? of permeate 1.2: Adalimumab Characterization Via Chromatography Step (mL) retentate (mL) (mg/mL) kg) (mg/mL) FIG. 1 shows a SEC chromatogram of an Adalimumab 1 500 54.66 305 30 reference standard (Adalimumab standard (bottom line)) 2 400 68.33 297 3.15 compared to the Adalimumab drug standard Solution before (middle line) and after (top line) the DF/UF processing pro 4 250 550 43.73 169 1.39 cedure. Note that all samples were frozen at -80° C. prior to 5 3OO 4.45 analysis. 6 250 550 47.27 93 2.58 35 7 250 Table 3 also contains the IEC chromatogram data (note all 8 250 500 samples were frozen at -80° C. prior to analysis). 9 250 10 250 500 TABLE 3 11 250 12 250 500 52.24 9 1.24 40 IEC Data of Various Adalimumab Samples 13 3OO 90.27 7.5 % Acidic 90 Acidic 14 130 213.87 4.08 Sample Name Region 1 Region 2 % O Lys % 1 Lys % 2 Lys Fields filled with “—” indicate that no DPC samples were pulled at that step. Reference 2.69 11.66 60.77 1942 S.40 45 standard The DF/UF processing was stopped after an approximate Adalimumab DS 2.51 11.38 62.OS 1914 4.83 5-fold Volume exchange (1 Volume exchange accounting for Adalimumab, after 2.26 11.81 61.97 1851 4.73 approx. 250 mL diafiltration medium). Assuming an ideal 100% excipient membrane permeability, the theoretical final excipient concentration reached by the experiment param 50 1.3: Impact of Excipients on Adalimumab Hydrodynamic eters applied is C,(250/500)=0.03125*Ci, with Ci being the Diameter (D) initial concentration. The maximum excipient reduction was It was previously determined that the hydrodynamic diam therefore 96.875% (if constant volume diafiltration would eter of JG95, as determined by dynamic light scattering (DLS) have been used, the theoretical excipient reduction with 5 measurements, was notably decreased when formulating 55 J695 into pure water. J695 in WFI had a D, of -3 nm, far diafiltration volumes would have been C, e=0.00674, i.e. an below the values that are expected for immunoglobulins. approximate 99.3% maximum excipient reduction). Adali Upon addition of low amounts of ionizable NaCl, the Dh mumab solution was drained from the TFF system to a 250 values increased to ~10 nm (independent of the NaCl con mL cell culture flask (low-volume rinse of the TFF system centration). Addition of non-ionizable mannitol increased was performed using WFI yielding a 175.05 mg/mL concen 60 J695 solution osmolality, but had no effect on J695 Dh. tration; without the rinse, the retentate concentration was In order to assess the impact of excipients on the hydrody 213.87 mg/mL). Samples were pulled for determination of namic diameter of Adalimumab that had been processed pH, osmolality and Adalimumab concentration. Additionally, according to the above DF/UF procedure, the Adalimumab samples were pulled for characterization by SEC and IEC. solution from the DF/UF experiment was used to formulate Characteristic parameters of the Adalimumab solution before 65 Adalimumab Solutions in pure water, but with varying levels and after DF/UF processing, respectively, are listed in Table of NaCl (0-50 mM) and sorbitol (0-40 mg/mL), respectively. 2. The impact of sorbitol (a non-ionizable excipient) and NaCl US 8,883,146 B2 45 46 (ionizable excipient) concentrations on Dh of Adalimumab TABLE 4 monomer is displayed in FIG. 2. The hydrodynamic diameter of Adalimumab monomer in Impact of DF/UF Processing. On JG95 Solution pure water was 2.65 nm. The Adalimumab Dhresponse to salt parameter Solution before DFUF solution after DFUF and non-ionic excipients was identical to the JG95 response pH 4.40 4.70 seen previously. Adalimumab Dh was virtually not impacted concentration (mg/mL) 122.9 1928 by the presence of sorbitol. Low concentrations of NaCl osmolality (mCSmol/kg) 26S 40 SEC data (% aggregate, O41 O.69 induced the monomer hydrodynamic diameter to increase to monomer, 98.42 98.11 expected levels of ~11 nm. These findings demonstrate that 10 fragment) 1.18 1.21 protein hydrodynamic diameters as measured by dynamic IEC data 92.00 92.11 light scattering are crucially impacted by the presence of (Sum of isoforms, 5.17 5.30 acidic species, 2.83 2.59 ionizable excipients. Absence of ionizable excipients also is basic species, 96) linked to solution viscosities. These findings have implications for high-concentrated 15 As with Adalimumab, the DF/UF experiments on J695 protein solutions: the lower the hydrodynamic diameter, the Substantiate the principal possibility of processing and for lower the spatial Volume proteins occupy. In high-concentra mulating J695 by using pure water as exchange medium in DF/UF operations. Both SEC and IEC data suggest no sub tion scenarios, the viscosities of protein solutions that are stantial impact on J695 stability while DF/UF processing for prepared by using water as DF/UF exchange medium will be an overall period of 1.5 days (process interruption overnight) substantially lower than the viscosities of traditional protein at ambient temperature using Milli-Q water as diafiltration formulations containing considerable amounts of ionizable medium. Throughout the experiment, the protein Solution buffer excipients. The Adalimumab data confirmed this, as remained clear, indicating no potential J695 solubility limi tations. viscosities of 200 mg/mL Adalimumab solutions in water for 25 1.5: J695 Characterization injection were found to be well below 50 mPas, independent Table 5 describes the percentages for aggregate, monomer of pH (e.g. pH 4, pH 5, and pH 6). More data on the effect of and fragment content for the three solutions as determined by pH on D, can be found in Example 17 below. SEC chromatogram. Overall, these findings are useful in high-concentration protein formulation activities, where viscosity related manu TABLE 5 facturing and dosing/delivery problems are well known. Fur Data from SEC chromatogram thermore, these findings show that the osmolality values of % Aggregate % Monomer % Fragment final Drug Product can be adjusted with non-ionizable excipi Sample Name content content content ents such as Sugars or Sugar alcohols as desired without induc 35 Reference standard O.45 98.00 1.56 ing an increase in protein Dhand Solution viscosity, respec J695 before DFAUF O41 98.42 1.18 tively. J695 after DFAUF O.69 98.11 1.21 1.4: DF/UF Processing of Jó95 (Anti-IL12 Antibody) Approximately 200 mL of JG95 solution were adjusted to FIG. 3 shows the IEC profile of Jó95 reference standard pH 4.4 with 1 M phosphoric acid and filled into the TFF 40 (bottom graph) and J695 DS, pH adjusted to pH 4.4 (top reservoir (pH adjustment was made to ensure a positive Zeta graph). potential of JG95 monomers and thus avoid a potential impact Only a small increase in aggregate content was observed in of uncharged protein monomer on data). Then, 300 mL of the JG95 samples after DF/UF processing. FIG. 4 shows the IEC profile of J695 after DF/UF with Milli-Q water were added to the TFF reservoir, and DF/UF 45 Milli-Q water, pH 4.7 (top graph), and J695 DS before processing was started in discontinuous mode. 250 mL res DF/UF. pH adjusted to pH 4.4 (bottom curve). ervoir volume, 250 mL of Milli-Q water were added, and As depicted in FIG. 4, the DF/UF step had no notable impact DF/UF processing was started again. The DF/UF processing on J695 stability when monitored by IEC. The differences was stopped after a total of 5 Volume exchange steps were between the two J695 reference standards (refer to FIG. 3) performed (1 Volume exchange accounting for approx. 250 50 can be attributed to differences in the manufacturing pro mL). cesses between the 3000 L and 6000 LDS campaigns. Table Assuming an ideal 100% excipient membrane permeabil 6 highlights more details on IEC data. ity, the theoretical final excipient concentration reached by the experiment parameters applied is Ci(250/500) TABLE 6 Ö=0.03125*Ci, with Ci being the initial concentration. The 55 maximum excipient reduction is therefore 96.875% (if con IEC Data of Various JG95 Samples stant volume diafiltration would have been used, the theoreti Sample Name Oglu (1) other isoforms acidic basic cal excipient reduction with 5 diafiltration volumes would Reference standard 43.57 SO.O6 4.47 1.90 have been Cie-5-0.00674, i.e. an approx. 99.3% maximum 60 J695 35.74 56.26 5.17 2.83 excipient reduction). JG95 solution was drained from the TFF J695 after DFAUF 36.59 55.52 5.30 2.59 system to a 250 mL cell culture flask (no rinse of the TFF system was performed). Samples were pulled for determina 1.6: Conclusion tion of pH, osmolality and J695 concentration. Additionally, The above example provides a diafiltration/ultrafiltration samples were pulled for characterization by SEC and IEC. 65 (DF/UF) experiment where water (Milli-Q water for HPLC) Characteristic parameters of the JG95 solution before and was used as diafiltration medium for monoclonal antibodies after DF/UF processing, respectively, are listed in Table 4. Adalimumab and J695. US 8,883,146 B2 47 48 Adalimumab was subjected to DF/UF processing by using pH probe (Metrohm pH-Meter, protein-suitable probe, pure water as DF/UF exchange medium and was formulated biotrode 46) at pH 5.2 at high concentrations (>200 mg/mL) without Laminar-Air-Flow bench, Steag LF-Werkbank, Mp.-No. inducing Solution haziness, severe opalescence or turbidity 12.5 formation. Upon one subsequent freeze/thaw step, SEC and 5 NaCl; mannitol IEC data suggested no notable difference between Adali Gemini 150 Peltier Plate Rheometer, Malvern mumab solution formulated in water via DF/UF processing Rheometer MCR 301 temperature-controlled P-PTD200 and the original Adalimumab DS. (plate with Peltiertempering) and cone/plate measure J695 also was also subjected to DF/UF processing by using ment system CP50/0.5° Ti as well as CP50/1 (stainless pure water as DF/UF exchange medium and was formulated 10 steel); Anton Paar at pH 4.7 without impacting J695 stability (visual inspection, Capillary viscometer, Schott, capillaries: type 53720, type SEC, IEC). 537 10, type 537 13 When formulated using such a DF/UF processing, the 1M hydrochloric acid (J. T. Baker) hydrodynamic diameter (Dh) of Adalimumab monomer was Analytics approx. 2.65 nm. The presence of non-ionic excipients such 15 as Sorbitol in concentrations up to 40 mg/mL was shown to UV/VIS spectrophotometry (OD 280 nm); Photon Corre have no impact on Dh data, whereas ionic excipients such as lation Spectroscopy (PCS): for approximately 10 NaCl already in low concentrations were demonstrated to mg/mL and approximately 20 mg/mL: 1.1 mPas, 3 runs, induce the Adalimumab monomer Dh to increase to approx. 30s, one measurement, 25°C., from approximately 30 11 nm (such Dh data are commonly monitored for IgG). mg/mL and above: 1.9 mPas, 30s, 3 runs, one measure Similar findings were made earlier for J695. ment, 25°C. In conclusion, processing and formulating proteins using Size Exclusion Chromatography (SEC) and Ion Exchange pure water as DF/UF exchange medium is feasible. Assuming Chromatography (IEC), as described below. an ideal 100% excipient membrane permeability, the theo viscosity measurement: different viscometers with indi retical final excipient concentration reached by the constant 25 vidual and different set-ups were used Volume diafiltration with 5 diafiltration volumes would be Ci Calculation of the Protein Concentration e-5-0.00674, i.e. an approx. 99.3% maximum excipient Calculation Formula: reduction. Using 6 diafiltration Volume exchanges, an theo retical ~99.98% maximum excipient reduction would result. Examples 2 to 5 describe experimental execution with 30 respect to three different proteins which were concentrated into an aqueous formulation, and Examples 6 to 11 describe analysis of each of the aqueous formulations. e—absorption coefficient Materials and Methods for Examples 2-11 c—concentration Adalimumab protein solution (10 mg/mL) in water for 35 d—length of cuvette that the light has to pass injection, Protein Drug Substance (DS) Material (49.68 E—absorbance mg/mL), DS contains Tween 80, Adalimumab, Adali Io initial light intensity mumab Drug Product (DP) (40 mg. solution for injec I—light intensity after passing through sample tion, filtered solution from commercial production). Protein absorption coefficient 280 nm: 1.39. 40 J695 protein solution (10 mg/mL) in water for injection, &Adalimumab = 1.39 Protein Drug Substance (DS) (54 mg/mL), DS contains mgXcm Tween 80. Absorption coefficient 280 nm: 1.42 8695 1.42 HSA protein solution (10 mg/mL) in water for injection, mgXcm DP without Tween 80, Grifols Human Serum Albumin 45 Grifols(R, 20%, 50 mL. Absorption coefficient 280 nm: &HSA = 1.042 1,042 6 R vial and 1 OR vial vial preparation: the vials were washed and autoclaved Viscosity Data and Calculation for Adalimumab stoppers, 19 mm, West, 4110/40/grey 50 Adalimumab commercial formulation (approximately 194 sample repositories (e.g., Eppendorf sample repository, mg/mL) density: Safe-Lock or simple Snap-fit, 1-2 mL) single-use Syringes, Sterile, 20 mL. Norm.Ject, 10 mL. single use filter units (filter Millex(R-GV. Syringe Driven p = 1,05475 Filter Unit, PVDF 0.22 um; Millex(R-GP. Syringe 55 Driven Filter Unit, PES 0.22 um, SteriveXR0.22 um Filter Unit) Adalimumab commercial formulation (approximately 194 Vivaspin concentrators (cut off 10 kDa, PES; cut off3 kDa, mg/mL) kinematic viscosity: PES) K—constant of the capillary Pipettes (e.g., Eppendorf, max.: 1000 uL) 60 t—the time the Solution needs for passing the capillary is Water for injection v—kinematic viscosity Centrifuge (Eppendorf) and Centrifuge No. 1 (tempera ture-controlled) Diafiltration equipment: Millipore LabscaleTM TFF Sys 2 2 tem, Millipore diafiltration membrane: Adalimumab: 65 y = Kxt = 0, 031.59-i-xS 279, 36 s = 8, 825- S - Polyethersulfone; J695: Polyethersulfone. HSA: regen erated cellulose US 8,883,146 B2 49 50 Adalimumab commercial formulation (approximately 194 tration of the initial drug substance material, followed by a mg/mL) dynamic viscosity: procedure to increase the concentration of the drug Substance m—dynamic viscosity in the solution. This may be done in separate procedures or p—density may be done in separate or coinciding steps within the same procedure. Diafiltration 2 9. A sufficient amount of Drug Substance (DS) material (de n = y Xp = 8, 825- S - x 1, 05475 is = 9,308 mPas pending on protein concentration of DS) was Subjected to diafiltration. Prior to diafiltration, the DS material was diluted Viscosity Data and Calculation for Human Serum Albumin 10 with water for injection ~10 mg/ml. Note that in total approxi HSA commercial formulation (approximately 200 mg/mL) mately 540 mL of a 10 mg/mL solution was needed for the density: experiment. Water for injection was used as diafiltration medium. The number of diafiltration steps performed was 5 to 7 (one dia 15 filtration step equals one total Volume exchange). In-Process p = 1,05833 Control (IPC) samples were pulled prior to diafiltration and after diafiltration step (200 uL for osmolality, 120 uL for HSA commercial formulation (app. 200 mg/mL) kinematic SEC). Viscosity: Diafiltration with TFF equipment is performed by applying the following parameters: K—constant of the capillary stirrer: position 2 t—the time the solution needs for passing the capillary is pump: position 2 v—kinematic viscosity pressure up-stream/inlet: max 20-30 psi pressure down-stream/outlet: max 10 psi 2 2 25 (Parameters used in this experiment were derived from y = Kxt = 0, 01024-, -X337, 69 s = 3, 46 manufacturer's recommendations. One with skill in the art S S would be able to alter the parameters of equipment operation to accommodate a particular protein or variances in equip HSA commercial formulation (approximately 200 mg/mL) ment, formulation, Viscosity, and other variables.) dynamic viscosity: 30 After diafiltration, protein concentration was assessed by m—dynamic viscosity means of OD280. If protein concentration was >10 mg/mL, p—density the protein concentration was adjusted to 10 mg/mL by appropriately diluting the Solution with water for injection. Concentration 2 35 20 mL of diafiltrated protein solution (e.g., Adalimumab, n = V Xp = 3, 46- x 1,05833 9. = 3,662 mPas S cm — J695, HSA) were put into a Vivaspin 20 Concentrator. The concentrator was closed and put into the centrifuge. The protein solution was centrifuged at maximum speed (5000 HSA in WFI (approximately 180 mg/mL) density: rpm). 40 Sample Pulls Samples of the concentrated solutions were pulled at: 10 p = 1,07905. mg/mL and then every 10 mg/mL (20, 30, 40 mg/ml etc.) or C until the protein aggregates visibly, and samples were ana lyzed as follows: HSA in WFI (approximately 180 mg/mL) kinematic viscos 45 The protein solution was homogenized in the Vivaspin ity: concentrator and filled in an adequate vial. K—constant of the capillary Optical appearance was inspected directly in the vial. t—the time the solution needs for passing the capillary is 300 uL was used for UV spectroscopy, 160 uL for PCS, 120 v—kinematic viscosity uL for SEC and 300 uL for IEX. 50 the samples for SEC and IEX were stored at -80°C. Dynamic Light Scattering (DLS) protocol 2 mm. Dynamic light scattering was performed using the Zeta y = Kxt = 0, 09573 - x 185, 3 s = 17, 72- - sizer Nano ZS (Malvern Instruments, Southborough, Mass.) equipped with Hellma precision cells (Plainview, N.Y.), HSA in WFI (approximately 180 mg/mL) dynamic viscosity: 55 suprasil quartz, type 105.251-QS, light path 3 mm, center m—dynamic viscosity Z8.5 mm, with at least 60 uL sample fill, using protein p—density samples as is and placed directly in measurement cell. Prior to measurement, the cell window was checked to verify that the solution was free of air bubbles or particles/dust/other con 2 60 taminants that may impact DLS measurement. Measure n = yx p = 17, 72 x 1,07905- ,3 = 19, 121 mPas ments were taken understandard operating procedures (“gen S C eral purpose' mode, 25° C., refractive index set to 1.45, measurement mode set to “manual. 1 run per measurement, General Experimental Execution for Arriving at High Con each comprising 3 measurements of 30 S each, type of mea centration Formulation 65 surement set to “size). Dispersion Technology Software, Generally, the process of the invention for arriving at a high version 4.10b 1, Malvern Instruments, was used to analyze concentration, salt-free, protein formulation includes diafil data. About 70 uLofa sample solution were filled in precision US 8,883,146 B2 51 52 cell for analysis of hydrodynamic diameters (Dh). Default Four Vivaspin 20 concentrators (10 kDa) were used. In sample viscosity was set 1.1 mPas for low concentrated pro three Vivaspins, 20 mL of Adalimumabsolution (10 mg/mL) tein solutions (e.g. <5 mg/mL). Underlying measurement were filled (in each). In the fourth Vivaspin device, water was principles concluded that minimal differences between real filled as counterbalance weight while centrifuging. The con Viscosity values of the sample solution to be measured and the centrators were closed and put into the centrifuge. The Adali use of default viscosities does not impact DLS data readout mumab Solution was centrifuged applying 4500xg centrifu substantially. This was verified by performing DLS measure gation force (in a Swing out rotor). ments of low protein concentration Solutions (<5 mg/mL) 2.3: Sample Pull where solution viscosities were determined and taken into Samples of the concentrated Adalimumab solution were account in Subsequent DLS measurements. For all samples 10 pulled when they reached a concentration of 10 mg/mL and at with higher protein concentration, Viscosities were deter each Subsequent 10 mg/mL concentration increment increase mined and taken into account during DLS measurements. (at 20 mg/mL, 30 mg/mL, 40 mg/mL etc. until approximately 200 mg/mL). At 40 minute intervals, the concentrators were Example 2 taken out of the centrifuge, the Solution was homogenized, 15 and the Solution and the centrifuge adapters were cooled for Formulation Comprising High TNFC. Antibody approximately 10 min on ice. After each 10 mg/mL concen Concentration trating increment, the solutions in the concentrators were homogenized, the optical appearance was checked and 2. 1: Diafiltration samples were pulled for analysis via UV (300 uL), PCS (160 Prior to diafiltration, Adalimumab (49.68 mg/mL) was uL), SEC (120 uL) and IEC (300 uL). After sample pulls, the diluted with water for injection to a concentration of approxi concentrators were filled up to approximately 20 mL with mately 15 mg/mL. Therefore 140.8 mL Adalimumab solution Adalimumab Solution (10 mg/mL). (49.68 mg/mL) were filled in a 500 mL volumetric flask. The Visual Inspection and PCS analysis of protein precipitation flask was filled up to the calibration mark with water for 25 were used to determine the solubility limit of Adalimumab injection. The Volumetric flask was closed and gently shaken protein (i.e. isoforms) in the solution. for homogenization of the solution. The TFF labscale system At a concentration of approximately 80 mg/mL it became was flushed with water. Then the membrane (PES) was obvious that the Adalimumab Solution was not opalescent adapted and was also flushed with 1 L distilled water. Next, anymore, opalescence being a known characteristic of Adali the TFF labscale system and the membrane were flushed with 30 mumab solutions having a high amount of fragment. There approximately 300 mL of water for injection. The diluted fore, it was suspected that fragmentation might have occurred Adalimumab solution was then filled in the reservoir of the during experiment execution. For further analysis, a sample TFF. A sample for an osmolality measurement (300 uL), UV of Adalimumab Solution (approximately 80 mg/mL) was ana spectrophotometry (500 uL) and a sample for SEC analysis lyzed by SEC. The remainder of the solution in each Vivaspin, (120 LL) were pulled. The system was closed and diafiltration 35 as well as the rest of Adalimumab Solution (10 mg/mL), was was started. The DF/UF (diafiltration/ultrafiltration) was fin removed to 50 mL falcontubes and stored at -80°C. The SEC ished after 5 Volume exchanges and after an osmolality value analysis showed a purity of 99.6% monomer. of 3 mosmol/kg was reached. The pH-value of the Adali The solution was thawed in water bath at 25°C. and sterile mumab solution after diafiltration was pH 5.25. filtered. Afterwards the solutions from 3 falcon tubes were Diafiltration with TFF equipment was performed by apply 40 place into each Vivaspin. The concentrators were filled to ing the following parameters: approximately 20 mL and the concentration was continued. stirrer: position 2 The experiment was finished as a concentration of approxi pump: position 2 mately 200 mg/mL was reached. pressure up-stream/inlet: max 20-30 psi All SEC and IEC samples were stored at -80° C. until pressure down-stream/outlet: max 10 psi 45 further analysis. UV and PCS were measured directly after After diafiltration, protein concentration was assessed by sample pull. The rest of the concentrated Adalimumab solu means of OD280. The concentration was determined to be tion was placed in Eppendorf repositories and stored at -80° 13.29 mg/mL. C. The Adalimumab solution was sterile filtered. Table 7 shown below describes calculation of volumes of The TFF and the membrane were flushed with approxi 50 protein solution to be refilled into the concentrators while mately 1 L distilled water and then with 500 mL 0.1MNaOH. concentrating Adalimumab solution. The scheme was calcu The membrane was stored in 0.1M NaOH, the TFF was again lated before experiment execution to define at which volume flushed with approximately 500 mL distilled water. samples have to be pulled. The duration of centrifugation is 2.2: Protein Concentration shown in Table 8. Prior to concentrating the antibody, the protein concentra 55 tion was again assessed by means of OD280. Adalimumab TABLE 7 concentration was determined to be 13.3 mg/mL. The Adali Centrifugation Scheme mumab solution was then diluted to 10 mg/mL.375.94 mL of Adalimumab solution (13.3 mg/mL) was filled in a 500 mL volume volumetric flask and the flask was filled up to the calibration 60 concentration 10 mg/ml mark with water for injection (WFI). 75.19 mL of Adali step volumeml mg/ml protein solution mumab solution (13.3 mg/mL) was also filled in a 100 mL O 2O 10 COC. 1 10 2O volumetric flask, and filled up to the calibration mark with Sampling 2 9 2O pure water, i.e., water for injection (WFI). Both flasks were filling 3 2O 14.5 11 gently shaken for homogenization. The solutions from both 65 COC. 4 9.66 30 flasks were placed in a 1L PETG bottle. The bottle was gently Sampling 5 8.66 30 shaken for homogenization. US 8,883,146 B2 53 54 TABLE 7-continued mumab (200 mg/mL) was diluted with 3 mL WFI to obtain the diluted solution (for a 50 mg/ml Adalimumab solution). Centrifugation Scheme For the rheometer Gemini 150 approximately 2 mL were volume needed and for the MCR 301 less than 1 mL was needed for concentration 10 mg/ml measurement. step volumeml mg/ml protein solution Adalimumab (approximately 194 mg/mL) in the commer filling 6 2O 1866 1.34 cial formulation was obtained by using Vivaspin tubes. The COC. 7 9.33 40 tubes were filled with Adalimumab solution in commercial Sampling 8 8.33 40 buffer and centrifugation was applied until a 194 mg/mL filling 9 2O 22.49 1.67 10 COC. 10 8.99 50 concentration was reached. Viscosity was measured with the Sampling 11 7.99 50 capillary viscometer Schott. filling 12 2O 25.98 2.01 2.5: Summary COC. 13 8.66 60 Sampling 14 7.66 60 In Sum, Adalimumab was concentrated from 50 mg/mL to filling 15 2O 29.15 2.34 15 approximately 194 mg/mL in Vivaspin 20 tubes in four dif COC. 16 8.32 70 ferent tubes. At the beginning, 20 mL of Adalimumab solu Sampling 17 7.32 70 filling 18 2O 31.96 2.68 tion (50 mg/mL) were in every tube (four tubes). At the end of COC. 19 7.99 8O the concentration, 5 mL of Adalimumab solution (approxi Sampling 2O 6.99 8O mately 194 mg/mL) were in every tube. The concentration filling 21 2O 34.46 3.01 step was performed at 5000 rpm (approximately 4500 g). COC. 22 7.65 90 After every hour, the oblong beakers and the protein solution Sampling 23 6.65 90 filling 24 2O 36.6 3.35 in the Vivaspin tubes were cooled in crushed ice for approxi COC. 25 7.32 100 mately 10 to 15 min. The density was measured with density Sampling 26 6.32 100 measurement device DMA 4500, Anton Paar. Further analy filling 27 2O 38.44 3.68 sis of the high Adalimumab concentration formulation is COC. 28 6.98 110 25 provided in Examples 5 to 11. Sampling 29 S.98 110 filling 30 2O 39.9 4.O2 COC. 31 6.65 120 Example 3 Sampling 32 5.65 120 filling 33 2O 41.07 4.35 Formulation Comprising High Concentration 11-12 COC. 34 6.31 130 30 Sampling 35 5.31 130 Antibody filling 36 2O 4.1.86 4.69 COC. 37 S.98 140 3.1: Diafiltration Sampling 38 4.98 140 Prior to diafiltration, IL-12 antibody J695 (54 mg/mL) was COC. 39 4.64 150 154.14 diluted with water for injection to a concentration of approxi 174.14 35 mately 15 mg/mL. This was done by placing 150 mL J695 solution (54 mg/mL) in a 500 mL volumetric flaskand filling the flask to the calibration mark with water for injection. The TABLE 8 Volumetric flask was closed and gently shaken for homogeni zation of the solution. The TFF labscale system was flushed Centrifugation times required for concentrating the Adalinumab solution 40 with water. Then the polyethersulfone membrane (PES) was adapted and was also flushed with 1 L of distilled water. concentration from -> to Afterwards the TFF labscale system and the membrane were mg/mL time min flushed with approximately 300 mL of water for injection. 10 to 20 15 Next, the diluted JG95 solution was placed in the reservoir of 20 to 30 2O the TFF. A sample for osmolality measurement (300 LL), UV 30 to 40 27 45 spectrophotometry (500 uL) and a sample for SEC analysis 40 to SO 30 SO to 60 40 (120 LL) were pulled. The system was closed and diafiltration 60 to 70 50 was started. After 200 mL of processing the DF volume, the 70 to 80 60 diafiltration was stopped and another sample for UV mea 80 to 90 67 surement was pulled. The DF/UF was stopped after 1800 mL 90 to 100 8O 50 diafiltration volume (approximately factor 3.5 volume 100 to 110 1OO exchange), reaching an osmolality value of 4 mosmol/kg. The 110 to 200 2O6 pH-value of the JG95 solution after diafiltration was pH 6.48. Diafiltration with TFF equipment was performed by apply Results from the concentration of Adalimumab are also ing the following parameters: 55 stirrer: position 2 shown below in Table 12. pump: position 2 2.4: Viscosity Measurement pressure up-stream/inlet: max 20-30 psi Adalimumab Solutions comprising either 50 mg/mL in pressure down-stream/outlet: max 10 psi WFI or 200 mg/mL in WFI were measured for viscosity. 50 After diafiltration, the protein concentration was assessed mg/mL and 200 mg/mL in WFI were measured using a 60 by means of OD280. The concentration was determined to be Gemini 150 Peltier Plate Rheometer, Malvern, and the 200 16.63 mg/mL. mg/mL in WFI solution was also measured via rheometer The J695 Solution was Sterile filtered. MCR 301 temperature-controlled P-PTD 200 (plate with The TFF instrument and the membrane were flushed with Peltier tempering) and cone/plate measurement system approximately 1 L of distilled water and then with 500 mL CP50/1 (stainless steel); Anton Paar). 65 0.1M NaOH. The membrane was Stored in 0.1M NaOH and Adalimumab solutions (200 mg/mL) in repository tubes the TFF was again flushed with approximately 500 mL of were thawed and homogenized in a 6R vial. 1 mL Adali distilled water. US 8,883,146 B2 55 56 3.2: Concentrating TABLE 9 Prior to concentrating, the JG95 solution was diluted to 10 mg/mL: 316 mL of JG95 solution (16.63 mg/mL) was placed Centrifugation Times used to Concentrate the JG95 Solution in a 500 mL volumetric flask and the flask was filled to the concentration from -> to calibration mark with water for injection (WFI). Additionally, mg/mL) time min 64 mL of JG95 solution (16.63 mL) was placed in a 100 mL 10 to 20 13 Volumetric flask and filled to the calibration mark with WFI. 20 to 30 22 30 to 40 27 Both flasks were gently shaken for homogenization. The 40 to SO 38 solutions from both flasks were placed in a 1 L PETG bottle. 10 SO to 60 45 The bottle was gently shaken for homogenization. 60 to 70 8O 70 to 80 90 Four Vivaspin 20 concentrators (10kDa cut-off) were used. 80 to 90 105 20 mL of Jô95 solution (10 mg/mL) were place in each of 90 to 100 16S three Vivaspins. In the fourth Vivaspin concentrator device, 100 to 200 270 water was filled as counterbalance weight while centrifuging. 15 The concentrators were closed and put into the centrifuge. The JG95 solution was centrifuged applying 4500xg centrifu 3.4: Impact of Excipients on the Hydrodynamic Diameter of gation force (in a Swing out rotor). J695 3.3: Sample Pull In this experiment the impact of sodium chloride and man nitol, separately, on the hydrodynamic diameter of JG95 was Samples of the concentrated JG95 solution were pulled analyzed. For this purpose, stock Solutions of Sodium chlo when they reached a concentration of 10 mg/mL and at each ride (12 mg/mL) and of mannitol (120 mg/mL) were pre Subsequent 10 mg/mL concentration increment increase (at pared. 1.2 g NaCl was weighed in a beaker, which was filled 20 mg/mL, 30 mg/mL, 40 mg/mL etc. until 200 mg/mL). with approximately 70 mL of WFI, and 12.002 g of Mannitol After every 40 minutes, the concentrators were taken out of 25 was weighed in a beaker which was filled with approximately the centrifuge, the solution was homogenized, and the solu 70 mL of WFI. The two solutions were stirred for homogeni tion and the centrifuge adapters were cooled for approxi Zation. Each Solution was placed in a Volumetric flask, which mately 10 min on ice. After every 10 mg/mL concentration was filled to the calibration mark with WFI. The flasks were increase, the solutions in the concentrators were homog 30 gently shaken for homogenization. enized, the optical appearance was checked and samples were Approximately 8 mL of J695 solution (approximately 200 pulled for UV (300 uL), PCS (160 uL), SEC (120 uL)and IEC mg/mL) was thawed at 37°C. The solution was filled in a 10R analysis (300 uL). After sample pulls, the concentrators were vial and homogenized. Seven2R vials were filled with 500LL filled up to approximately 20 mL with J695 solution (10 J695 solution (approximately 200 mg/mL) each. The filling mg/mL). scheme is described in Table 10 below. TABLE 10 Filling Scheme for Preparation of Jó95 Solutions Containing Different Concentrations of NaCl or Mannitol

vol. NaCl stock vol. mannitol concentration volume ABT-874 solution stock solution wo. WFI excipient excipient (200 mg/ml) LL (12 mg/mL) IL (12 mg/mL) LL LIL 500 500 NaCl 2 mg/mL. 500 167 333 NaCl 4 mg/mL 500 333 167 NaCl 6 mg/mL. 500 500 mannitol 20 mg/mL 500 167 333 mannitol 40 mg/mL 500 333 167 mannitol 60 mg/mL 500 500

Visual Inspection and PCS analysis were used to determine The 2R vials were gently homogenized via shaking. There the solubility (i.e., to check for potential precipitation) and after, PCS and osmolality measurements were taken of the stability of Jó95. different J695 solutions (100 mg/mL). 55 To prepare samples for PCS analysis, the cuvettes were first At the conclusion of the experiment, a concentration of flushed with 50 uL of the sample. Then measurements were approximately 200 mg/mL was reached. taken using 100 uL of the sample. Further analysis of the high J695 concentration formula All SEC and IEC samples were stored at -80°C. for further tion is provided in Examples 5 to 11. analysis (see below). UV spectrophotometry and PCS mea- 60 Example 4 surements were taken directly after each sample pull. The rest of the concentrated JG95 solution was placed in Eppendorf High Concentration Human Serum Albumin (HSA) repositories and stored at -80° C. Formulation

Details regarding the centrifugation scheme are provided 65 4.1: Diafiltration above in Table 7. The duration of the JG95 centrifugation are Prior to diafiltration, HSA solution (200 mg/mL, commer provided in Table 9. cial formulation) was diluted with water for injection to a US 8,883,146 B2 57 58 concentration of 15.29 mg/mL. To achieve this, 38 mL HSA homogenized, the optical appearance was checked and (200 mg/mL) were filled in a 500 mL volumetric flask. The samples were pulled for analysis via UV (300 uL), PCS (160 flask was filled to the calibration mark with water for injec uL), SEC (120 uL) and IEC (300 uL). After the sample pull, tion. The Volumetric flask was closed and gently shaken for HSA solution (9.52 mg/mL) was added to the concentrators, homogenization of the solution. The TFF labscale system was up to approximately 20 mL. flushed with water. Then the membrane (regenerated cellu When the projected concentration for the HSA solution in lose) was adapted and was also flushed with 1 L of distilled the concentrator reached approximately 20 mg/mL, permeate water. Afterwards the TFF labscale system and the membrane was measured via OD280, revealing a concentration of were flushed with approximately 300 mL water for injection. 0.5964 mg/mL. The concentration of the HSA solution was Next, the diluted HSA solution was filled in the reservoir of 10 only 15.99 mg/mL, which was less than expected. A sample the TFF. Samples for osmolality measurement (300 LL), UV of concentrated HSA in WFI (10 mg/mL) was analyzed via spectrophotometry (500 uL) and a sample for SEC analysis SEC to scrutinize for potential fragmentation. The HSA solu (120 LL) were pulled. The system was closed and diafiltration tion (15.99 mg/mL) in the Vivaspins was placed in falcon was started. After diafiltration of approximately 300 mL of tubes and stored at -80°C. The remainder of the original HSA volume, a UV measurement of the permeate was taken. The solution (9.52 mg/mL) used to fill the concentrators was also permeate revealed a concentration of 2.74 mg/mL, indicating Stored at -80° C. that protein was passing through the membrane. The diafil SEC analysis was performed to determine whether the tration was stopped after approximately 500 mL of DF, and HSA protein underwent degradation, producing Small frag another sample for UV measurement was pulled (HSA con ments that could pass through the membrane. The SEC analy centration 11.03 mg/mL). The DF/UF was finished after 950 sis, however, revealed a monomer amount of 92.45% for 10 mL of diafiltration volume (approximately 2 volume mg/mL HSA in WFI with virtually no fragments. exchanges) and after reaching an osmolality value of 4 mos The solutions that were stored at -80° C. were thawed at mol/kg. The pH-value of the HSA solution after diafiltration 25° C. and sterile filtered. The solutions in the falcon tubes was pH 7.13. 25 were transferred in one Vivaspin 20 concentrator each (3 kDa UV spectrophotometric measurement of the permeate was cut-off). The Vivaspins were filled up with HSA solution performed three times (n=3). (9.52 mg/mL) and centrifuged (refer to 3.2 concentration Diafiltration with TFF equipment was performed by apply process described above). ing the following parameters: Visual Inspection and PCS analysis were used to determine 30 the solubility limit of HSA. stirrer: position 2 At the completion of the experiment, a concentration of pump: position 2 approximately 180 mg/mL HSA was reached. pressure up-stream/inlet: max 20-30 psi All SEC and IEC samples were stored at -80° C. until pressure down-stream/outlet: max 10 psi further analysis. UV and PCS measurements were performed After diafiltration, protein concentration was assessed by 35 directly after sample pull. The rest of the concentrated HSA means of OD280. The concentration was determined 9.41 Solution was placed in Eppendorf repositories and stored at mg/mL. -80° C. The HSA Solution was sterile filtered. The TFF and the An overview of the centrifugation scheme is described membrane were flushed with approximately 1 L of distilled above in Table 7. The duration of the centrifugation used to water. Afterwards an integrity test was done (see Operating 40 concentrate HSA is described in Table 11. Instructions LabscaleTMTFF System, page 5-3 to 5-5, 1997). The volume flow was 1.2 mL/min, thus, the integrity test was TABLE 11 passed (acceptable maximal limit 3 mL/min). The membrane was once more flushed with 500 mL of distilled water and Centrifugation Times Necessary to Concentrate HSA Solution then with 500 mL of 0.05 M. NaOH. The membrane was 45 concentration from -> to stored in 0.05 M. NaOH, the TFF was again flushed with mg/mL) time min approximately 500 mL of distilled water. 10 to 20 9 4.2: Concentration Process 20 to 30 30 Prior to concentrating the HSA protein solution, the con 30 to 40 40 50 40 to SO 50 centration was assessed by means of OD280 and was deter SO to 60 8O mined to be 9.52 mg/mL. Four Vivaspin 20 concentrators (10 60 to 70 90 kDa) were used. 20 mL of HSA solution (9.52 mg/mL) were 70 to 80 110 placed in each of 3 Vivaspin concentrators. In the fourth 80 to 90 130 Vivaspin, water was filled as counterweight balance while 90 to 100 170 100 to 18O 360 centrifuging. The concentrators were closed and put into the 55 centrifuge. The HSA Solution was centrifuged applying 4500xg centrifugation force (in a Swing out rotor). 4.4: Impact of the pH Value on the Hydrodynamic Diameter 4.3: Sample Pull of HSA Samples of the concentrated HSA solution were pulled The following part of the experiment was performed to when the concentration reached 10 mg/mL and Subsequently 60 evaluate a potential impact of pH on the hydrodynamic diam after every 10 mg/mL concentration increment increase (at 20 eter of HSA when the protein is dissolved in WFI. Four 6R mg/mL, 30 mg/mL, 40 mg/mL etc. until approximately 180 vials were filled with 5.09 mL HSA solution (9.83 mg/mL), mg/mL). Every 40 minutes the concentrators were taken out and pH values from 3 to 6 were set up with 1M HCl (actual of the centrifuge, the solution was homogenized, and the pH: 3.04, 3.99, 5.05, 6.01). These solutions were each trans Solution and the centrifuge adapters were cooled for approxi 65 ferred to a separate 10 mL volumetric flask. The flasks were mately 10 min on ice. After every 10 mg/mL concentration then filled to the calibration mark and gently shaken for increment increase, the Solutions in the concentrators were homogenization. US 8,883,146 B2 59 60 The HCl solutions were placed in 10R vials and analyzed It was a Surprising observation that (in addition to being via PCS. The solutions were sterile filtered and measured soluble at all at Such a high protein concentration) Adali again via PCS. Also, 5.09 mL HSA solution (9.83 mg/mL) mumab in WFI appeared to have a low viscosity, even at were transferred in a 10 mL volumetric flask and this was higher concentrations such as 200 mg/mL. Depending on the concentration, the optical characteris filled with WFI to the calibration mark. The flask was gently tics/color of HSA solutions changed from clear and slightly shaken for homogenization and then the solution was sterile yellow (10 mg/mL in WFI) to clear and yellow (approxi filtered and measured via PCS. mately 180 mg/mL in WFI). Sample Preparation for PCS Measurement: During the concentration process, no precipitation was The cuvettes were flushed with 50 uL of sample. Measure observed for the Adalimumab solution and HSA solution. ment was performed with 100 uL of sample volume. 10 Precipitation would have been an indication for solubility 4.5: Viscosity Measurement limitations. The solutions stayed clear until the experiment For HSA in commercial formulation (200 mg/mL) and for was finalized. It is to be highlighted that the experiments were HSA in WFI (approximately 180 mg/mL) the viscosity was not finished because potential solubility limits were measured with a capillary viscometer (Schott, MP.-No. 33.2). approached and precipitation occurred, but were finished A 15 mL aliquot was pulled from a 50 mL bottle of com 15 because the solution Volumes remaining in the concentrators mercial formulation HSA. HSA in WFI was thawed at were not sufficient to proceed with concentrating (i.e. lack of approximately 20° C. and approximately 9 mL were aliquot material). It appears very likely that the solubility limits of Adalimumab, J695, and HSA are well beyond 220 mg/mL. ted in a Falcon tube. The density was measured with density In the JG95 solution a crystal like precipitate was observed measurement device DMA 4500, Anton Paar. when the high-concentrated Solution was stored over night at Further analysis of the high HSA concentration formula 2-8°C. in the concentrators (approximately 120 mg/mL). The tion is provided in Examples 5 to 11. crystal like precipitate redissolved after approximately 3-5 min when the solution was stored at ambient temperature. Example 5 Thus the environment created by dissolving J695 at high concentration in pure water provides conditions where pro Analysis of High Protein Formulations—Optical 25 tein crystallization might be performed by mere temperature Appearance cycling (e.g., from ambient temperature to 2-8°C.). In contrast to Adalimumab in the commercial formulation, Example 6 Adalimumab in WFI did not reveal opalescence. J695 also did not reveal any opalescence phenomena when dissolved in 30 Analysis of High Protein Formulations—Protein WFI. Despite the fact that the protein concentration of Adali Concentration mumab was 80 mg/mL and 200 mg/mL in WFI, there was virtually no opalescence observed. In contrast, the commer The calculation of the protein concentrations is provided cial formulation comprising 50 mg/mL Adalimumab above in the Materials and Methods section. revealed notable opalescence in the commercial formulation. 35 An overview of the concentration of Adalimumab, J695, Thus, the use of pure water, i.e., WFI, as a dissolution medium and HSA into pure water, high protein formulation is pro had a positive effect on protein solution opalescence. vided below in Tables 12-14: TABLE 12 Concentrations of Adalimumab as Assessed Via OD280 during the Concentration Process sample name absorbance average value dilution concentration imumab in WF 10 mg/mL O.68O O.6SO 2O 9.35 O.695 O.575 imumab in WF 20 mg/mL 1 1.064 O.813 40 23.40 O688 O.687 imumab in WF 20 mg/mL 2 O.781 O.788 40 22.68 0.793 O.791 imumab in WF 20 mg/mL 3 O.870 O.824 40 23.71 O.883 O.719 imumab in WF 30 mg/mL 1 O.817 O.807 60 34.84 O812 0.793 imumab in WF 30 mg/mL 2 O.839 O.827 60 3569 O.813 O.829 imumab in WF 30 mg/mL 3 0.770 O.744 60 32.10 O.729 0.732 imumab in WF 40 mg/mL 1 O494 O491 100 35.35 O493 O488 imumab in WF 40 mg/mL 2 O499 OSO1 100 36.06 O.S16 O489 US 8,883,146 B2 61 62 TABLE 12-continued Concentrations of Adalimumab as Assessed Via OD280 during the Concentration Process sample name absorbance average value di ution concentration Adalimumab in WF 40 mg/mL 3 O495 OO 36.81 O.S23 0.517 Adalimumab in WF 50 mg/mL 1 O.S74 0.585 OO 42.11 0.579 O.603 Adalimumab in WF 50 mg/mL 2 O.671 O.634 OO 45.63 O.630 O.6O1 Adalimumab in WF 50 mg/mL 3 0.579 OO 41.27 O.S74 O.S68 Adalimumab in WF 60 mg/mL 1 O.838 O.837 OO 60.21 O.833 O840 Adalimumab in WF 60 mg/mL 2 0.793 0.777 OO 55.89 0.767 0.770 Adalimumab in WF 60 mg/mL 3 O.802 OO 56.10 0.759 0.779 Adalimumab in WF 70 mg/mL 1 O.911 O.878 OO 63.15 O.866 0.857 Adalimumab in WF 70 mg/mL 2 1.012 O.996 OO 71.68 O.976 1.OO1 Adalimumab in WF 70 mg/mL 3 O.879 O.871 OO 62.66 O.874 O861 Adalimumab in WF 80 mg/mL 1 O.S12 200 73.45 O489 O.S31 Adalimumab in WF 80 mg/mL 2 O.S.42 200 75.64 O.S19 0.517 Adalimumab in WF 80 mg/mL 3 0.551 200 76.42 O.S11 O.S31 Adalimumab in WF 90 mg/mL 1 0.550 200 78.64 0.550 O.S39 Adalimumab in WF 90 mg/mL 2 O.S49 200 78.8O 0.551 O.S43 Adalimumab in WF 90 mg/mL 3 O.S32 200 76.81 O.S33 0.537 Adalimumab in WF 00 mg/mL 1 O640 O.628 200 90.36 O621 O.623 Adalimumab in WF 00 mg/mL 2 O.748 0.747 200 107.41 0.735 O.757 Adalimumab in WF 00 mg/mL 3 O.625 O621 200 89.39 O616 O.623 Adalimumab in WF 10 mg/mL 1 O.674 O669 200 96.19 O.671 O660 Adalimumab in WF 10 mg/mL 2 O.693 O668 200 96.OS O.690 O.62O Adalimumab in WF 10 mg/mL 3 O604 O640 200 92.05 O664 O.652 Adalimumab in W 200 mg/mL 1 O.863 O.698 400 201.00 O612 O621 Adalimumab in W 200 mg/mL 2 1.055 O.791 400 227.53 O.658 O.659 US 8,883,146 B2 63 64 TABLE 12-continued Concentrations of Adalimumab as Assessed Via OD280 during the Concentration Process sample name absorbance average value dilution concentration Adalimumab in WFI 200 mg/mL 3 0.732 O.66S 400 191.44 O648 O.615

TABLE 13 Concentrations of Jó95 as Assessed Via OD280 during the Concentration Process sample name absorbance average value dilution concentration ABT-874 in WFI 10 mg/mL 0.715 O.703 2O 9.90 O.708 0.705 ABT-874 in WFI 20 mg/mL O686 40 19.31 ABT-874 in WFI 20 mg/mL 2 O.684 40 1926 ABT-874 in WFI 20 mg/mL 3 O.685 40 19.29 ABT-874 in WFI 30 mg/mL O.7OO 60 29.59 ABT-874 in WFI 30 mg/mL 2 O.703 60 29.70 ABT-874 in WFI 30 mg/mL 3 O.684 60 28.91 ABT-874 in WFI 40 mg/mL O.S39 1OO 37.97 ABT-874 in WFI 40 mg/mL 2 O.S4O 1OO 38.02 ABT-874 in WFI 40 mg/mL 3 O.S2O 1OO 36.65 ABT-874 in WFI 50 mg/mL O.698 1OO 49.15 ABT-874 in WFI 50 mg/mL 2 O.653 1OO 45.95 ABT-874 in WFI 50 mg/mL 3 O.623 1OO 43.87 ABT-874 in WFI 60 mg/mL O.834 1OO 58.75 ABT-874 in WFI 60 mg/mL 2 O.781 1OO 55.02 ABT-874 in WFI 60 mg/mL 3 0.778 1OO 54.76 ABT-874 in WFI 70 mg/mL 1.103 1OO 77.69 ABT-874 in WFI 70 mg/mL 2 1.102 1OO 77.62 ABT-874 in WFI 70 mg/mL 3 1110 1OO 78.13 ABT-874 in WFI 80 mg/mL O.671 2OO 94.45 ABT-874 in WFI 80 mg/mL 2 O.746 2OO 105.06 ABT-874 in WFI 80 mg/mL 3 O664 2OO 93.45 ABT-874 in WFI 90 mg/mL O.826 2OO 116.37 ABT-874 in WFI 90 mg/mL 2 O.809 2OO 113.92 ABT-874 in WFI 90 mg/mL 3 O.804 2OO 113.27 ABT-874 in WFI 100 mg/mL 1 O861 2OO 121.21 ABT-874 in WFI 100 mg/mL 2 O.993 2OO 139.80 ABT-874 in WFI 100 mg/mL 3 O.98S 2OO 138.73 ABT-874 in WFI 200 mg/mL 1 O681 O.805 400 226.67 O864 O869 ABT-874 in WFI 200 mg/mL 2 O.690 0.767 400 216.10 O828 O.784 ABT-874 in WFI 200 mg/mL 3 O.708 O.745 400 209.83 O.789 O.738

50 Tables 14a and 14b: Concentrations of HSA as Assessed Via TABLE 14a-continued OD280 during the Concentration Process sample name absorbance dilution concentration TABLE 14a HSA in WFI 60 mg/mL 3 O.S68 100 54.52 sample name absorbance dilution concentration HSA in WFI 70 mg/mL 1 O645 100 61.89 HSA in WFI 70 mg/mL 2 O.623 100 59.76 HSA in WFI 10 mg/mL 0.515 2O 9.88 HSA in WFI 70 mg/mL 3 O618 100 59.28 HSA in WFI 30 mg/mL 1 O.398 60 22.94 HSA in WFI 80 mg/mL 1 O.393 200 75.37 HSA in WFI 30 mg/mL 2 O.395 60 22.73 HSA in WFI 80 mg/mL 2 O436 200 83.69 HSA in WFI 30 mg/mL 3 O400 60 23.00 o HSA in WFI 80 mg/mL3 O.363 200 69.67 HSA in WFI40 mg/mL 1 O.383 100 36.78 HSA in WFI 90 mg/mL 1 O484 200 92.90 SA I WE s IEE : 9. g 3. HSA in WFI 90 mg/mL 2 O439 200 84.22 HSA in WFI 50 mg/mL 1 O.479 100 45.97 HSA in WFI90 mg/mL3 O419 200 8O.SO HSA in WFI 50 mg/mL 2 O496 100 47.61 HSA in WFI 100 mg/mL 1 O604 200 115.93 HSA in WFISO mg/mL 3 O465 100 44.61 HSA in WFI 100 mg/mL 2 0.573 200 110.00 HSA in WFI 60 mg/mL 1 O.609 100 58.47 65 HSA in WFI 100 mg/mL 3 0.585 200 112.30 HSA in WFI 60 mg/mL 2 O.653 100 62.69 US 8,883,146 B2 65 66 TABLE 1.4b. 7.2: Human Serum Albumin Viscosity HSA commercial formulation (approximately 200 mg/mL) average Viscosity: concentra K—constant of the capillary sample name absorbance value dilution tion t—the time the solution needs for running through the capil HSA in WFI 180 mg/mL 1 O.946 O.952 2OO 182.79 lary is O.9SO v—kinematic viscosity O.961 m—dynamic viscosity HSA in WFI 180 mg/mL 2 O.994 O.929 2OO 178.24 p—density O.906 10 O.886 HSA in WFI 180 mg/mL 3 O.843 O896 2OO 172.05 O.963 times K mm2/s2) v mm2/s) p g/cm3 mm Pas O.884 337.69 O.O1024 3.46 105475 3.66 15 All three proteins evaluated remained soluble in the con HSA in WFI (approximately 180 mg/mL) viscosity: centration ranges evaluated (i.e. >200 mg/mL for Adali K—constant of the capillary mumab and J695, >175 mg/mL for HSA). No indications of t—the time the solution needs for running through the capil insolubility, e.g., the clouding phenomena or precipitation lary is v—kinematic viscosity occurring in the solution, were observed. For Adalimumab, m—dynamic viscosity the results indicate that, over the concentration range evalu p—density ated, all Adalimumab isoforms (i.e. lysine variants) remained soluble, as no precipitation occurred at all. This observation is also consistent with ion exchange chromatography data 25 times K mm2/s2) vmm2/s) p g/cm3 mm Pas described in Example 11, which describes that the sum of lysine variants stayed virtually consistent regardless of Adali 185.3 0.09573 17.72 107905 1912 mumab concentration. The viscosity of HSA in WFI (approximately 180 mg/mL) Example 7 30 was determined to be approximately 19.121 mPas. The vis cosity of HSA in the commercial formulation (approximately 194 mg/mL) was determined to be 9.308 mPas (measured Analysis of High Protein Formulations Viscosity with the capillary viscometer from Schott). 7.3: Analysis of the Viscosities of Adalimumab and HSA 35 The dynamic viscosity of Adalimumab 50 mg/mL in WFI 7.1: Adalimumab Viscosity was lower than the viscosity of Adalimumab 200 mg/mL in The viscosity of Adalimumab (approximately 50 mg/mL) WFI and in commercial buffer, respectively. For HSA the in water for injection was determined to be around 1.5-2 dynamic viscosity for a concentration of 180 mg/mL in WFI mPas. For Adalimumab (approximately 200 mg/mL) in WFI, was about six-fold higher than for a concentration of 200 two values were determined. The one value determined with 40 mg/mL in commercial buffer. Thus, it seems that the intensity cone/plate rheometer from Malvern (Gemini 150) was of viscosity change (i.e. increase and decrease, respectively) approximately 6-6.5 mPas, while the other value (measured due to effects conveyed by pure water as dissolution medium with cone/plate rheometer from Anton Paar, MCR 301) was may depend on the individual protein. approximately 12 mPas. 45 Example 8 Adalimumab commercial formulation (approximately 194 mg/mL) viscosity: Analysis of the Hydrodynamic Diameters of High K—constant of the capillary Protein Formulations—Photon Correlation Spectroscopy (PCS) t—the time the solution needs for passing the capillary is 50 v—kinematic viscosity The following example provides an analysis of the hydro m—dynamic viscosity dynamic diameter (Dh) (the Z-average of the mean hydrody p—density namic molecule diameter) of various proteins in aqueous formulations obtained using the DF/UF methods of the inven 55 tion. 8.1: Adalimumab Hydrodynamic Diameter times K mm2/s2) vmm2/s) p g/cm3 mm Pas As shown in FIGS. 5 and 6, a trend can be observed where 279.36 O.O31.59 8.82S 105475 9.31 the hydrodynamic diameter (D) increases with increasing Adalimumab concentration. FIG. 5 shows the correlation 60 between hydrodynamic diameter (Z-average) and the concen The viscosity of Adalimumab in WFI (approximately 200 tration of Adalimumab in WFI. FIG. 6 shows the correlation mg/mL) was determined to be approximately 12 mPas with between hydrodynamic diameter (peak monomer) and the the viscosimeter from Anton Paar and approximately 6 mPas concentration of Adalimumab in WFI. determined with the viscometer from Malvern. In contrast, The difference between the D determined from the 23.27 the viscosity of Adalimumab in the commercial formulation 65 mg/mL sample compared to the 34.20 mg/mL sample exists (approximately 194 mg/mL) is higher, at 9.308 mPas (mea because of assumptions made in the Standard Operating Pro sured with the capillary viscometer from Schott). cedure (SOP) for hydrodynamic diameter measurement. For US 8,883,146 B2 67 68 Adalimumab samples having s23.27 mg/mL concentration, of the protein. Without salt being present, the hydrodynamic PCS measurements were performed with a SOP that assumes diameter of JG95 was dramatically lower than what normally a 1.1 mPas value for the viscosity of the samples. For Adali is expected for J695 (usually values around 10 nm are deter mumab samples having 34.20 mPas, a SOP assuming a 1.9 mined). mPassample viscosity was used. It is known that PCS data are As illustrated in Table 15, the osmolality increased linearly strongly influenced by the given viscosity of the sample solu with an increase in concentration of mannitol in the protein tion, as PCS data is based on random Brownian motion of the Solution. In contrast, the hydrodynamic diameter did not sample specimen, which is impacted by sample viscosity. show a dependence on mannitol concentration. Mannitol is a Thus, the increase in the hydrodynamic diameter with non-ionic Sugar alcohol/polyol. Mannitol or polyols are used increasing protein concentration can be explained, as increas 10 as stabilizers during parenteral formulation development and ing protein concentration raises the Viscosity of the Solution (higher viscosity leads to lower Brownian motion and higher in final formulations. Mannitol stabilizes the protein by pref calculated D, data). The protein molecules experience a erential exclusion. As other osmolytes, mannitol is preferen lower random Brownian motion and thus, for a given viscos tially excluded from the surface of the protein and it is outside ity, the hydrodynamic diameters of the sample specimen are 15 of the hydrate shell of the protein. Thus the folded state of the calculated higher. Overall, the Z-average based D, values and protein is stabilized because the unfolded state, which has a the D, values of the monomer match well. Additionally, no larger Surface, becomes thermodynamically less favorable increase in D, indicative for protein insolubility is observed (Foster, T. M., Thermal instability of low molecular weight with increasing concentration (i.e. high molecular weight urokinase during heat treatment. III. Effect of salts, sugars aggregates and precipitate (if present) would induce a Sub and Tween 80, 134 International Journal of Pharmaceutics stantial increase in D). 193 (1996); Singh, S, and Singh, J. Effect of polyols on the 8.2: J695 Hydrodynamic Diameter conformational stability and biological activity of a model FIGS. 7 and 8 show that the hydrodynamic diameter of protein lysozyme, 4 AAPS PharmSciTech, Article 42 (2003)). J695 was relatively independent from the protein concentra However, it is interesting that the osmolality can be adjusted tion until a 114.52 mg/mL concentration was reached. 25 basically as desired which would be an important feature of Increasing the J695 concentration from 114.52 mg/mL to the protein findings described herein without impacting the 133.25 mg/mL induced a rapid increase in D. The hydrody D, of the protein. These findings may be useful in high namic diameter at the 217.53 mg/mL concentration was concentration protein formulation, where viscosity related higher than at 114.52 mg/mL. This finding was not Surprising manufacturing and dosing issues may be present, as the as both protein solutions were measured using the same SOP 30 osmolality adjustment with mannitol is not mirrored by an (assuming same viscosity of 1.9 mPas), when in reality the increase in protein D, (meaning viscosity is expected to viscosity increases as the protein concentration increases. remain constant). The strong increase from 114.52 mg/mL to 133.25 mg/mL thus can be explained as an artifact. TABLE 1.5 8.3: Human Serum Albumin Hydrodynamic Diameter 35 Impact of Excipients on J695 Osmolality and Z-Average The hydrodynamic diameter of HSA in WFI was found to NaCl decrease as concentrations rose from 9.88 mg/mL to 112.74 concentration oSmolality Z-average mg/mL. From 112.74 mg/mL to 177.69 mg/mL, however, the mg/mL mosmol/kg nm. hydrodynamic diameter was found to increase. HSA showed a general tendency of increasing hydrody 40 O 16 4.19 namic diameters (peak monomer) with increasing protein 2 92 12.2 concentration which is in-line with underlying theoretical 4 158 16.2 principles. The D, decrease from 9.88 mg/mL to 22.89 6 230 17 mg/mL is caused by a change in the measurement SOP mannitol (Switching from assumed Viscosity of 1.1 mPasto anassumed 45 concentration oSmolality Z-average viscosity of 1.9 mPas). mg/mL mosmol/kg nm. Numerical data describing the above is provided in Appen O 16 4.19 dix A. 2O 148 S.49 40 276 3.22 Example 9 50 60 432 3.54 J695: Impact of Excipients of the Hydrodynamic Diameter Example 10

Having found the Surprising result that proteins can be 55 Analysis of High Protein Formulations with Size dissolved in high concentrations in pure water, the impact of Exclusion Chromatography (SEC) ionizable and non-ionizable excipients typically used in parenteral formulations on the hydrodynamic diameter was For the SEC analysis, samples of Adalimumab, JG95 and evaluated. J695 was used as a model protein. HSA were diluted to 2 mg/mL before injection. The injection Table 15 shows that the solution osmolality is directly 60 volume for Adalimumab was 20 uL. For J695 and HSA, a 10 proportional to the concentration of sodium chloride. The LL injection Volume was used. osmolality in the protein solution rises along with the NaCl 10.1: SEC Analysis of Adalimumab concentration (an almost linear correlation). Interestingly, the The amount of monomer of Adalimumab tended to slightly hydrodynamic diameter of JG95 protein increased with decrease from 99.4% to 98.8% while concentrating from 9.35 increasing salt concentration. NaCl is an ionic excipient and 65 mg/mL to 206.63 mg/mL. That decrease of monomer is asso dissociated into positively charged sodium ions and nega ciated with an increase in the amount of aggregate in Adali tively charged chloride ions which might adsorb at the surface mumab solution from 0.4% to 1.1% while concentrating from US 8,883,146 B2 69 70 23.27 mg/mL to 206.62 mg/mL, respectively. The amount of Numerical data describing the above IEC experiments is fragment remained constant at 0.1%, independent of protein provided in Appendix C. concentration (see table in Appendix B). Thus, Adalimumab Summary of Findings in Examples 2-11 was stable in WFI. It was initially thought that transferring proteins, such as Overall, the increase in protein aggregation with increasing antibodies, into WFI would likely induce protein precipita protein concentration is deemed only minor A similar trend in tion by concentrating the protein beyond its solubility limit in monomer decrease would be expected when either the protein pure water. The above studies demonstrate that proteins, was formulated in a buffer system and when additionally including antibodies, not only can be transferred into pure Surfactants are added. Adalimumab protein appears to be WFI at lower concentrations without encountering any pre Surprisingly stable when formulated in pure water. 10 cipitation phenomena and solubility limitations, but that, Sur 10.2: SEC Analysis of JG95 prisingly, Adalimumab (as well as the other two test proteins) The amount of J695 monomer slightly decreased from can be concentrated in pure water to ultra-high concentrations 99.4% to 98.6% with increasing protein concentration from beyond 200 mg/mL using UF/DF and centrifugation tech 9.99 mg/mL to 217.53 mg/mL. The decrease of monomer was 15 niques (e.g., TFF equipment, Vivaspin devices). In addition, associated with an increase in aggregate from about 0.4% to Adalimumab opalescence was unexpectedly found to be Sub about 1.2% with increasing protein concentration from 9.99 stantially reduced when the protein was formulated in WFI. mg/mL to 217.53 mg/mL. Independent from protein concen Osmolality was monitored to ensure that the Adalimumab tration, the amount of fragment was almost constant with buffer medium was completely exchanged by pure, salt free 0.17% to 0.23% with increasing protein concentration from water (i.e. WFI). Moreover, freeze-thaw processing was per 9.99 mg/mL to 217.53 mg/mL. formed during sample preparation for analysis, and virtually Overall, the increase in protein aggregation with increasing no instability phenomena were observed with SEC and IEC protein concentration was deemed only minor A similar trend analysis. in monomer decrease would be expected when the protein is The approach of formulating proteins, e.g., Adalimumab, formulated in buffer systems and when additional surfactants 25 at high concentrations in WFI revealed the potential to reduce are added. Thus, JG95 protein appears to be surprisingly Viscosity phenomena, which often impedes Straightforward stable when formulated in pure water. Drug Product development at high protein concentrations. 10.3: SEC Analysis of HSA Finally, the hydrodynamic diameter (determined via pho The amount of monomer HSA decreased from 95.9% to ton correlation spectroscopy, PCS) of Adalimumab was 92.75% while concentrating from 9.88 mg/ml to 112.74 30 found to be notably lower in WFI than in commercial buffer mg/mL. For the sample with 177.69 mg/mL, an increase in (indicative of lower viscosity proneness). monomer up to 94.5% was determined. The decrease of the Overall, it was concluded that the findings of antibodies amount of monomer goes along with an increase in protein and the globular model protein HSA being soluble in pure aggregate from 4.1% to 7.25% while concentrating from 9.88 water in ultra-high concentrations have potential to provide mg/mL to 112.74 mg/mL. Thus, HSA protein also appears to 35 new insight into fundamental protein regimes and to poten be stable when formulated in pure water. tially offer new approaches in protein drug formulation and Numerical data describing the above-mentioned SEC manufacturing, for instance by: experiments is provided in Appendix B. reducing opalescence of high-concentrated protein formu lations Example 11 40 reducing viscosity of high-concentrated protein formula tions Analysis of High Protein enabling to adjust osmolality as desired in protein-WFI Formulations—Ion-Exchange-Chromatography Solutions by adding non-ionic excipients such as man (IEC) nitol without changing features such as Viscosity and 45 non-opalescence (it was demonstrated for J695 that For the IECanalysis the samples of Adalimumab, J695 and hydrodynamic diameter and opalescence did not change HSA were diluted to 1 mg/mL before injection. The injection when mannitol was added, but increased dramatically volume for all proteins was 100 uL. when NaCl was added) 11.1: IEC Analysis of Adalimumab providing a new paradigm in Drug Substance formulation As shown in FIG.9, Adalimumab was stable in WFI. FIG. 50 and processing, as it was demonstrated that proteins can 9 shows a slight trending which may be interpreted as indi be subjected to operations such as DF/UF for concen cating that the Sum of lysine variants (lysine 0, 1 and 2) trating the protein in WFI to ultra-high concentrations decreases with an increase concentration of Adalimumab in and to freeze and thaw without substantial stability WFI. Overall, however, the percentage of lysine variants var implications. Given the background that it is well ied less than 0.25%. 55 known that during DF/UF the composition of protein 11.2: IEC Analysis of Jó95 formulations, especially during processing to high con FIG. 10 shows that the sum of the Jó95 peaks 1 to 7 is centrations, necessarily changes (Stoner, M. et al., Pro slightly decreasing with increasing JG95 concentration. With tein-solute interactions affect the outcome of ultrafiltra the decrease in peak 1-7 the Sum of acidic and basic peaks is tion/diafiltration operations, 93 J. Pharm. Sci. 2332 slightly increasing, with the increase in the acidic peaks being 60 (2004)), these new findings could beneficially be applied a little more pronounced (see FIGS. 11 and 12). The sum of by either adjusting the Drug Substance concentration by acidic peaks slightly increases from approximately 10.2% to DF/UF of the protein in pure water, and add excipients at 10.6% and the sum of basic peaks from 0.52% to 0.59%, high DS concentrations Subsequently (by this avoiding respectively. the risk of DS formulation changes during process unit Overall, it can be stated that no major instability effects or 65 operations). Alternatively, the excipients could be added insolubility effects of JG95 formulations in pure water were to Drug Substance during final Drug Product fill-finish observed via IEC. 1ng. US 8,883,146 B2 71 72 Example 12 Table 16a shows the results of analysis of highly concen trated Adalimumab in water (DF/UF processed) bulk drug Preparation of Adalimumab in Water Formulation solutions after Step 3 of the procedure.

The following example illustrates the scaling up the DF/UF TABLE 16a procedures resulting in large scale production of adalimumab in water. 12. 1: Evaluation of Process Parameters Osmolality and Conductivity Data for DF/UF Dialysis process evaluation studies were performed on a Bulk-Processed Adalimumab laboratory scale to define suitable parameters for the dialysis 10 of Bulk Adalimumab Drug Solution formulated in a phos oSmolality conductivity density Adalimumab phate/citrate buffer system containing other excipients, e.g., mosm/kg mScm pH gicm3 conc mg/ml mannitol and sodium chloride (FIGS. 13 and 14). Conductivity measurements may be taken with any com mercially available conductivity meter suitable for conduc 15 62 O.95 S.28 1.0764 277.8 tivity analysis in protein Solutions, e.g. conductivity meter Model SevenMulti, with expansion capacity for broad pH range (Mettler Toledo, Schwerzenbach, Switzerland). The 12.3: Freeze/Thaw (F/T) Procedure Simulating Manufactur instrument is operated according to the manufacturers ing Conditions instructions (e.g., if the conductivity sensor is changed in the Freezing was conducted using an ultra-low temperature Mettler Toledo instrument, calibration must be performed freezer (Revco Ultima II, 20 cu. ft.) with a manufacturing again, as each sensor has a different cell constant; refer to scale load of 47 kg of liquid to be frozen at a temperature Operating Instructions of Model SevenMulti conductivity below -50° C., typically -70° C. to -80°C. The liquid was meter). If the instructions are followed, conductivity mea packaged in individual bottles of 1.6 kg fill weight (e.g., Surements can be taken by directly immersing the measuring 25 probe into the sample solution. Nalgene 2 L PETG square media). Freezing was completed FIG. 13 shows the efficiency of the dialysis procedure, in after 48 hours. Thawing was conducted in a circulating water terms of the reduction of components responsible for the bath (e.g., Lindergh/Blue) with a manufacturing scale load of osmolality and the conductivity of the formulation containing 24 kg at a temperature between 20° C. and 40°C., typically adalimumab at 74 mg/ml. After a reduction of the solutes in 30 30°C., until the material was completely thawed. the antibody solution by a factor of 100, osmolality and 12.4: Bottle Mapping During Freeze and Thaw conductivity measurements largely stabilized at levels far Individual horizontal solution layers in the bottle volume below the original measurements of these parameters from were isolated and analyzed. At protein concentrations of 250 the commercial formulation. mg/ml and 200 mg/ml, only minimal gradient formation was FIG. 14 shows the stability of pH in dialyzed Adalimumab 35 bulk solutions. pH levels before and after dialysis against detected in the Adalimumab water solution, as seen in FIGS. deionized water (1:1,000,000) are shown for Adalimumab 15 through 19. Freezing and thawing of formulated Adali solutions with a range of different initial pH readings. pH mumab solutions (solutions with a Phosphate/Citrate Buffer levels remained nearly the same in the retentate before and system containing other excipients, e.g., Mannitol and after dialysis. 40 Sodium Chloride) at 250 mg/ml and 200 mg/ml, however, led 12.2: Production of High-Concentrated Adalimumab in to the formation of precipitate on the bottom of the bottle. Water Bulk Drug Solution 12.5: Gradient Formation in Commercial and Low-Ionic For In a first step, the formulated bulk drug solution (Phos mulations of Adalimumab phate? Citrate Buffer system containing other excipients, e.g., The formation of gradients by freeze thaw procedures in Mannitol and Sodium Chloride) was up-concentrated by 45 Ultrafiltration/Diafiltration to a concentration of approxi commercial and low-ionic (water) formulations of Adali mately 100 mg/ml (12 L scale, Millipore Pellicon 2 Mini mumab was compared. Table 16b shows the results of visual Bio-AMWCO 10k columns). In a second step, the up-con inspection of commercial Adalimumab solutions of various centrated Solution was dialyzed against deionized water concentrations after a fit step. The formation of precipitates (SpectraPor7 MWCO10k, dilution factor 1:100,000). As a 50 indicates that instability was created in the solution by the fit third step the dialyzed solution was up-concentrated by Ultra procedure. Above 100 mg/ml, significant precipitate forma filtration/Diafiltration to a concentration of approximately tion was observed. Table 17 shows the analytical data of two 250 mg/ml using Millipore Pellicon 2 Mini Bio-A MWCO 50 mg/ml solutions and one 100 mg/ml low-ionic formulation 10k columns. before the freeze-thaw experiment. TABLE 16b

Observed Precipitation in Commercial Adalimumab Solutions after FT 250 mg/ml 220 mg/ml 200 mg/ml 150 mg/ml 120 mg/ml 100 mg/ml 60 mg/ml

Commericial Precipitate Precipitate Precipitate Precipitate Partial Clear Clear freezing process Precipitate in ultra low temp freezer: -70° C.23° C. US 8,883,146 B2 73 74 TABLE 17 Solution Analytical Data before Freeze-Thaw Density Osmolality Protein conc formulation pH (g/cm) (mosmol/kg) (mg/mL) E167 130 O1 CL low-ionic 5.18 1.0121 5 49.3 50 mg/mL in water E167 14001 CL Commercial 5.20 1.0224 28O 48.7 50 mg/mL in buffer formulation 100 mg/mL in water low-ionic 5.32 1.0262 12 99.8

About 1600 mL (50 mg/ml solutions) or 800 ml (100 equipped with a 30 kDa RC membrane. The protein concen mg/ml solution) of each formulation were placed into PETG tration after DF/UF was determined approx. 192 mg/mL. pH bottles and subjected to a conventional freeze (-80°C.) thaw 15 4.7. (23°C., water bath) procedures. Samples were then pulled 13.1: Size Exclusion Analysis (SEC) Experimental Proce dures from top, center and bottom of the PETG bottles and analyzed A size exclusion method was developed for the purity for pH, density, osmolality, and protein concentration. Analy assessment of Jô95. Size exclusion chromatography (SEC) sis results are shown in Table 18. separates macromolecules according to molecular weight. The resin acts as a sieving agent, retaining Smaller molecules TABLE 18 in the pores of the resin and allowing larger molecules to pass through the column Retention time and resolution are func Analysis of Bottle-Mapped Layers from Frozen Thawed Solutions tions of the pore size of the resin selected. Each sample was diluted to 2.5 mg/mL with purified water protein 25 content (Milli-Q) based on the stated concentration. 50 lug of each density osmolality (volumetric) sample was injected onto the column in duplicate. A Tosoh sample pH g/cm3 mOsmol/kg mg/mL Bioscience G3000swxl, 7.8 mmx30 cm, 5um (Cat #08541) SEC column is used for separation. For Buffer A, 211 mM 50 mg/mL in water NaSO/92 mM NaHPO, pH 7.0 was used. Detection was top 5.20 1.01.19 6 48.72 30 performed at 280 nm and 214 nm. The column was kept at middle 5.19 1.O120 8 49.35 room temperature with a flow rate of 0.3 mL/min. bottom 5.17 1.O120 6 49.76 This chromatography utilized an isocratic gradient with a commercial formulation 100% mobile phase A solvent for 50 minutes duration. 13.2: SEC Data top S.16 1.016S 236 37.9 middle S.13 1.0221 306 45.58 35 Table 19 describes data from size exclusion chromatogra bottom S.12 1.0257 368 SS.48 phy experiments. 100 mg/mL in water TABLE 19 top S.29 1.0259 13 98.7 middle 5.3 1.O262 16 99.9 SEC Analysis Data for Jó95 Reference Standard, DS and bottom S.28 1.O262 14 101.2 40 Post-DFUF (in water ABT-874 load HM Monom Frag The commercial formulation of Adalimumab revealed sig nificant gradients upon freeze? thaw with regard to density BF RefSt. SOL O49 97.9 1.28 O.26 (indicating heterogeneities/gradients of protein and excipi BF RefStd dup SOL O41 98.0 1.29 0.27 45 BF Ref std avg O4S 98.0 1.29 0.27 ents), osmolality (indicating excipient gradients), and protein stol O.O6 O.OS O.O1 O.O1 % RS 12.5 O.OS 0.55 2.67 content. In contrast, no gradients were found in the 50 mg/ml DS in Buffer pH 4.4 SOL O42 98.3 1.04 O16 low-ionic Adalimumab formulation upon freeze/thaw. DS in Buffer pH 4.4 dup SOL O4O 98.4 1.01 O.13 At higher protein concentrations, gradient formation may DS in Buffer pH 4.4 avg O41 98.4 1.03 O.15 stol O.O1 O.O6 O.O2 O.O2 Sometimes be expected to become worse. However, no gra 50 % RS 3.45 O.O6 2.07 14.6 dients were found in the 100 mg/ml low-ionic Adalimumab DSUF/DF in Water pH 4.7 50 u O.69 98.1 1.04 O.14 formulation upon freeze/thaw with regard to pH, density, UF/DF in Water pH 4.7 dup 50 u O.69 98.0 1.07 O16 osmolality and protein concentration. DSUF/DF in Water pH O.69 98.1 1.06 O.15 4.7 avg stol O.OO O.04 O.O2 O.O1 Example 13 55 % RS O.OO O.04 2.01 9.43

Stability of J695 after DF/UF 13.3: SEC Analysis Conclusions The data in Table 19 shows that the commercial formula The following example provides data on the stability of tion of Jó95 (DS PFS, pH-44) has comparable levels of J695 after DF/UF processing in accordance with the methods 60 fragments and aggregate as the Jô95 reference standard. of the invention. There was a difference noted in the aggregate amount Protein samples from J695 in normal DS buffer were ana between the commercial formulation J695 control and the lyzed, either after pH adjustment or after diafiltration. pH was J695 that had undergone DF/UF, in water (DS in H2O, adjusted to pH 4.4 with 0.1M phosphoric acid, protein con pH=4.7, 192 mg/ml): an increase from 0.4% to 0.7% in aggre centration 112 mg/mL. For concentrated samples in WFI, 65 gation was seen. This was not a significant increase and may protein samples were diafiltered (DF/UF) against water for be due to time spent at room temperature during the UF/DF. approximately 1.5 days at ambient temperature, using a TFF There is no change to the fragments. US 8,883,146 B2 75 76 13.4: IEC (WCX-10) Experimental Procedure 13.5: IEC Data A cation exchange method was developed for the assess Table 21 provides results from experiments comparing the ment of the heterogeneity of JG95 using the Dionex WCX-10 J695 Reference Standard to J695 in commercial buffer (DS column. Generally, cation exchange chromatography sepa pH-4.4), as well as a comparison of the commercial buffer rates protein isoforms according to the apparent pI and the formulation to J695 after DF/UF (DF/UF H2O, pH=4.7). TABLE 21 IEC Data for Jó95 Reference Standard, Commercial Formulation (DS) and after DFUF (in water Oglu Oglu 1 glu 1 glu 1 glu 2 glu 2 glu (1) (2 + 2a) (3) (4) (5) + (5a) (6) (7) acidic basic ref std 43.77 7.SS 8.OO 21.87 4.28 4.82 3.75 4.OS 1.92 ref std dup 43.49 7.49 7.98 21.70 4.26 4.81 3.75 4.61 1.90 ref std dup 43.44 7.49 8.OO 21.65 4.24 4.81 3.74 4.7S 1.89 ref std avg 43.57 7.51 7.99 21.74 4.26 4.81 3.75 4.47 1.90 SD 0.2O O.04 O.O1 O.12 O.O1 O.O1 OOO O4O O.O1 % RSD O.45 O.S6 0.18 0.55 O.33 O.15 O.OO 8.86 0.74 ABT-874 DS pH = 4.4 35.65 14.74 7.26 18.06 6.76 5.32 3.98 5.55 2.70 ABT-874 DS pH = 4.4. Dup 35.82 14.73 7.29 18.14 6.82 5.39 4.06 4.79 2.95 ABT-874 DS pH = 4.4 avg 35.74 14.74 7.28. 18.10 6.79 5.36 4.02 S.17 2.83 SD 0.12 O.O1 O.O2 O.O6 O.04 O.OS OO6 O-54 O.18 % RSD 0.34 O.OS 0.29 O.31 O.62 O.92 1.41 10.39 6.26 ABT-874 DF/UF H2O pH = 4.7 36.57 14:51 7.26 18:09 6.57 S.22 3.91 S.28 2.61 ABT-874 DF/UF H2O pH = 4.7 Dup 36.6O 14.43 7.25 18:02 6.66 5.18 4.OO 5.31 2.56 ABT-874 DF/UF H2O pH = 4.7 36.59 14.47 7.26 18.06 6.62 S.2O 3.96 S.30 2.59 SD O.O2 O.O6 O.O1 O.OS O.O6 O.O3 OO6 O.O2 O.04 % RSD 0.06 O.39 O.10 0.27 O.96 O.54 1.61 O4O 1.37 surface charge interaction with the resin. The protein of inter 13.6: IEC Analysis Conclusions est is bound to the column under specific low salt starting 30 There were some differences noted between the J695 Ref erence Standard and the commercial formulation (DS, pH conditions and is eluted from the column by increasing the 4.4). These differences were noted in the initial run of the DS salt concentration through a gradient. Proteins with lower engineering run sample and are attributed to differences in the apparent pI bind less tightly to a cation exchange column and manufacturing processes between the 3000 L and 6000 L are the first to elute and proteins with a higher apparent pI 35 campaigns. There were no notable differences between the bind tighter and are the last to elute. DS, pH 4.4 control and the Jó95 in HO pH=4.7, 192 mg/ml Cation exchange chromatography using WCX-10 was sample. used in quality control as a lot release assay. The assay con Example 14 ditions were modified to improve separation of known JG95 isoforms. 40 Stability of Adalimumab after DF/UF and The sample was diluted to 1.0 mg/mL with purified water Long-Term Storage at 2-8°C. (Milli-Q). Reference standard was run in triplicate as a com parison and was diluted to 1 mg/ml in purified water (Milli The following example provides data showing the stability Q). of Adalimumab in an aqueous formulation in accordance with 45 the methods of the invention, after 22.5 months storage at Dionex Propac WCX-10 columns (p/n 054993), along with 2-89 C. corresponding guard columns (p/n 054994), were used for Adalimumab samples for SEC and WCX-10 analysis were separation. Buffers used in the procedure included Buffer A diafiltered against water and concentrated to about 177 (10 mMNaHPO, pH=6.0) and Buffer B (10 mMNaHPO, mg/mL. Samples were stored and analyzed at various time 500 mM NaCl, pH=6.0). Column temperature was main points for stability. tained at 35°C. and column flow rate was 1 mL/min Injection Standard Adalimumab solution (DS, pH approx. 5.2) in volumes were 100 ul for a 100 ug load and detection was commercial Humira buffer was used as a starting material for performed at 280 nm. Buffer gradients over the course of the generating a concentrated Solution in water. Protein solution chromatographic separation are provided in Table 20. samples were diafiltered (DF/UF) against water for approxi mately 1.5 days at ambient temperature, using a TFF 55 equipped with a 30kDa RC membrane. Protein concentration TABLE 20 after DF/UF was determined approximately 177 mg/mL, pH Buffer Gradients used in IEC Analysis of JG95 5.2. The sample was stored at 2-8°C. for 22.5 months before analysis. Time (min) % MPA % MPB 14.1: SEC Experimental Procedure O 75 25 60 A size exclusion method was previously developed to 3 60 40 check for the presence of antibody fragments and aggregates. 33 40 60 Size exclusion chromatography (SEC) separates macromol 36 O 1OO ecules according to molecular weight. The resin acts as a 41 O 1OO 43 75 25 sieving agent, retaining Smaller molecules in the pores of the 48 75 25 65 resin and allowing larger molecules to pass through the col umn. Retention time and resolution are functions of the pore size of the resin selected. US 8,883,146 B2 77 78 Each sample was diluted to 1.0 mg/mL with milli Q water reduced self-association tendencies (Liu, J. et al., 94 Journal and 50 mg of each sample was injected onto the column. For of Pharmaceutical Sciences 1928 (2004)). SE-HPLC, a Sephadex 200 column (Pharmacia catil 175175 Thus, it is likely that differences in sample preparations 01, S/N 0504057) or a TSK gel G3000SW (cath.O8541; for and different lag-times between Adalimumab solution dilu analysis of 22.5 month samples) were used. The mobile phase 5 of the column comprised 20 mMSodium phosphate and 150 tion (from 177 mg/mL to 1 mg/mL) and Subsequent sample mM Sodium chloride, pH 7.5. Detection was performed at analysis by SEC are the reason for the differences in aggre 280 nm and 214 nm. Columns were kept at ambient tempera gate content of the 8.5 month and the 9 month stability ture and the flow rate was 0.5 mL/min (Sephadex column), or samples. 0.3 mL/min (TSK column). 10 14.4; IEC Experimental Procedure 14.2: SEC Data A cation exchange method was developed for the assess FIG. 20 and Table 22 contain results of the analysis of a ment of antibody charge heterogeneity using the Dionex low-ionic Adalimumab solution stored as a liquid at 2-8°C. WCX-10 column Cation exchange chromatography sepa for 8.5 months compared to the same solution stored at -80° rates protein isoforms according to the apparent pI and the C. Table 23 contains analysis data for a low-ionic Adali 15 mumab solution stored at 2-8°C. for 22.5 months compared surface charge interaction with the resin. The protein of inter to a reference standard sample of Adalimumab. est is bound to the column under specific low salt starting conditions and is eluted from the column by increasing the TABLE 22 salt concentration through a gradient. Proteins with lower apparent pI bind less tightly to a cation exchange column and SEC Analysis Data Comparing Adalimumab from are the first to elute and proteins with a higher apparent pI Frozen Storage versusAdalimumab from Long-Tenn Refrigerated Storage bind tighter and are the last to elute. Before the procedure, samples were diluted to 1.0 mg/mL % % % with milli Q water. Dionex Propac WCX-10 columns (p/n Sample Load HMW Monomer LMW 25 054993), along with a corresponding guard columns (p/n DF/UF against water, 177 50 g O.1 99.6 O.3 05499), were used for separation. Two mobile phase buffers mg/mL, 4.5 months at -80° C. DF/UF against water, 177 50 g O.2 99.5 O.3 were prepared, 10 mMSodium phosphate, pH 7.5 (Buffer A) mg/mL, 9 months at 2-8°C. and 10 mMSodium phosphate, 500 mMSodium chloride, pH 5.5 (Buffer B). Columns were kept at ambient temperature 30 and the flow rate was 1.0 mL/min Injection volumes were 100 ul for a 100 ug load and detection was performed at 280 nm. TABLE 23 Buffer gradients over the course of the chromatographic sepa SEC Analysis Data Comparing Adalimumab Reference ration are provided in Table 24. Standard against Adalimumab from 35 Long-Tern Refrigerated Storage TABLE 24 % % % Buffer Gradients used in IEC Analysis of Adalinumab Sample Load HMW Monomer LMW Time (min) % MPA % MPB Reference Stod. Adalimumab 50 g O.31 98.85 O.84 40 DF/UF against water, 177 50 g 1.42 97.59 O.98 O.OS 94 6 mg/mL, 22.5 months 2O 84 16 at 2-8° C. 22 O 100 26 O 100 28 94 6 34 94 6 As can be seen in Table 22, SEC analysis revealed that 45 adalimumab in water was stable even after 9 months at 2-8°C. 35 94 6 or for 4.5 months at -80° C., as the percent aggregate (% HMW) and percent fragment (% LMW) were minimal over 14.5: Ion Exchange Data time. Table 25 shows the ion exchange chromatographic data for 14.3: SEC Analysis Conclusions 50 After 8.5 months storage at 2-8°C., the Adalimumabsolu the Adalimumab reference standard, commercial formulation tion (DF/UF against water) revealed a small fraction of high (150 mg/ml) and post-DF/UF low-ionic solution before stor molecular weight (HMW) species (0.2%) and a small fraction age. Table 26 shows data for the reference standard compared to the low-ionic solution after 22.5 months of storage at 2-8 of fragment (0.3%). Storage for 4.5 months at -80° C. and C. subsequent thaw (water bath, 23°C.) did not impact Adali 55 mumab stability (0.1% aggregate, 0.3% fragment). Analysis of a sample stored for 22.5 months at 2-8°C. also TABLE 25 shows comparable fragment content to Adalimumab refer IEC Analysis Data of Adalimumab Reference Standard, ence standard (Table 23). However, the aggregate levels DS. Commercial Formulation and After DFUF (in water detected in the 22.5 month stability sample (1.66%) are some 60 what higher than aggregate levels detected in the reference % Acidic 96 Acidic 96 0 %1 % 2 standard. Sample Name Region 1 Region 2 Lys Lys Lys It is known that self-association of antibodies is highly Adalimumab Ref. Std. 2.69 11.66 60.77 1942 S.40 dependent on the antibody concentration, i.e. the formation of Adalimumab DS 150 mg/ml 2.51 11.38 62.05 1914 4.83 Adalimumab diafiltered 2.26 11.81 61.97 18.51 4.73 non-covalent aggregate and associate complexes is most pro 65 against water, 177 mg/ml nounced at high protein concentration. This self-association is reversible, and dilution with buffer solution results in US 8,883,146 B2 79 80 TABLE 26 IEC Analysis Data Comparing Reference Standard to DF/UF Sample from Long-Tern Refrigerated Storage

% Acidic 90 Acidic % O %. 1 %2 Sample Name Region 1 Region 2 Lys Lys Lys Adalimumab Ref. Std. 2.1 10.9 63.8 18.4 4.6 Adalimumab DF/UF against water, 2.7 13.4 62 16.7 4.1 177 mg/mL, 22.5 months at 2-8°C.

14.6: Ion Exchange Analysis Conclusions mg/mL), sucrose (10 mg/mL), NaCl (100 mM), and polysor For the T0 samples, data show no significant difference in bate 80 (0.01%). 1D4.7 was also formulated in water (by the percentage of acidic region 1, 2,0Lys, 1 LyS, or 2 Lys (i.e., dialysis) with no excipients added at all. Water for injection charge heterogeneity) between reference standard Adali 15 was also subjected to fit cycling and subvisible particle test mumab, commercial formulation Adalimumab (used as DS to ing to evaluate a potential impact of material handling, fit, and formulate Adalimumab into water by DF/UF), and Adali sample pull on particle load. mumab diafiltered against water and concentrated to 177 The stability of 1D4.7 formulated in water upon f7t mg/ml (Table 25). exceeded the stability of 1D4.7 solutions formulated with Also, after 22.5 months storage of the 177 mg/mL Adali excipients typically used in protein formulations. Mannitol, mumab sample in water, only slight differences in O Lys, 1 Sucrose, and Sorbitol are known to act as lyoprotectant and/or Lys and 2 Lys fractions can be seen when compared to the cryoprotectant, and polysorbate 80 is a non-ionic excipient Adalimumab reference standard. In Summary, no significant prevalently known to increase physical stability of proteins chemical instability tendencies are observed when Adali 25 upon exposure to hydrophobic-hydrophilic interfaces such as mumab is formulated into water by DF/UF processing and air-water and ice-water, respectively. Thus, 1D4.7 solutions stored for 22.5 months at 2-8° C. at a concentration of 177 formulated in water appeared to be stable when analyzed with mg/mL. other methodologies applied (e.g. SEC, visual inspection, etc.). Example 15 30 Example 16 Freeze/Thaw Stability of Low-Ionic 1D4.7 Solution Freeze/Thaw Stability of Low-Ionic 13C5.5 1D4.7 protein (an immunoglobulin G1) anti-IL 12/anti-IL Antibody Solution 23 was formulated in water by dialysis (using slide-a-lyzer 35 cassettes, used according to operating instructions of the 13C5.5 anti IL-13 protein formulated in water was dem manufacturer, Pierce, Rockford, Ill.) was demonstrated to be stable during repeated freeze/thaw (ft) processing (-80° onstrated to be stable during repeated freeze? thaw processing C./25°C. water bath) at 2 mg/mL concentration, pH 6. Data (-80° C./25°C. water bath) at 2 mg/mL concentration, pH 6. were compared with routine formulations (2 mg/mL, pH 6), 40 Data were compared with routine formulations (2 mg/mL, pH and it was found that the stability of 1D4.7 formulated in 6), and it was found that the stability of 13C5.5 formulated in water exceeded the stability of 1D4.7 formulated in routinely water exceeded the stability of 13C5.5 formulated in rou screened buffer systems (e.g. 20 mM histidine, 20 mM gly tinely screened buffer systems (e.g. 20 mM histidine, 20 mM cine, 10 mM phosphate, 10 mM citrate) and even exceeded glycine, 10 mM phosphate, 10 mM citrate) and even the stability of 1D4.7 formulations based on universal buffer 45 exceeded the stability of 13C5.5 formulations based on uni (10 mM phosphate, 10 mM citrate) with a variety of excipi versal buffer (10 mM phosphate, 10 mM citrate) with a vari ents that are commonly used in protein formulation, e.g. 10 ety of excipients that are commonly used in protein formula mg/mL mannitol. 10 mg/mL Sorbitol, 10 mg/mL Sucrose, tion (e.g. 10 mg/mL mannitol. 10 mg/mL Sorbitol, 10 mg/mL 0.01% polysorbate 80, 20 mM. NaCl. sucrose, 0.01% polysorbate 80,20mMNaCl, 200mMNaCl). SEC, DLS and particle counting was performed to monitor 50 Sample preparation, experiment processing, sample pull protein stability, and particle counting was performed by and sample analysis was performed in the same way as out using a particle counting system with a 1-200 um measure lined in Example 15 for 1D4.7. ment range (e.g. particle counter Model Syringe, Markus 13C5.5 formulated in water compared with formulations Klotz GmbH, Bad Liebenzell, Germany). Experiment details listed above are as follows: 55 4 freeze/thaw cycles applied 1D4.7 formulated in water compared with formulations 30 mL PETG repository listed above 2 mg/mL, pH 6 4 freeze/thaw cycles applied sampling at T0, T1, T2, T3, and T4 30 mL PETG repository, about 25 mL fill, 2 mg/mL, pH 6 analytics: visual inspection, SEC, DLS, subvisible particle sampling at T0, T1 (i.e. after one fit step), T2, T3, and T4 60 measurement analytics: visual inspection, SEC, DLS, subvisible particle FIG.22 shows 13C5.5 stability during repeated fit cycling measurement (-80° C./25° C.), mirrored by formation of subvisible par FIG. 21 shows 1D4.7 stability during repeated fit cycling ticles >10 um. 13C5.5 was formulated in either 10 mM phos (-80° C./25° C.), mirrored by formation of subvisible par phate buffer, 10 mM citrate buffer, 20 mM glycine buffer, and ticles >1 um. 1D4.7 was formulated in universal buffer (10 65 20 mM histidine buffer. 13C5.5 was also formulated in water mM citrate, 10 mM phosphate) and then the following excipi (by dialysis) with no excipients added at all. Water for injec ent variations were tested: sorbitol (10 mg/mL), mannitol (10 tion was also subjected to fit cycling and subvisible particle US 8,883,146 B2 81 82 testing to evaluate a potential impact of material handling, ft, UV/VIS spectrophotometer for protein concentration mea and sample pull on particle load (blank). Surement The stability of 13C5.5 formulated in water upon f7t Photon Correlation Spectroscopy (PCS) exceeded the stability of 13C5.5 solutions formulated in buff Viscosity measurement ers typically used in protein formulations. No instabilities of 5 Turbidity measurement 13C5.5 solutions formulated in water have been observed Size Exclusion Chromatography (SEC) with other analytical methodologies applied (e.g. SEC, visual Fourier transform mid infrared spectroscopy (FT-M-IR) inspection, etc.) 17.1 Overview of Preparation for DF/UF of Adalimumab FIG. 23 shows 13C5.5 stability during repeated fit cycling Commercial Formulation (-80° C./25° C.), mirrored by formation of subvisible par 10 The Adalimumab DS solution (120 mg/mL) was divided ticles >1 um. 13C5.5 was formulated in universal buffer (10 mM citrate, 10 mM phosphate) and then the following excipi into 7 volume portions which were adjusted to pH3, pH4. ent variations were tested: sorbitol (10 mg/mL), mannitol (10 pH5, pH6, pH7, pH8, pH9 with 0.25NNaOH and 0.25NHC1, mg/mL), sucrose (10 mg/mL), NaCl (200 mM), NaCl (20 respectively. Then the samples were diluted with Adali mM) and polysorbate 80 (0.01%). 13C5.5 was also formu 15 mumab buffer of the respective pH to 100 mg/mL. The solu lated in water (by dialysis) with no excipients added at all for tions revealed a slight cloudyness that disappeared after ster comparison (pure water). Water for injection was also Sub ile filtration (0.22um, PVDF sterile filter). After dilution, the jected to fit cycling and Subvisible particle testing to evaluate pH value were monitored again (see Table 27 below). a potential impact of material handling, fit, and sample pull on The following samples of the 100 mg/mL solutions were particle load. pulled from each solution: The stability of 13C5.5 formulated in water upon f7t 4 mL for turbidity and Subsequent Zetapotential measure exceeded the stability of 13C5.5 solutions formulated with ment excipients typically used in protein formulations. Mannitol, 1 mL for viscosity measurement (using dropping-ball vis Sucrose, and Sorbitol are known to act as lyoprotectant and/or cometer) cryoprotectant, and polysorbate 80 is a non-ionic excipient 25 0.15 mL for osmolality measurement prevalently known to increase physical stability of proteins 2 mL for density measurement upon exposure to hydrophobic-hydrophilic interfaces such as 0.15 mL for PCS (sample viscosity taken into account for air-water and ice-water, respectively. measurements) No instabilities of 13C5.5 solutions formulated in water 1 mL for FT-M-IR have been observed with other analytical methodologies 30 2 mL for viscosity and static light scattering measurements applied, (e.g. SEC, visual inspection, etc.). The samples for Zetapotential, viscosity and static light DLS analysis of 13C5.5 solutions after fit procedures was performed as described above. An 13C5.5 solution with scattering measurements were frozen (-80°C.). The remain 0.01% Tween-80 contained significant high molecular weight ing volumes of pH 4, pH 5, pH 6, pH 7, and pH 8 solutions (HMW) aggregate forms after only 1 fit step, whereas 13C5.5 35 were Subjected to continuous mode diafiltration using water in water contained no HMW aggregate forms, even after 3 fit for injection as exchange medium. The samples were first steps. frozen at -80° C. Before DF/UF, the samples were thawed at 25°C. in a Julabo water bath. Example 17 17.2 DF/UF and Concentration Procedures 40 Adalimumab solutions in commercial formulation, with Impact of Solution pH on Adalimumab in WFI concentrations of 100 mg/ml, with pH levels of 4, 5, 6, 7 and 8, were subject to DF/UF processing and further subject to The following experiments were performed to determine concentration process with UF in a centrifuge. This section the impact of Solution pH on physico-chemical characteris describes the processing of the pH 6 Adalimumab solution as tics of highly concentrated Adalimumab formulated in WFI. 45 an example. Processing for the other Solutions was done in a The following concentrations were tested: 2 mg/mL, 50 similar manner. mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, and 250 The Adalimumab solution (100 mg/mL, pH 6) was thawed mg/mL. in a water bath at 25° C. and then homogenized. Then, the Materials Solution was subjected to diafiltration using water for injec Adalimumab Drug Substance (DS), commercial material 50 tion as exchange medium with TFF equipment M.P. 33.4 by 25°C. water bath (circulating) used for thawing applying the following parameters: Diafiltration equipment: Sartorius Sartocon Slice, mem stirrer: speed 2 brane: PES 50 kD, 1000 cm pump: Speed 1 Diafiltration equipment: Millipore LabscaleTM TFF Sys pressure up-streamfinlet: 2-2.4 bar tem, membrane: PLCTK 30 kD. regenerated Cellulose, 55 size: 50 cm pressure down-stream/outlet: 0.6-0.8 bar Eppendorf Centrifuge 5810 R membrane: regenerated Cellulose, cut off 30 kD Amicon Ultra-15 repositories for centrifugation, Ultracel continuous mode DF/UF 30k, Regenerated Cellulose 30,000 MWCO about 6-fold volume exchange applied during DF/UF Millex GV 0.22 um, Millipore for sterile filtration of 60 operation samples After applying 6-volume exchange steps, the concentration Sample repositories (Eppendorf sample repository 1.5 mL, of Adalimumab was determined by means of OD280, pho Roth cryovials 5 mL, PETG bottle 125 mL) tometer M.P. 9.7. The osmolality of permeate and retentate Analytics: was checked. pH measurement using Biothrode 65 concentration: 125.1 mg/mL Density measurement osmolality permeate: 57 mCSmol/kg Osmolality measurement osmolality retentate: 12 mosmol/kg US 8,883,146 B2 83 84 The Adalimumab solution in water after DF was diluted 17.3 Visual Inspection of Adalimumab Solutions with water for injection to 100 mg/mL and sterile filtered. The After DF/UF and concentration to 250 mg/mL, the Adali following samples were pulled from 100 mg/mL solution mumab solutions in water at various pH appeared less opal after the DF/UF process: escent than the Adalimumab solution in buffer (commercial 4 mL for turbidity and Subsequent Zetapotential measure formulation). All of the Adalimumab solutions in water ment appeared as clear solutions at each pH value. None of the 1 mL for Viscosity measurement Adalimumab Solutions revealed opalescence after dilution. 0.15 mL for osmolality measurement Overall, during concentration and dilution procedures, no 2 mL for density measurement precipitation was observed in Adalimumab solutions in water. 0.15 mL for PCS (viscosity taken into account during mea 10 17.4 Viscosity Surement) The Viscosity measurements were performed taking into 0.15 mL for SEC account the density of pH 5 Adalimumab solutions at each of pH-measurement the respective concentrations. A dropping-ball viscometer 1 mL for FT-M-IR was used. Viscosities higher than 200 mPas were measured 15 using capillary viscometer. 2 mL for viscosity and static light scattering measurements FIG. 24 provides an overview of viscosity data of Adali A portion of the 100 mg/mL Adalimumab solution was mumab Solutions in water with pH ranging from 4 to 8, at diluted with water for injection to create 50 mg/mL and 2 various concentrations (2 mg/mL to 250 mg/mL, in 50 mg/mL Solutions. The following samples were pulled from mg/mL concentration steps). There is a clear correlation both solutions: between solution pH, concentration and Viscosity. The vis 4 mL for turbidity and Subsequent Zetapotential measure cosity increases with increases of protein concentration, inde ment pendent of the solution pH. At solution pH values close to the 2 mL for viscosity measurement pI of Adalimumab (i.e. pH 7 and pH 8), increases in solution 0.15 mL for osmolality measurement Viscosity were most pronounced, especially at higher protein 2 mL for density measurement 25 concentrations (i.e. 200 mg/mL, 250 mg/mL). 0.15 mL for PCS (viscosity taken into account) 17.5 Turbidity pH-measurement As seen in FIG. 25, the same trend was found for turbidity Adalimumab solutions (pH 6, 100 mg/mL) in water were data, (i.e., the turbidity increased with increasing concentra Subjected to concentration experiments using centrifugation. tion and with increasing pH). All samples were sterile filtered Centrifugation was performed with Eppendorf Centrifuge 30 (0.22 um) before turbidity measurement. (5810R M.P. 33.57). Each centrifugation step was applied for 17.6 Hydrodynamic Diameter (PCS) 15 min. at 4000 rpm. After that, homogenization of sample The PCS measurements were performed taking into Solution in the centrifuge concentration device was per account the Viscosity for each sample, at each concentration formed by gentle upside-down rotation in order to homog and at each pH value. Solutions at 200 mg/mL and 250 enized the solution and thereby to avoid gel formation in areas 35 mg/mL were measured but were outside the testing param immediately adjacent to the membrane. Temperature during eters of the Zetasizer nano series (Malvern Instruments) concentration was 15° C. The centrifugation was performed equipment, and consequently the data from these measure to about 250 mg/mL. The concentration was determined by ments was not analyzed. means of measuring OD280, photometer M.P. 9.7. The Adali The hydrodynamic diameter (Dh) was found to be notably mumab solutions were then diluted to concentrations of 250 40 decreased when Adalimumab was formulated in water (Dh mg/mL, 200 mg/mL and 150 mg/mL. about 2 nm at 50 mg/mL, pH 5) in comparison to Adali The following samples were pulled after the concentration mumab formulated into commercial formulation (Dhabout 7 procedure and after each individual step of dilution. Sample nm). FIG. 26 illustrates the PCS data (also found in Table 39). volumes pulled from 250 mg/mL and 150 mg/mL solutions Corresponding data tables are shown below in part 17.11. Were 45 As shown in FIG. 26, for solutions at pH values of 4, 5 and 2 mL for viscosity-measurement 6, the Dh of Adalimumab monomer decreased constantly 0.15 mL for PCS (viscosity taken into account) with increased protein concentration. In contrast, Solutions 0.15 mL for osmolality measurement with pH values closer to the p of Adalimumab (i.e., at pH 7 0.15 mL for SEC and pH8) showed considerable increases in Dhas concentra pH - measurement 50 tion increased from 2 mg/mL to 50 mg/mL. As concentrations Sample volumes pulled from the 200 mg/mL solution were: rose beyond 50 mg/mL in pH 7 and 8 solutions, however, Dh 4 mL for turbidity and Subsequent Zetapotential measure decreased. At a concentration of 150 mg/mL, all of the solu ment tions had lower Dhvalues than the corresponding pH solution 1 mL for Viscosity measurement at 2 mg/mL. FIG. 27 shows Dh size distributions for pH 5 0.15 mL for osmolality measurement 55 solutions of various concentrations. FIG. 28 shows Dh size 2 mL for density measurement distributions for five Adalimumab solutions formulated in 0.15 mL for PCS (viscosity taken into account) water, each having a 100 mg/mL protein concentration and a 0.15 mL for SEC different pH value. FIG. 29 shows data similar to data in FIG. pH-measurement 28, except that the five Adalimumab solutions were formu 2 mL for analytical work to be performed at ABC (viscosity 60 lated in buffer. and static light scattering measurements) 17.7 pH Measurement The concentration processing of Adalimumab Solution in Measurements of solution pH were performed at 100 water was halted at approximately 250 mg/mL at each pH mg/mL before and after DF/UF using water (i.e., performed value because the viscosity of Adalimumab solution in water on Adalimumab formulated in buffer and in water, respec at higher concentrations, and especially at pH values close to 65 tively). Table 27 shows the results. The pH values stay con the pl (about pH 8.5 for Adalimumab), increased dramatically stant at pH 5, pH 6 and pH 7 before and after DF/UF. The (viscosities approaching gel formation). Solution pH does not change because of a medium change. US 8,883,146 B2 85 86 The pH value at pH 4 slightly increases and at pH 8 slightly 17.10 Conclusions decreases after DF/UF using water. This experiment was designed to examine the impact of Solution pH and protein concentration on viscosity and Dh TABLE 27 (hydrodynamic diameter) of Adalimumab Solutions formu 5 lated in water by DF/UF processing. Such solutions are pH values before and after DFIUF with water referred to as low-ionic solutions. A pH range of 4-8 was evaluated, and protein concentrations tested were in a range pH 4 pH 5 pH 6 pH 7 pH 8 between 2 and 250 mg/mL. Adalimumab 100 mg/mL in 4.OO 4.99 6.OO 7.03 8.00 With regard to viscosity (Section 17.4), it was found that buffer low-ionic Adalimumab solutions have the same characteris 10 Adalimumab 100 mg/mL in 4.29 4.98 S.98 7.02 7.67 tics as Adalimumab solutions formulated in the presence of Water ions (i.e. ionic excipients such as organic buffer components or salts): The higher the protein concentration, the higher Solution 17.8 Osmolality Measurements viscosity. This concentration-viscosity correlation was During DF/UF of the pH 5 solution samples, solution 15 more pronounced for solutions with pH values close to osmolality was measured after each Volume exchange step the Adalimumab pi (i.e., pH 7 and pH8). Conversely, for (i.e., after 100 mL permeate, 200 mL permeate, etc.) to check Solutions at a constant concentration, Viscosity corre whether a 5-fold volume exchange is sufficient to reduce lated with the closeness of the solution's pH value to the osmolality to values below 15 mCSmol/kg. Table 28 shows pI of Adalimumab. the results. With regard to DLS data (Section 17.6), the following conclusions can be drawn: TABLE 28 Adalimumab Dh values determined by DLS of low ionic Adalimumab solutions were found to be lower than Dh OSmolality change during DFIUF using water, pH S Solution values measured in Adalimumab commercial formula Volume exchange Retentate Permeate 25 tions, especially at very low solution pH. step in mL. mOSmol/kg mOSmol/kg The lower the solution pH, the lower Dhvalues determined 100 96 166 by DLS. 200 28 115 The higher the protein concentration, the lower the Dh 300 29 89 values in low-ionic Adalimumab solutions of a given 400 12 67 30 pH. 500 15 49 The explanation for this behavior is that the ionic strength (i.e. the presence of ions and ionizable excipients) in protein At pH 4, pH 6, pH 7 and pH 8, the osmolality was measured Solutions is crucial for the extent of protein-protein interac at the end of the DF/UF process only. Table 29 shows the tions. Especially at lower solution pH, charge-charge repul osmolality results (in mCSmol/kg units) for each pH. 35 sions are more pronounced in low ionic Adalimumab solu tions. When a protein is formulated in water by using water as TABLE 29 exchange medium in DF/UF processing, the amount of ion izable counter ions present that can compose both the Helm OSmolality at various pH values, before and after DFIUF with water holtz layer and the Gouy-Chapman layer is notably reduced. 40 Consequently, intermolecular charge-charge interactions pH 4 pH 5 pH 6 pH 7 pH 8 (due to the charges of amino acid residues present at the Adalimumab 100 mg/mL in 287 298 297 286 279 protein's Surface) may be more pronounced than in an envi buffer Adalimumab 100 mg/mL in 40 13 11 5 5 ronment where ionizable counter ions (e.g. ionizable excipi Water ents) are abundant, and charge-charge repulsion between pro 45 tein monomers (leading to molecule motion in case of charge charge repulsion) and random Brownian motion contribute to The osmolality measurements were performed with a the mobility/motion of the protein molecule measured by freezing point Viscometer. DLS. In DLS experiments, greater molecule mobilities are 17.9 Fragmentation (SEC) translated into greater molecular diffusion coefficients, which The SEC data show a relative pronounced fragmentation of 50 usually are assigned to molecules with Smaller hydrodynamic the protein in ph 4 solutions over the whole concentration sizes via using the Stokes-Einstein equation. This can explain range (100-250 mg/mL), while there almost no fragmentation why the hydrodynamic diameter of proteins is reduced in detected at pH ranging from 5 to 8 over the same concentra low-ionic formulations. tion range. Consequently, the monomer content of pH 4 solu Charge-charge interactions between antibody molecules tions decreased accordingly (FIG.30). Aggregate values were 55 can be repulsive (at lower solution pH) and attractive (at found to increase with increasing pH values (from pH4 to pH higher solution pH close to the protein's pl). 8), independent of the concentration (FIG. 31). 17.11 Data Tables TABLE 30

Adalimumab 100 mg/mL in buffer before DF versus water pH3 pH4 pH5 pH6 pH7 pH8 pH9

turbity (NTU) 9.9 15.4 28.5 36.3 4S.O 48.4 46.5 viscosity (mPas) 2.5197 2.7935 3.2062 3.1512 3.S116 3.5494 3.5844 viscosity (mm2/s) 2.4366 2.6991 3.0969 3.0444 3.389.3 3.4261 3.4589 US 8,883,146 B2 87 88 TABLE 30-continued Adalimumab 100 mg/mL in buffer before DF versus water pH3 pH4 pH5 pH6 pH7 pH8 pH9 density (g/cm3) 1.0341 1.O3SO 1.O3S3 1.0351 1.0361 1.0360 1.0363 oSmolality (mCSmol/kg) 293 287 298 297 286 279 285 Z-Aved (nm) PCS 4.3 4.3 6.0 7.3 7.7 8.0 7.8 pH 4.OO 4.99 6.OO 7.03 8.00 9.03

TABLE 31 TABLE 32-continued Adalimumab after DF versector concentration, diluted Adalimumab after DF versus water, before concentration, diluted 15 with water to pH 4 pH 4 pH 4 2 mg/mL. 50 mg/mL 100.5 mg/mL pH 6 pH 6 pH 6 2 mg/mL. 50 mg/mL 100 mg/mL turbity (NTU) O.296 1.46 3.30 viscosity (mPas) O.9653 14471 2.2411 density (g/cm3) O.9989 1.012 1.O2S1 yies.2. O.966S 14298 2.1834 20 osmolality (mosmol/kg) 3 + 11 = 14:2 = 7 27 11 oSmolality (mCSmol/kg) 40 Z-Aved (nm) PCS 30.8 2.78 2.48 Z-Aved (nm) PCS 3.37 2.24 1.81 pH 5.72 5.95 S.98 pH 4.29

Adalimumab after concentration and dilution with water to 25 pH 4 pH 4 pH 4 TABLE 33 150.5 mg/mL 219.0 mg/mL 251.8 mg/mL Adalimumab after concentration and diluted with water to turbity (NTU) 3.56 viscosity (mPas) 4.0283 13304 48.642 p H 6 pH 6 pH 6 viscosity (mm2/s) 3.8712 12.614 45.567 30 146.6 mg/mL 201.8 mg/mL 248.5 mg/mL density (g/cm3) turbity (NTU) 9.29 sty (mCSmol/kg) 64 96 141 viscosity (mPa's) 9.0193 32.352 126.06 Z-Aved (nm) PCS 1.32 O.458 O.162 viscosityty (mm2's 8.6775 30.709 118.07 pH 4.32 4.54 density (g/cm3) 1.0394 1.0535 10677 lali O k 37 58 95 Adalimumab after DF verwater, before concentration, diluted 35 st t g) O.989 0.355 O.108 with Water to pH S.92 6.OS 6.03 pH 5 pH 5 pH 5 2 mg/mL. 50 mg/mL 97.5 mg/mL turbity (NTU) O.O2 1.66 3.54 40 TABLE 34 viscosity (mPas) 1.0563 16664 2.8661 viscosity (mm2/s) 1.0576 16465 2.7924 Adalimumab after DF versus water, before concentration, diluted density (g/cm3) O.9988 1.01.21 1.0264 with water to oSmolality (mCSmol/kg) 13 Z-Aved (nm) PCS 157 32.4 1.3 pH 7 pH 7 pH 7 pH 4.SS 4.83 4.98 2 mg/mL. 50 mg/mL 103.2 mg/mL 45 Adalimumab after concentration and dilution with water to turbity (NTU) O.1 7.13 14.9 pH 5 pH 5 pH 5 viscosity (mPa's) 1.1252 16898 4.2257 150.7 L 200.2 mg/mL 253.0 mg/mL viscosity (mm2/s) 1.1268 16688 4.1.146 mg/m mg/m. mg/m. density (g/cm3) O.9986 1.O126 1.027 osmolality (mCSmol/kg) O 2 5 turbity (NT). 7.24 so Z-Aved (nm) PCS 3.31 4.16 2.89 viscosity (mPa's) 7.0866 19539 79.272 pH 6.63 6.93 7.02 viscosity (mm2/s) 6.81O2 18.525 74.26 density (g/cm3) 1.04O6 1.OS47 1.0675 oSmolality (mCSmol/kg) 78 8O 96 Z-Aved (nm) PCS O.727 O.335 0.255 pH S.O.3 5.05 S.O8 55 TABLE 35 Adalimumab after concentration and diluted with water to pH 7 pH 7 pH 7 TABLE 32 143.0 mg/mL 203.4 mg/mL 251.7 mg/mL Adalimumab after DF versus water, before concentration, diluted 60 turbity (NTU) 19.3 with water to viscosity (mPa *s) 14.024 74.987 343.881 viscosity (mm2/s) 13492 70.928 321-144 pH 6 pH 6 pH 6 density (g/cm3) 1.0571 1.0708 2 mg/mL. 50 mg/mL 100 mg/mL osmolality 65 106 160 (mCSmol/kg) turbity (NTU) O.458 2.24 2.95 Z-Aved (nm) PCS 1.27 O.346 O.O876 viscosity (mPas) 1.O696 18003 3.1147 65 pH 6.9 7.01 7.2 viscosity (mm2/s) 1.0708 1.7789 3.0385

US 8,883,146 B2 91 92 TABLE 40 Experimental Groups

SEC data O1 Control (vehicle) COC. % % % Area O2 50 mg/ml Afelimomab, liquid, standard formulation pH mg/mL aggregate OOle fragmente (mVs) O7 200 mg/ml Afelimomab, liquid, water formulation Group A Observation period 2 days 4 OO O.28 67.95 31.76 45.195.348 Group B Observation period 7 days 4 50 O.26 66.O7 33.68 44492.8O3 Group C Observation period 14 days 4 200 O.30 64.59 35.11 52558.050 4 250 O.29 64.40 35.31 484.91.299 5 OO 1.46 98.44 O.11 481.27.249 10 5 50 1.33 98.56 O.11 43226.397 Grouping and Rat Identification (N=1 Per Group) 5 200 1.39 98.50 O.11 43634.282 5 250 1.38 98.52 O.11 41643.062 6 OO 2.00 97.90 O.10 44338.373 Animal number 6 50 2.52 97.37 O.11 41899.182 6 200 2.52 97.37 O.11 43.869.183 15 Group Group A Group B Group C 6 250 2.39 97.50 O.11 34969.456 7 OO 2.78 97.12 O.10 46194824 O1 1 2 3 7 50 4.24 95.65 O.11 47443.014 O2 4 5 6 7 200 3.61 96.29 O.10 41916.220 O7 19 2O 21 7 250 3.39 96.50 O.11 38.185.208 8 OO 3.24 96.65 O.12 423.34.491 8 50 3.64 96.18 O.18 4O305.890 The animals were repeatedly observed for clinical signs 8 200 3.63 96.25 O.13 4O280.342 and mortality on day 1 at 15 min, 1, 3, 5, and 24 hours past 8 250 3.76 96.OS O.19 32O67.297 administration and at least once daily afterwards. Body weights were measured on the days of dosing (day 1) and necropsy (day 3, 15 or 21, respectively) and twice weekly, if Example 18 25 applicable. Blood samples for drug analysis were collected on Day 1 (4 hours past administration), and on Days 2, 3, 5, 8, Impact of pH on J695 Viscosity and 15 as applicable. Prior to necropsy blood was collected and hematological and clinical chemistry parameters were Viscosity data were generated for J695 after DF/UF pro 30 evaluated. Blood Smears were prepared of each animal prior cessing using water as exchange medium. Jó95 DS (see to necropsy. At necropsy, macroscopy of body cavities was Example 1) was diafiltered against water, applying at least 5 performed. Organ weight measurement was performed on DF/UF steps. Viscosity was then determined at various tem liver, kidneys, thymus, spleen, and lymph nodes. Preliminary peratures using a plate-plate viscometer, 100 rpm shear rate, histopathology was performed on the injection site and on 150 um gap, 60mm plate diameter (equipment: Bohlin Gemi 35 liver, kidneys, thymus, spleen, and lymph nodes. nim viscometer (Malvern Instruments, Southborough, All animals survived the study until scheduled necropsy. Mass.), temperature range evaluated 8-25°C.). The rat administered the water formulation showed crusts in As seen in FIG. 32, at concentrations of 179 mg/mL and the cervical region from Day 14 to 15. No test item-related 192 mg/mL, respectively, J695 solution viscosities were effect on body weight was observed. Hematology and clinical below 70 cPat 12°C., below 40 cB at 20°C., and below 30 cP 40 chemistry values were variable. No clearly test item-related at 25° C. changes were identified in haematology or clinical chemistry. No test item-related changes were noted in urinalysis. Mea Example 19 Surement of organ weights resulted in high variability and no clearly test item-related changes in organ weights. Pharmacokinetics (PK) of an Antibody in Pure Water 45 At gross observation reddening of the Subcutis at the area of injection was noted in the rat receiving the water formula The goal of this study was to evaluate potential impact of tion at Day 3. All other changes belonged to the spectrum of formulation parameters (i.e. low ionic protein formulation spontaneous findings commonly seen in Sprague-Dawley containing water VS conventional protein formulations using rats of this strain and age. ionic excipients such as buffers and salts) on local tolerability 50 Microscopic Findings were as follows: and PK after sub-cutaneous (s.c.) dosing with Afelimomab. In No findings in Groups 01 02 addition, Systemic toxicity and toxicokinetic data of the for Minimal diffuse subcutaneous inflammation in Group 07 mulations was investigated. Protein concentrations used Focal Subcutaneous hemorrhage correlating with redden ranged from 50 mg/mL to 200 mg/mL and ionic strengths ing on gross pathology in Group 07 (Day 3), thought to 55 be administration related ranged from 3 mOsm/kg to 300 mOsm/kg. Preliminary immunohistochemistry results of pan-T, Sup A single (s.c.) dose feasibility study was carried out with pressor/cytotoxic T cells/natural killer cells, pan-B cells Afelimomab (MAK195F mouse anti human TNF F(ab')2 and pan-macrophage markers on the local reactions (Abbott Laboratories)) in male Sprague-Dawley rats to assess indicate mainly macrophages and natural killer cells the local tolerance and toxicity of Afelimomab in rats follow 60 involved in the subcutaneous inflammations/infiltra ing s.c. administration of liquid formulations at 50 and 200 tions. Thus, so far there are no hints for a local immu mg/kg. Single s.c. doses were followed by an observation/ nogenic response to the formulations used. recovery period. Limited blood sampling was carried out to All other changes belonged to the spectrum of spontaneous measure circulating Afelimomab levels and assess absorption findings commonly seen in Sprague-Dawley rats of this strain and half-life. The administered dose volume was 1 mL/kg 65 and age. body weight. The experimental groups included the follow Following Subcutaneous administration Afelimomab 1ng: absorption appeared to be fast with maximum serum levels US 8,883,146 B2 93 94 reached 0.2-3 days after injection. The absolute levels of In this study, the observed absolute levels of MAK195F in Afelimomab in all samples tested were low. Large variability low ionic Solution (water) provided a better exposure, longer was observed between the samples, likely because of the detectable serum levels and half-life’ than in conventional limited sampling frequency and the low number of animals MAK195F liquid formulation. used. In most samples, no Afelimomab could be detected in Afelimomab half-lives were in the range of 1-2 days in serum after 5-8 days. This drop in serum levels is probably standard formulation in agreement with previous observa due to the high clearance of the F(ab'). The observed T'/2 for tions for F(ab') molecules. However, a seemingly longer most samples were in the range of 1-2 days in agreement with half-life was observed for the low-ionic formulation (7.8 d). previous observations. The longer half-life of the low-ionic 10 Accordingly, the mean residence time of MAK 195F in this formulation (7.8 d) may represent a protracted absorption of formulation appeared to be longer compared to the standard the sample. Data are presented in Tables 41 and 42. formulation tested. TABLE 41 Plasma exposure levels of MAK195F Average Time (day) Concentration (g/ml) (g/ml) STD Rat 4 Rat 5 Rat 6

50 mg/kg liquid O.167 140 1.17 1.38 1.32 O.13 standard 2 O.76 0.97 O.66 O.8O O16 formulation 3 O.45 O.67 O.47 O.S3 O.12 5 LLOQ LLOQ LLOQ 8 LLOQ LLOQ LLOQ 15 LLOQ LLOQ

Rat 19 Rat 20 Rat 21

200 mg/kg O.167 1.27 3.01 3.17 2.48 water formulation 2 O.17 1.57 1.53 1.09 3 LLOQ 1.54 1.56 1.55 5 O.64 O.66 O.65 8 O4O 0.37 O.38 15 0.25 0.25 LLOQ = below quantitation limit 35 There were no aggregation state findings for liquids, nei Example 20 ther Afelimomab or control substance. For the low-ionic strength formulation, minimal diffuse S.c. injection site inflammation was seen. Inflammation, DF/UF of 2.5(E)mg1 (Anti IL-18 Antibody) either minimal to slight, or slight to moderate, was seen with 40 increased protein concentration (50 mg/mL and 200 mg/mL, Diafiltration/ultrafiltration (continuous mode) of respectively). Some local s.c. hemorrhage was seen, correlat 2.5(E)mg1 bulk solution (59.6 mg/mL) was performed, ing with reddening on gross pathology; this was considered to be the consequence of blood vessel puncture during injection. applying an about 4-fold Volume exchange using water for Some Subcutaneous reddening at the injection site was 45 injection (in the following referred to as “water). The DF/UF observed at Day 3 for the water formulation, but was not operation was controlled by monitoring turbidity, protein considered detrimental. Overall, the formulation was toler concentration (OD280), pH and osmolarity of retentate, and ated locally. DLS measurements. During DF/UF, permeate osmolarities In Table 42 below, PK data of conventional liquid formu were also monitored to control the excipient reduction of the lation vs. the water formulation is presented. 2.5(E)mg1 bulk solutions. TABLE 42 Pharmacokinetic parameters of MAK 195F after Subcutaneous dosing in different formulations.

Last Mean Time of Last Detectable Dose Half-life Tmax Cmax AUC Residence Detectable Conc. (mg/kg) Formulation (day) (day) (1gml) (day 1gml) Time (day) Conc. (day) (ig/ml) 50 liquid O.S O.2 1.32 3.3 1.5 3 O.S3 200 water 5.9 O.2 2.48 11.8 7.5 15 O.25 formulation

An increase in duration of detectable serum levels was seen Materials and Methods 65 with low ionic formulation (i.e. Afelimomab formulated in 2.5(E)mg1 Bulk Drug Substance (methionine, histidine, water), as seen in Table 42. free of polysorbate 80) (Abbott Bioresearch Center, US 8,883,146 B2 95 96 Worcester, Mass.): 2 PETG bottles with a total of 589.12 TABLE 45 g solution, Solution concentration 59.6 mg/mL. Ampuwa (water for injection) (Fresenius Medical Care, DF/UF process parameters for solution concentration Waltham, Mass.). Labscale TFFUF Settings Millipore Labscale TFF DF/UF unit including 2x Pelicon Pump speed 1.5-2 XL filter cassettes, Millipore, PLCTK 30 kDa mem Pressure of pump max. 30 psi brane, regenerated cellulose Stirring speed -3 UV/VIS spectrophotometer, Specord 50 using 280 nm Experiment duration 51 min. wavelength 10 Final weight of solution: 257.83 g Metrohm pH-meter, type 744 with Biotrode probe No. 57 Osmometer: Knauer, K-7400 The concentrated solution (-130 mg/mL) was subjected to density measurements using equipment of Paar, DMA 0.2 um filtration. The solution was cooled to 2-8°C. and then 4100 15 Stored at -80° C. Laminar air flow box Hereaus 20.2 Data Collected During DF/UF of 2.5(E)mg1 turbidity measurements: Hach, 2100AN TABLE 46 viscometer: Paar, AMVn In Process control data scales: Mettler Toledo, AT261 and 33.45 Temper filters: Millex AP 20 (fiberglass) and Minisart High Flow ature Filter (celluloseacetate), 0.20 um pore size. Solution Room Osmo 20.1 Experimental Procedures 25 temper- tur- lality COC Thawing of 2.5(E)mg1 DS samples: 2 L PETG bottles DF Volume ature bidity mOSmolf mg/ containing frozen DS were thawed within 2 hrs using a cir steps mL time C. (NTU) pH kg) ml culating water bath at 23° C. The thawed DS was clear, 2.5 (E)mg1 14.5 S.91 150 59.6 slightly opalescent, and free from visible particles. Ol O 08:00 19.024.1 NA. S.91 125 65.2 1 575 10:02 24.4, 24.4 10.1 S.92 50 70.1 30 Concentration of DS by DF/UF: due to the DF/UF unit 2 11SO 11:SO 24.3.24.7 6.67 S.94 16 72.8 reservoir volume limit of 530 mL, the 2.5(E)mg1 DS was 3 1730 13:SO 25.O.24.8 6.55 5.97 6 74.6 concentrated to a final volume of 525 mL. ca. 4 2200 15:35 25.8.25.5 10.1 5.97 5 76.7 DF/UFusing water (bufferexchange): the DS (methionine, histidine, 2.5(E)mg1) was subjected to DF/UF, applying a 35 4-fold volume exchange. Table 43 gives the amounts of water TABLE 47 that were used throughout the experiment and Table 44 pro OSmolalty of permeate (fractionated and measured during process vides the experimental parameters. Temper ature TABLE 43 40 solution Room No. DFIUF water volume exchanges temper- Permeat Of osmolality Sample ature Permeate cumulative DF/UF mCosmol/ Volume of water used lO. time C. ml ml stops kg) Volume exchange (cumulative) 45 O3 07:35 NA 90 NA NA 125 1-fold 576 mL. 1 08:00 19.024.1 200 2OO O.3 124 2-fold 1152 mL. 2 08:SO 23.0.24.1 200 400 0.7 82 3-fold 1728 mL. 3 09:27 24.024.2 200 600 1.O 53 4-fold (end of experiment) 2304 mL. 4 10:12 24.424.4 200 800 1.4 37 5 10:SO 24.S. 24.3 200 1OOO 1.7 25 50 6 11:25 24.624.3 200 1200 2.1 16 7 12:10 24.724.3 230 1430 2.5 7 TABLE 44 8 12:SS 24.724.4 170 1600 2.8 4 9 13:25 24.824.4 200 1800 3.1 2 DFIUF procedure parameters 10 14:15 25.124.8 200 2OOO 3.5 O 11 14:55 25.8.25.5 200 2200 3.8 1 Labscale TFF DF Settings 55 Pump speed 1.5-2 Pressure of pump 20-30 psi TABLE 48 Stirring speed -3 Experiment duration 8 hrs Concentration of 2.5(E)ngll solution after buffer exchange 60 Solution Volume in reservoir Osmolarity measurement of permeate was performed at Temperature (i.e. retentate) about every 200 mL of permeate processed. time C. ml pH 15:54 25.9 450 5.94 After DF/UF against water, the volume of the 2.5(E)mg 1 16:02 26.1 400 S.96 solution was 450 mL and the protein concentration 76.6 65 16:07 26.1 375 S.96 mg/mL. This solution, containing 2.5(E)mgl dissolved 16:20 26.2 350 S.96 essentially in water, was then concentrated. US 8,883,146 B2 97 98 TABLE 48-continued bidity units (NTU) of DS starting solution 14.5, after 3-fold volume exchange 6.55, after 4-fold volume exchange 10.5. Concentration of 2.5(E)ngll Solution after buffer exchange As seen with other antibodies, the hydrodynamic diameter Solution Volume in reservoir as determined by DLS decreased due to excipient reduction Temperature (i.e. retentate) (intermolecular charge-charge repulsion adding to random time C. ml pH Brownian motion, resulting in higher molecule mobility, 16:28 26.3 3OO S.98 translates to lower Dh values calculated). The pH of the 16:35 26.5 275 S.98 2.5(E)mg1 solution was basically the same before (pH 5.94) 16:45 26.5 250 5.99 and after (pH 5.99) the DF/UF operation. 10 As shown by DLS monitoring, the 2.5(E)mg1 remained stable during the DF/UF operation. No substantial increase in TABLE 49 high molecular weight specimen was detected. Analytical characterization of concentrated 2.5(E)mg1 Solution Example 21 before and after 0.2 in filtration): 15 lot Preparation of Adalimumab Formulated in Water and parameter before filtration after filtration Stability Studies Thereof turbidity 15.4 9.58 The following example describes the stability of a formu (NTU) oSmolality 6 NA lation comprising adalimumab originating from processes mOSmol/kg described in the above examples, i.e., adalimumab was suc density 1.0346 NA cessfully dialyzed into water. g/ml Materials and Methods pH 5.99 NA Dyn. Viscosity (25°C.) NA 7.9998 25 3323.6 g Adalimumab solution (71.3 mg/mL) were diafil mPas tered using pure water. After a 7-fold volume exchange with pure water (theoretical excipients reduction, 99.9%), the pro tein solution was diluted/ultrafiltered to final target concen trations of 220 and 63 mg/mL, respectively. PH, osmolality, TABLE SO 30 Viscosity, conductivity, PCS, visual inspection and protein Dynamic light scattering data (determination concentration measurements (OD280) were performed to of Dh of monomer and Z-average value of monitor the status of the protein after DF/UF processing. Dh = Dh of all specimen present in solution) during DF/UF After DF/UF processing, the protein solutions were sterile Sample pull filtered (0.22 um Millipak-60 and Millipak-200 membrane 35 filters) and subsequently filled into BD HyPak SCFTM 1 mL After After long syringes, equipped with 27.5G RNS needles and sterile DV DV DV DV COCC- fil BD HyPak BSCF 4432/50 stoppers. The filling volume was DLS data 1-fold 2-fold 3-fold 4-fold tration tration around 0.825 mL per syringe. Peak 1 After filling the syringes were stored at 2-8°C., 25°C. and 40 40°C., respectively, and analyzed as indicated in the sample diameter 4.32 3.68 3.54 3.48 2.03 2.13 pull scheme depicted below. OOle 1OO.O 1OOO 1OOO 89.6 87.O 1OOO Adalimumab Drug Substance (Adalimumab extinction intensity 3.95 3.28 3.2O 3.53 2.12 1.89 coefficient 280 nm: 1.39 mL/mg cm): Drug Substance %) did not contain polysorbate 80. DS buffer, pH 5.38. Z-Average O.077 O.106 0.094 O.245 O.287 O.113 45 nm. Sortorius Sartocon Slice diafiltration system, equipped Pd with Ultrasert PES membrane cassettes (50 kDa and 30 Peak 2 kDa cutoff). The Sartocon Slice system was operated in continuous mode at ambient temperature according to diameter NA NA NFA 984 411 NA nm. Sartorius Operating Instructions. intensity 10.4 11.8 50 pH electrodes %) PerkinElmer UV visible spectrophotometer, Lambda 35, Peak 3 was used for protein concentration measurements (280 diameter NA NA NA NiA 4260 NA nm wavelength). Disposable UV cuvettes, 1.5 mL, semi nm. micro, Poly(methyl methacrylate) (PMMA), were used intensity 1.2 55 for the concentration measurements. %) Sterilized water for injection Ph.Eur./USP was used as DF/UF medium. 20.3 Discussion AVogel Osmometer OM815, was used for osmolality mea The experiment demonstrated that 2.5(E)mg1 (buffered in surements (calibrated with 400 mOsmol/kg NaCl cali methionine, histidine) can be formulated in essentially water 60 bration solution, Art. No. Y 1241, Herbert Knauer at higher concentration (no solubility limitations observed at GmbH, Berlin, Germany). 130 mg/mL). After 3 Volume exchanges using water osmola Anton Paar MicroViscosimeter, type AWVn, was used for lity of permeate and retentate were below 10 mCsmol/kg, viscosity assessment of the protein Solutions according demonstrating that buffer excipients have been effectively to Anton Paar Operating Instructions. Viscosity was reduced. The opalescence of the 2.5(E)mg1 solution was 65 assessed at 20° C. reduced during DF/UF using water (optimal appearance), An InoLab Cond Level2 WTW device was used for con mirrored also by reduces turbidity values (nephelometric tur ductivity measurements normalized to 25°C. US 8,883,146 B2 99 100 A Malvern Instruments Zetasizer nano ZS, was used for determination of Z-average values, applying a standard Temp. method. Measurements were performed at 25°C., using viscosity data obtained by falling ball viscosimetry (An 50 C. ton Paar MicroViscosimeter, type AWVn, at 25°C.). 25o C. HPLC Methods 40° C. Adalimumab, SEC analysis: Sephadex 200 column (Phar macia Cat. No. 175175-01). Mobile phase 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow rate, ambient temperature, detection 10 Test parameter Test method UV 214 nm and 280 nm Each sample was diluted to 1.0 Visible particles analogous DAC (EA 4.43) mg/mL with Milli-Q water, sample injection load 50 ug Subvisible particles analogous (duplicate injection). Ph. Eur, USPEA 444 Adalimumab, IEC analysis: Dionex, Propac WCX-10 col Turbidity analogous Ph. Eur. (EA 4.42) 15 Color (visual) Ph. Eur. (EA 4.50) umn (p/n 054993) along with a corresponding guard pH Ph. Eur. (EA 4.24) column (p/n 054994). Separation conditions: mobile Size exclusion HPLC Desribed in the text above phase A: 10 mM sodium phosphate, pH 7.5: mobile Cation exchange HPLC Desribed in the text above phase B 10 mM Sodium phosphate, 500 mM Sodium chloride, pH 5.5. 1.0 mL/min flow rate, ambient tem perature. Each sample was diluted to 1.0 mg/mL with DF/UF Processing of Adalimumab Milli-Q water, sample injection load 100 ug, duplicate Table 51 describes the adalimumab characteristics after injection. diafiltration. TABLE 51

Protein Concentration Osmolality Viscosity Visual Conductivity Sample mg/mL pH mosmol/kg cP Inspection IS/cm High 220 5.57 26 27.9 Slightly 1167 O.34 concentration opalescent, essentially free from visible particles Low 63 5.44 1.8 Slightly 522 1.85 concentration opalescent, essentially free from visible particles

40 Calculation of the Protein Concentration Adalimumab characterization upon storage, including clarity and opalescence, degree of coloration of liquids, SEC, at Calculation Formula: different temperature degrees is described in Appendix D. Conclusion The above example provides a diafiltration/ultrafiltration 45 (DF/UF) experiment where water (sterilized water for injec tion Ph.Eur./USP) was used as diafiltration medium for the monoclonal antibody Adalimumab. Adalimumab was subjected to DF/UF processing by using pure water as DF/UF exchange medium and was formulated e—absorption coefficient 50 at pH 5.57 at high concentration (220 mg/mL) and at pH 5.44 c—concentration at lower concentration (63 mg/mL) without inducing Solution haziness, severe opalescence or turbidity formation. d-length of cuvette that the light has to pass Adalimumab from the DF/UF experiments was stored in E -absorbance SCF syringes at 2-8°C., 25° C. and 40° C. for 3 months. Data 55 obtained points at favorable overall stability of the protein. Io initial light intensity In conclusion, processing and formulating proteins using I—light intensity after passing through sample pure water as DF/UF exchange medium is feasible. Assuming an ideal 100% excipient membrane permeability, an approx. 99.9% maximum excipient reduction can be estimated. 60 &Adalimumab = 1.39 mgXcm Example 22 Stability Studies of Adalimumab Formulated in Sample Pull Scheme Water Using Non-Ionic Excipients Samples of the prepared solutions are stored at the tempera 65 The following example describes stability studies of a for tures listed below and pulled (x) at the indicated time points mulation containing an antibody, i.e., adalimumab, in water after study start. with additional non-ionic excipients. US 8,883,146 B2 101 102 Materials and Methods HPLC Methods Adalimumab material was the same as in example 21 (DF/ Adalimumab, SEC analysis: Sephadex 200 column (Phar UF processing). After DF/UF processing, the protein solu macia Cat. No. 175175-01). Mobile phase 20 mM tions were formulated as denoted in Table 52. Mannitol was sodium phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow rate, ambient temperature, detection chosen as example from the group of Sugar alcohols, like UV 214 nm and 280 nm Each sample was diluted to 1.0 mannitol, Sorbitol, etc. Sucrose was chosen as example from mg/mL with Milli-Q water, sample injection load 50 g the group of Sugars, like Sucrose, trehalose, raffinose, mal (duplicate injection). tose, etc. Polysorbate 80 was chosen as example from the Adalimumab, IEC analysis: Dionex, Propac WCX-10 col group of non-ionic Surfactants, like polysorbate 80, polysor umn along with a corresponding guard column Separa bate 20, pluronic F68, etc. ~10.7 mL were prepared for any 10 tion conditions: mobile phase A: 10 mM sodium phos formulation. Osmolality, viscosity and PCS measurements phate, pH 7.5: mobile phase B 10 mM Sodium were performed for any formulation after preparation. phosphate, 500 mM Sodium chloride, pH 5.5. 1.0 mL/min flow rate, ambient temperature. Each sample TABLE 52 was diluted to 1.0 mg/mL with Milli-Q water, sample 15 injection load 100 ug, duplicate injection. Final protein Mannitol Sucrose Polysorbate 80 Sample Sample Pull Scheme concentration (mg/mL) (mg/mL) (% w/w) Name Samples of the prepared solutions were stored at 5°C., 25° 50 mg/mL 50 LISO.O1 C., and 40°C. and pulled at either 1 minute (5° C. and 40°C.) 50 mg/mL 8O LISO.O2 or at T0 and 1 minute (25°C.) after study start. Test param 50 mg/mL 50 O.O1 LISO.O3 eters were measured according to appropriate methods, e.g., 50 mg/mL 8O O.O1 LISO.O4 color was determined visually, turbidity was determined at an 50 mg/mL 50 O.1 LISO,05 50 mg/mL 8O O.1 LISOO6 absorption at 344 nm. 50 mg/mL O.O1 LISOO7 Initial Formulation Characterization 50 mg/mL O.1 LISO.08 Table 53 described the initial formulation osmolalities and 200 mg/mL 50 LI2OOO1 25 Viscosities. 200 mg/mL 8O LI2OOO2 200 mg/mL 50 O.O1 LI2OOO3 200 mg/mL 8O O.O1 LI2O004 TABLE 53 200 mg/mL 50 O.1 LI2OOOS 200 mg/mL 8O O.1 LI2OOO6 oSmolarity viscosity LOt. comp. moSmol mPas 200 mg/mL O.O1 LI2OOO7 30 200 mg/mL O.1 LI2OOO8 LI 50/01 50 mg/mL mannitol 309 .9796 Polysorbate 80 stock solution 0.5% and 5% in sterile water for injection: Addition in 1:50 LI 50/02 80 mg/mL Sucrose 272 2.1284 ratio (210 L to 10.5 mL Adalimumab solution, addition of 210 L water for injection to LI 50/03 50 mg/mL mannitol; 0.01% Tween 307 98.43 samples formulated without surfactant to assure equal protein concentration in all samples) 8O Addition of mannitol, sucrose in solid form (525 mg/840 mg, respectively). LI 50/04 80 mg/mL Sucrose; 0.01% Tween 80 269 2.1.194 35 LI 50/05 50 mg/mL mannitol; 0.1% Tween 80 307 .998O The preparations were sterile filtered (Millex GV, Milli LI 50/06 80 mg/mL Sucrose: 0.1% Tween 80 272 2.1235 pore, 0.22 um, Ø 33 mm, Art. SLGV033RS) and subse LI 5007 0.01% Tween 80 8 7335 LI 5008 0.1% Tween 80 8 8162 quently filled into BD HyPak SCFTM 1 mL long syringes, LI 200/01 50 mg/mL mannitol 396 21.395 equipped with 27.5G RNS needles and sterile BD HyPak LI 200/02 80 mg/mL Sucrose 351 21744 BSCF 4432750 stoppers. The filling volume was around 0.6 40 LI 200/03 50 mg/mL mannitol; 0.01% Tween 387 21.233 mL per Syringe. 8O LI 200/04 80 mg/mL Sucrose; 0.01% Tween 80 350 21.701 After filling the syringes were stored at 2-8°C., 25°C. and LI 200/05 50 mg/mL mannitol; 0.1% Tween 80 387 21.592 40°C., respectively, and analyzed as indicated in the sample LI 200/06 80 mg/mL Sucrose: 0.1% Tween 80 355 21943 pull scheme depicted below. LI 20007 0.01% Tween 80 27 21.296 LI2O008 0.1% Tween 80 28 21.889 Adalimumab Drug Substance (Adalimumab extinction 45 coefficient 280 nm: 1.39 mL/mg cm): Drug Substance did not contain polysorbate 80. DS buffer, pH 5.38. All formulations of one concentration demonstrated equal PH electrodes Viscosities. Those of Sucrose containing formulations were Sterilized water for injection Ph.Eur./USP was used as slightly higher. The reduced viscosities of the highly concen DF/UF medium. 50 trated formulations in comparison to the highly concentrated Mannitol, polysorbate 80, and Sucrose, all matching formulation in water (example A, viscosity 27.9cP) is Ph.Eur. quality explained by sample dilution with polysorbate 80 stock solu AVogel Osmometer OM815, was used for osmolality mea tions or plain water, leading to a final concentration of ~215 surements (calibrated with 400 mOsmol/kg NaCl cali mg/mL vs. 220 mg/mL in example 21. bration solution, Art. No. Y 1241, Herbert Knauer 55 Table 54 describes the PCS data determined for each GmbH, Berlin, Germany). sample. Anton Paar MicroViscosimeter, type AWVn, was used for Viscosity assessment of the protein Solutions according TABLE 54 to Anton Paar Operating Instructions. Viscosity was assessed at 20° C. 60 PCS Fluostar Optima, BMG Labtech (absorption measurement Sample Z-averaged. Inm at 344 nm in well plates, assessment of turbidity) LISO.O1 2.58 LISO.O2 2.22 A Malvern Instruments Zetasizer nano ZS, was used for LISO.O3 2.13 determination of Z-average values, applying a standard LISO,04 2.22 method. Measurements were performed at 25°C., using 65 LISO/OS 2.25 viscosity data obtained by falling ball viscosimetry (An LISOO6 2.55 ton Paar MicroViscosimeter, type AWVn, at 25°C.). US 8,883,146 B2 103 104 TABLE 54-continued Example 23 PCS Preparation of Jó95 Formulated in Water with Sample Z-averaged. Inm Non-Ionic Excipients LISO/O7 2.87 LISO.08 1.94 LI2OOO1 OSO The following example describes the preparation of a for LI2OOf O2 O43 mulation containing an antibody, i.e., adalimumab, in water LI2OOO3 O.36 with additional non-ionic excipients. The example also LI200,04 O.38 describes the stability (as measured for example by SE-HPLC LI2OOOS 0.37 10 LI2OOO6 O41 and IEC) of JG95 formulated in water with additional non LI2OOO7 O3S ionic excipients. LI2OO.08 O.36 Materials and Methods 2x30 mLJ695 solution (~125 mg/mL) at different pH were dialyzed using pure water applying Slide-A-Lyzer dialysis The data provided in Table 54 shows that Z-average values 15 do not significantly differ from the values obtained from cassettes. Dialysis of the samples was performed for 3 times Adalimumab Solutions in non-ionic excipient free systems against 3 L pure water, respectively (theoretical excipients (63 mg/mL, 1.85 dinn, 220 mg/mL, 0.34 dinn, example 21). reduction, 1:1,000,000). The protein solutions were ultrafil tered to final target concentrations of 200 mg/mL, by using Adalimumab Characterization Upon Storage Vivaspin 20 concentrators. PH, osmolality, viscosity, conduc Appendix E provides data on Adalimumab stability upon tivity, PCS, visual inspection, HPLC and protein concentra Storage. tion measurements (OD280) were performed to monitor the Conductivity of Placebo Solutions status of the protein during and after processing. After processing, the protein solutions were formulated as Table 55 describes the influence of non-ionic excipients on 25 denoted in the following. Mannitol was chosen as an example the conductivity of the various adalimumab formulations. All to use from the group of Sugar alcohols, like mannitol, Sorbi placebo Solutions were prepared using sterilized water for tol, etc. Sucrose was chosen as an example to use from the injection Ph.Eur./USP. group of Sugars, like Sucrose, trehalose, raffinose, maltose, etc. Polysorbate 80 was chosen as an example to use from the TABLE 55 30 group of non-ionic Surfactants, like polysorbate 80, polysor Polysorbate 80 Conductivity bate 20, pluronic F68, etc. A volume of 0.5 mL was prepared Mannitol (mg/mL) Sucrose (mg/mL) (% w/w) (IS/cm) for each of these formulations. PH, osmolality, visual inspec 1.1 tion, and HPLC analysis were performed to monitor the status 50 1.2 of the protein after sample preparation. 35 8O 2.2 50 O.O1 2.3 TABLE 56 8O O.O1 1.4 50 O.1 2.6 Description of various JG95 formulations 8O O.1 3.6 O.O1 1.2 Final protein Mannitol Sucrose Polysorbate 80 O.1 2.6 40 concentration (mg/mL) (mg/mL) (% w/w) Sample Name* 200 mg/mL 50 LI2OOO1 S Conclusion 200 mg/mL 8O LI2OOO2S 200 mg/mL 50 O.O1 LI2OOO3:S The preparations were stored in SCF syringes at 2-8°C., 200 mg/mL 8O O.O1 LI2OOO4;S 45 200 mg/mL 50 O.1 LI2OOOSS 25°C. and 40°C. for 1 month. Data obtained from the storage 200 mg/mL 8O O.1 LI2OOO6S study showed that there was overall stability of the protein in 200 mg/mL O.O1 LI2OOO7. S all formulations tested. The stability data was comparable to 200 mg/mL O.1 LI2OOO8.5 200 mg/mL 50 LI2OOO1.6 the stability of samples from example 21. Measurement of the 200 mg/mL 8O LI2OOO2.6 conductivity of non-ionic excipient containing placebo Solu 50 200 mg/mL 50 O.O1 LI2OOO3.6 tions demonstrates a marginal increase of conductivity for 200 mg/mL 8O O.O1 LI200,046 200 mg/mL 50 O.1 LI2OOOS6 Some excipients in the range of some uS/cm. PCS measure 200 mg/mL 8O O.1 LI2OOO6.6 ments demonstrate no significant increase in hydrodynamic 200 mg/mL O.O1 LI2OOO7.6 diameters in comparison to non-ionic excipient free systems. 200 mg/mL O.1 LI2OOO8.6 In conclusion, processing proteins using pure water as 55 sm “5” or “6” is added to any sample name to differentiate between samples at pH 8iC to, DF/UF exchange medium and formulation with non-ionic Polysorbate 80 stock solution 0.5% and 5% in sterile water for injection: Addition in 1:50 excipients is feasible. Adalimumab was also assessed by PCS ratio (10 ul to 0.5 mL J695 solution, addition of 10 uI water for injection to samples formulated without surfactant to assure equal protein concentration in all samples) in a buffer of following composition: 10 mM phosphate Addition of mannitol sucrose in solid form (25 mg 40 mg, respectively), buffer, 100 mM sodium chloride, 10 mM citrate buffer, 12 mg/mL mannitol, 0.1% polysorbate 80, pH 5.2. The Adali 60 J695 Drug Substance (J695 extinction coefficient 280 nm: mumab concentration was 50 mg/mL and 200 mg/mL, 1.42 mL/mg cm): Drug Substance did not contain respectively. The Z-average values were 11.9 d-nm for the 50 polysorbate 80. DS buffer, pH 6.29. mg/mL formulation and 1.01 d-nm for the 200 mg/mL for pH electrodes mulation, respectively. Thus, it was clearly demonstrated that Demineralized and sterile filtered water was used as dialy hydrodynamic diameters at a given protein concentration are 65 sis medium. dependent on the ionic strength (clearly higher diameters in Mannitol, polysorbate 80, and Sucrose, all matching salt containing buffers). Ph.Eur. quality US 8,883,146 B2 105 106 AVogel Osmometer OM815, was used for osmolality mea J695, IEC analysis: Dionex, Propac WCX-10 column (p/n surements (calibrated with 400 mOsmol/kg NaCl cali 054993) along with a corresponding guard column (p/n bration solution, Art. No. Y 1241, Herbert Knauer 054994). Separation conditions: mobile phase A: 10 GmbH, Berlin, Germany). mM NaHPO, pH 6.0; mobile phase B 10 mM Anton Paar MicroViscosimeter, type AWVn, was used for 5 NaHPO 500 mMNaCl, pH 6.0.1.0 mL/min flow rate, Viscosity assessment of the protein Solutions according 35° C. temperature. Each sample was diluted to 1.0 to Anton Paar Operating Instructions. Viscosity was mg/mL with Milli-Q water, sample injection load 100 assessed at 20° C. l2. Fluostar Optima, BMG Labtech (absorption measurement Calculation of the Protein Concentration at 344 nm in well plates, assessment of turbidity) 10 Calculation Formula: Eppendorf Centrifuge 5810 R Slide-A-Lyzer dialysis cassettes, Pierce Biotechnology (Cat No 66830) Vivaspin 20 concentrators, 10 KDa PES membranes (Viva science, Product number VS2001), used according to 15 standard Operating Instructions PerkinElmer UV visible spectrophotometer, Lambda 35, e—absorption coefficient was used for protein concentration measurements (280 c—concentration nm wavelength). Disposable UV cuvettes, 1.5 mL, semi d—length of cuvette that the light has to pass micro, Poly(methyl methacrylate) (PMMA), were used 20 E—absorbance for the concentration measurements. Io initial light intensity An InoLab Cond Level2 WTW device was used for con I—light intensity after passing through sample ductivity measurements normalized to 25°C. A Malvern Instruments Zetasizer nano ZS, was used for determination of Z-average values, applying a standard 25 &Adalimumab = 1.42 method. Measurements were performed at 25°C., using mgXcm viscosity data obtained by falling ball viscosimetry (An ton Paar MicroViscosimeter, type AWVn, at 25°C.). Processing of Jó95 HPLC Methods J695 in water was characterized prior to the addition of any J695, SEC analysis: Tosoh Bioscience G3000swxl, 7.8 30 non-ionic excipients. Table 57 provides details of the JG95 mmx30 cm, 5um (Cat. No. 08541). Mobile phase 211 characterization during dialysis/ultrafiltration. TABLE 57

Protein PCS Concentration Osmolality Viscosity Conductivity Z-average Sample mg/mL PH mosmol/kg cP Visual Inspection IS/cm d nm) Starting ~125 mg/mL 6.29 (for NA N/A Slightly opalescent, NA NA material low pH essentially free samples, from visible adjusted to particles 4.77 with O.O1M hydrochloric acid) After dialysis, 42.5 mg/mL. S.21 7 1.60 Slightly opalescent, 6O2 1.5 low pH essentially free rom visible particles After dialysis, 56.9 mg/mL 6.30 6 2.11 Slightly opalescent, 500 2.7 high pH essentially free rom visible particles After 206 mg/mL S.40 50 39.35 Slightly opalescent, 1676 O.21 concentration, essentially free low pH rom visible particles After 182 mg/mL. 6.46 39 47.76 Slightly opalescent, 1088 O.21 concentration, essentially free high pH rom visible particles

mM NaSO/92 mM NaHPO, pH 7.0. 0.25 mL/min Characterization of Formulations with Non-Ionic Excipients flow rate, ambient temperature, detection UV 214 nm Following the addition of the various non-ionic excipients

and 280 nm. Each sample was diluted to 2.0 mg/mL with 65 to the JG95 formulation (see description in Table 56), each Milli-Q water, sample injection load 20 Lug (duplicate formulation was analysed. Results from osmolality and injection). visual inspection, and pH are described below in Table 58. US 8,883,146 B2 107 108 TABLE 58 Osmolality Sample pH mosmol/kg Visua Inspection

LI2OOO1 S 5.39 473 Slightly opalescen , essen ia ree rom visi e particles LI2OOO2AS 5.38 402 Slightly opalescen , essen ia ree rom visi e particles LI2OOO3.5 5.37 466 Slightly opalescen , essen ia ree rom visi e particles LI2OOO4, S. S.37 397 Slightly opalescen , essen ia ree rom visi e particles LI2O005.5 5.37 458 Slightly opalescen , essen ia ree rom visi e particles LI2OOO6.S. S.37 396 Slightly opalescen , essen ia ree rom visi e particles LI2OOO7. S S36 50 Slightly opalescen , essen ia ree rom visi e particles LI2OOO8,S S36 48 Slightly opalescen , essen ia ree rom visi e particles LI20001.6 6.43 428 Slightly opalescen , essen ia ree rom visi e particles LI200,026 6.42 40S Slightly opalescen , essen ia ree rom visi e particles LI20003f6 6.43 348 Slightly opalescen , essen ia ree rom visi e particles LI200,04f6 6.43 383 Slightly opalescen , essen ia ree rom visi e particles LI2OOOS, 6 6.42 432 Slightly opalescen , essen ia ree rom visi e particles LI200,066 6.42 402 Slightly opalescen , essen ia ree rom visi e particles LI2OOO7.6 6.43 38 Slightly opalescen , essen ia ree rom visi e particles LI200,086 6.43 39 Slightly opalescen , essen ia ree rom visi e particles

HPLC Data TABLE 59-continued Each of the non-ionic excipient containing J695 formula tions were also examined using SE-HPLC and IEX. The data SE-HPLC results of various J695 formulations from these analyses are provided in Tables 59 and 60 and Sum Sum Aggregates Monomer Fragments provide an overview of JG95 stability during processing and 25 formulation. sample name % % % in Water 1.083 98.195 O.721 TABLE 59 bH 6 O.998 98.350 O.652 50 mg/mL mannitol 1.OO2 98.364 O.634 SE-HPLC results of various J695 formulations LI2OOO1.6 1.OOO 98.357 O643 30 bH 6 1.028 98.243 O.729 Sum Sum 80 mg/mL Sucrose O.983 98.355 O.662 Aggregates Monomer Fragments LI 200026 1.OO6 98.299 0.695 sample name pH 6, 50 mg mannitol 1.OOS 98.322 O.673 O.O1% Tween 80 1.008 98.317 O.676 H5 O.608 98.619 0.773 LI2OOO3f6 1.OO6 98.319 O.674 25 mg/mL. O.619 98.598 O.783 35 pH 6, 80 mg Sucrose O.987 98.363 O649 arting mat. O.614 98.608 0.778 O.O1% Tween 80 O.987 98.321 O.692 H6 O.427 98.809 O.764 LI 20004f6 O.987 98.342 O.671 25 mg/mL. O.392 99.005 O.603 pH 6, 50 mg mannitol O.996 98.326 O.678 arting mat. O.409 98.907 O.683 O.1% Tween 80 O.996 98.338 O666 H5 O.654 98.604 O.742 LI2OOOSF6 O.996 98.332 O.672 0.677 98.560 O.763 .5 mg/mL. 40 pH 6, 80 mg Sucrose O.998 98.305 O.697 er dialysis O. 666 98.582 0.753 O.1% Tween 80 O.984 98.345 O.671 H6 O.748 98.541 O.711 LI2OOO6.6 O.991 98.325 O.684 6 .9 mg/mL. 0.739 98.597 O.66S bH 6 1.OOO 98.325 0.675 er dialysis O.743 98.569 O688 O.O1% Tween 80 O.994 98.347 O.659 H5 O.913 98.416 O.671 LI2OOO7.6 O.997 98.336 O.667 6 mg/mL. O.923 98.356 O.721 Water O.918 98.386 O.696 45 bH 6 1.OO3 98.314 O.682 5 O.928 98.312 O.760 O.O1% Tween 80 O.998 98.338 O664 o mg/mL mannitol O.926 98.339 O.736 LI2OOO8.6 1.OO1 98.326 O.673 2OOO1 S O.927 98.325 O.748 5 O.925 98.319 O.755 o mg/mL Sucrose O.929 98.332 O.738 2OOO2 S O.927 98.326 0.747 50 TABLE 60 5, 50 mg mannitol O.942 98.326 0.732 O1% Tween 8O O.942 98.300 0.758 IEX results of various J695 formulations 2OOO3 S O.942 98.313 O.745 H5, 80 mg Sucrose O.944 98.315 O.741 Sum Acicid Sum Sum Basic O1% Tween 8O O.944 98.339 0.717 Peaks Glutamine Peaks 2OOO4;S O.944 98.327 O.729 55 sample name % % % 5, 50 mg mannitol O.941 98.348 O.711 % Tween 80 O.967 98.299 O.734 pH 5 4.598 3.303 200/05/5 O.9S4 98.323 O.722 125 mg/mL. 4.599 3.177 5, 50 mg mannitol O.944 98.346 O.710 starting mat. 4.599 92.162 3.240 % Tween 80 O.948 98.340 O.712 pH 6 4.597 3.159 2OOO6.5 O.946 98.343 O.711 125 mg/mL. 4.629 3.156 5, 50 mg mannitol O.946 98.348 O.7O6 60 starting mat. 4.613 92.229 3.158 % Tween 80 O.953 98.328 O.719 pH 5 4.7O6 3.177 200,075 O.949 98.338 O.713 42.5 mg/mL. 4.725 3.2OS 5, 50 mg mannitol O.987 98.313 O.701 after dialysis 4.715 92.094 3.191 % Tween 80 O.994 98.283 0.723 pH 6 4.739 3.182 2OOO8.5 O.991 98.298 O.712 56.9 mg/mL 4.752 3.167 6 1.091 98.169 0.739 65 after dialysis 4.746 92.08O 3.174 82 mg/mL 1075 98.221 O.703 pH 5 4.6SS 3.167 US 8,883,146 B2 109 110 TABLE 60-continued TABLE 60-continued

IEX results of various J695 formulations IEX results of various J695 formulations

Sum Acicid Sum Sum Basic Sum Acicid Sum Sum Basic Peaks Glutamine Peaks 5 Peaks Glutamine Peaks sample name % % % sample name % % % 206 mg/mL 4.676 3.210 LI2OOO7.6 4.916 91681 3.402 in Water 4.666 92.146 3.189 pH 6 4.884 3.410 bH S 4.721 3.321 O.1% Tween 80 4.934 3.366 50 mg/mL mannitol 4.733 3.356 10 LI2OOO8.6 4.909 91.703 3.388 LI2OOO1 S 4.727 91.935 3.338 bH S 4.715 3.299 80 mg/mL Sucrose 4.687 3.338 Conclusion LI2OOO2FS 4.701 91.981 3.31.8 The above example provides an experiment where water pH 5, 50 mg mannitol 4.767 3.246 (demineralised and sterile filtered water) was used as dialysis O.01% Tween 80 4.736 3.253 15 LI2OOO3FS 4.752 91.999 3.2SO medium for the monoclonal antibody J695. pH 5, 80 mg Sucrose 4.751 3.257 J695 was subjected to dialysis and concentration process O.01% Tween 80 4.742 3.229 ing by using pure water as exchange medium and was formu LI2OOO4fS 4.746 92.011 3.243 lated at pH 5.40 as well as 6.46 at high concentration (206 and pH 5, 50 mg mannitol 4.78O 3.42O 182 mg/mL, respectively) without inducing Solution hazi O.1% Tween 80 4.720 3.394 LI2OOOSFS 4.7SO 91.843 3.407 2O ness, severe opalescence or turbidity formation. pH 5, 80 mg Sucrose 4.756 3.421 J695 from the processing experiment was characterized, O.1% Tween 80 4.894 3.375 and formulated with various non-ionic excipients. Data LI2OOO6.5 4.82S 91.777 3.398 obtained points at favorable overall stability of the protein in bH S 4.813 3.425 the formulations tested. O.01% Tween 80 4.757 3.413 LI2OOO7 S 4.785 91.796 3.419 25 In conclusion, processing proteins using pure water as bH S 4.769 3.361 exchange medium and formulation with non-ionic excipients O.1% Tween 80 4.842 3.335 is feasible. Assuming an ideal 100% excipient membrane LI2OOO8.5 4.806 91.846 3.348 permeability, an approx. 99.9% maximum excipient reduc bH 6 4.882 3.452 tion can be estimated. 82 mg/mL 4.886 3.451 in Water 4.884 91.664 3.451 30 bH 6 4.843 3.456 Example 24 50 mg/mL mannitol 4.833 3.393 LI2OOO1.6 4.838 91.737 3.425 Syringeability of Adalimumab Formulated in Water bH 6 4.923 3.407 80 mg/mL Sucrose 4.896 3.491 The formulations from example 21 (63 and 220 mg/mL LI2OOO2.6 4.909 91.642 3.449 35 pH 6, 50 mg mannitol 4.864 3.423 Adalimumab) were subjected to force measurements upon O.01% Tween 80 4.899 3.392 Syringe depletion. 220 mg/mL samples of Adalimumab were LI2OOO3f6 4.882 91.711 3.408 diluted to 200 mg/mL, 150 mg/mL and 100 mg/mL, respec pH 6, 80 mg Sucrose 4.870 3.32O O.01% Tween 80 4.928 3.369 tively, and were also assessed. A Zwick Z2.5/TN1S was used LI 20004f6 4.899 91.756 3.345 at a constant feed of 80 mm/min Finally, viscosity data of the pH 6, 50 mg mannitol 4.905 3.385 40 formulations was assessed using an Anton Paar MicroVisco O.1% Tween 80 4922 3.489 simeter, type AWVn, at 20°C. The following data collection LI2OOOSF6 4.914 91649 3.437 Suggests that both needle and Syringe diameters have a sig pH 6, 80 mg Sucrose 4.973 3.443 O.1% Tween 80 4.962 3.335 nificant effect on the gliding forces upon Syringe depletion. LI2OOO6.6 4.968 91.644 3.389 Surprisingly, the highly concentrated Solution at 220 mg/mL bH 6 4.934 3.413 45 (viscosity 27.9 cP at 20° C.) can be delivered by applying O.01% Tween 80 4.899 3.392 equivalent depletion forces as with the lower concentrated formulation at 63 mg/mL (viscosity 1.8 cF at 20° C.). TABLE 61 Gliding Force Values obtained for Adalimumabsolutions in different packaging Systems. D. HyPak SCF TM 1 mL long Syringes, equipped with

BD HyPak 25 G x 26 G x 27 G X SCFTM1 mL 5/8" 1A" 1/2" 1 mL Soft-Ject (R) long Syringes, needles needles needles Tuberkulin equipped with (Sterican) (Sterican) (Sterican) syringes (Smaller 27.5 G. RNS BD BD BD diameter than BD needles HyPak HyPak HyPak HyPak Syringes, Adaliumab BD HyPak BSCF BSCF BSCF equipped with Concentration BSCF 4432; SO 4432 SO 4432; SO 4432 SO 27 Gx 1/2" (Viscosity) stoppers stoppers stoppers stoppers needles (Sterican) 63 mg/ml 3.9N (1.8 cP) 100 mg/mL 3.3ON 1.O2N 1.33N 1.69N 1.OON (2.9 cP) US 8,883,146 B2

TABLE 61-continued Gliding Force Values obtained for Adalimumabsolutions in different packaging Systems. D. HyPak SCF TM 1 mL long Syringes, equipped with

BD HyPak 25 G x 26 G x 27 G X SCFTM1 mL 5/8" 1/2" 1A" 1 mL Soft-Ject (R) long Syringes, needles needles needles Tuberkulin equipped with (Sterican) (Sterican) (Sterican) syringes (Smaller 27.5 G. RNS BD BD BD diameter than BD needles HyPak HyPak HyPak HyPak syringes, Adaliumab BD HyPak BSCF BSCF BSCF equipped with Concentration BSCF 4432; SO 4432 SO 4432; SO 4432 SO 27 Gx 1/2" (Viscosity) stoppers stoppers stoppers stoppers needles (Sterican) 150 mg/mL 4.63N 1.16N 1.58N 2.93N 1.33N (7.4cP) 200 mg/mL 7.25N 2.16N 3.24N 6.25N 2.55N (15.7 cP) 220 mg/mL 14.SN 2.99N 3.97N 9.96N 3.16N (27.9 cP)

The above Suggests that even with high concentrations of Example 25 protein, Such formulations are conducive to administration using a Syringe, e.g., Subcutaneous. 25 Freeze/Thaw Stability of Adalimumab Formulated in Water and with Non-Ionic Excipients Examples 25-28 The following example describes the stability of an anti Examples 25-28 describe freeze/thaw stability experi body, e.g., adalimumab, in a water formulation and in water ments of various antibody formulations containing the anti 30 formulations in which non-ionic excipients have been added. body formulated in water (referred to in examples 26-28 as Aliquots of samples from examples 21 and 22 were Subjected low-ionic strength protein formulations). The freeze thaw to freeze/thaw experiments and analyzed by SE-HPLC. Data behavior of a number of antibodies was evaluated by cycling was compared to SE-HPLC results derived from freeze/thaw various protein formulations up to 4 times between the frozen experiments using Adalimumab in a buffer of the following 35 composition: 10 mM phosphate buffer, 100 mM sodium chlo state and the liquid State. Freezing was performed by means ride, 10 mM citrate buffer, 12 mg/mL mannitol, 0.1% of a temperature controlled-80° C. freezer, and thawing was polysorbate 80, pH 5.2. Adalimumab in this latter buffer was performed by means of a 25°C. temperature controlled water used at 50 mg/mL and 200 mg/mL, respectively. Freeze/thaw bath. About 25 mL of antibody solution each were filled in 30 cycles were performed in Eppendorf caps, by freezing to -80° mL PETG repositories for these experiment series. C. and storage in the freezer for 8 hours, followed by thawing Formation of subvisible particles presents a major safety 40 at room temperature for 1 hour and Subsequent sample pull. concern in pharmaceutical protein formulations. Subvisible Each formulation was subjected to 5 cycles, i.e., cycles 0, 1, protein particles are thought to have the potential to nega 2, 3, 4, and 5 described in the tables below. tively impact clinical performance to a similar or greater HPLC Method degree than other degradation products. Such as soluble 45 Adalimumab, SEC analysis: Sephadex 200 column (Pharma aggregates and chemically modified species that are evalu cia Cat. No. 175175-01). Mobile phase 20 mM sodium phos ated and quantified as part of product characterization and phate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow quality assurance programs (Carpenter, JF et al. Commen rate, ambient temperature, detection UV 214 nm and 280 nm. tary: Overlooking subvisible particles in therapeutic protein Each sample was diluted to 1.0 mg/mL with Milli-Q water, products: baps that may compromise product quality. J. 50 sample injection load 50 ug (duplicate injection). Pharm. Sci., 2008). As demonstrated in the examples listed Adalimumab Characterization. Upon Freeze/Thaw Cycling below, a number of antibodies were surprisingly stable— Table 62 describes Adalimumab purity during the freeze/ especially with regard to subvisible particle formation— thaw experiments. For sample composition, refer to examples when formulated in the formulation of invention. 21 and 22. TABLE 62 Freezethaw - Low ionic Adalimumab - 50 mg/mL cycle LI 50/01 LISO/O2 LI50/03 LI50/04 LI50/05 LI50/06 LI50/07 LI 50/08 Fraction monomere 96

O 99.6OS 99.629 99.632 99.626 99.619 99.626 99.653 99.6SS 1 99.743 99.768 99.739 99.759 99.731 99.725 99.686 99.669 2 99.715 99.726 99.571 99.661 99.721 99.721 99.6O1 99.619 4 99.668 99.689 99.632 99.678 99.523 99.724 99.491 99.542 5 99.627 99.771 99.539 99.772 99.525 99.773 99.357 99.445 US 8,883,146 B2 113 114 TABLE 62-continued Fraction aggregate %

O O. 106 O.104 O. 119 O.136 O.139 O.145 O.158 O.159 1 O. 149 O.134 O.153 O.1SO O. 149 O.166 O.2O6 O.2O1 2 O.192 O.178 O.32O O.242 O.178 O.184 O.319 O.261 4 O.213 O.185 O.239 O.187 0.357 O.170 O.393 O.353 5 O.301 O.151 O.384 O.1SO O.398 O.1SO O.S68 O484 Fraction fragmente (%)

O O.289 O.267 O.249 O.238 O.242 O.229 O. 189 O.186 1 O.108 O.098 O.108 O.091 O. 120 O.108 O.107 O.130 2 O.093 O.097 O.108 O.097 O.100 O.O94 O.08O O. 119 4 O. 119 O.126 O.130 O.13S O. 120 O. 106 O. 116 O.1OS 5 O.O72 O.078 O.078 O.078 0.077 0.077 0.075 O.O71 Freezethaw - Low ionic Adalimumab - 200 mg/mL cycle LI 200/01 LI 200/02 LI 200/03 LI 200/04 LI 200/05 LI 200/06 LI 200/07 LI 200/08 Fraction monomere 96 O 99.294 99.296 99.348 99.333 99.313 99.349 99.32O 99.290 1 99.286 99.267 99.259 99.256 99.120 99.254 98.999 99.126 2 99.305 99.311 99.249 99.214 99.296 99.288 99.149 99.128 4 99.303 99.272 99.261 99.301 99.296 99.283 99.004 99.061 5 99.32O 99.322 99.330 99.331 99.327 99.333 98.939 98.949 Fraction aggregate %

O O.489 OSO9 O.491 O484 O492 O488 0.515 O.575 1 O.S90 O.S74 O.S84 O.S86 O.68O O.S82 0.785 O.718 2 O. 604 O604 O. 616 O.630 O.6O7 O607 O.731 O.736 4 O.S91 O.S92 O.612 O.S81 O. 604 O.S96 O.868 O.836 5 O.S93 O.S86 O.S83 O.S96 0.597 O.S89 O.98S O.981 Fraction fragmente (%)

O O.218 O.196 O. 161 O.183 O.195 O.163 O.16S O.13S 1 O.124 O.159 0.157 O.159 O.200 O.164 O.216 0.157 2 O.091 O.O85 O.13S O.156 O.097 O.1OS O. 120 O.136 4 O. 106 O.136 O.127 O.118 O.100 O.121 O.128 O.103 5 O.087 O.092 O.087 O.O73 0.075 O.078 O.O76 O.O70

Freezethaw - Adalimumab Commercial and in water from example A, from example A, Standard, Standard, cycle low conc. high conc. 50 mg/mL 200 mg/mL Fraction monomer 96 O 99.733 99.286 99.374 99.227 1 99.689 99.212 99.375 99.215 2 99.614 99.130 99.370 99.218 4 99.489 99.029 99.361 99.196 5 99.430 98.945 99.362 99.177 Fraction aggregates 96

O O.186 O.63S O.358 O.SO2 1 O.226 O.7O6 O.359 O.S16 2 O.304 O.78O O.364 O.S13 4 O428 O.888 0.372 0.535 5 O.485 O.971 0.373 0.553 Fraction fragments (9%)

O O.08O O.O79 O.268 O.272 1 O.O85 O.083 O.266 O-269 2 O.O82 O.O90 O.266 O-269 4 O.083 O.083 O.267 O.270 5 O.O85 O.O85 O.26S O-269

Conclusion freeze? thaw cycles, with minimal amounts of aggregate or The above example provides an experiment where Adali- 60 fragments as cycles continued. mumab DF/UF processed into water (Sterilized water for Example 26 injection Ph.Eur./USP) and formulated with various non ionic excipients was subjected to freeze? thaw cycling. Data Freeze/Thaw Stability of Low-Ionic 1D4.7 Solutions obtained (described in Table 62) indicates favorable overall as stability of the protein in all formulations tested. All formu- 1D4.7 protein (an anti-IL 12/anti-IL 23 IgG1) was formu lations contained above 98.5% monomeric species after 5 lated in water by dialysis (using slide-a-lyZer cassettes, used US 8,883,146 B2 115 116 according to operating instructions of the manufacturer, lated in buffer systems often used in parenteral protein for Pierce, Rockford, Ill.) and was demonstrated to be stable mulations (e.g. 20 mM histidine, 20 mM glycine, 10 mM during repeated freeze/thaw (ft) processing (-80° C./25°C. phosphate, or 10 mM citrate) and even exceeded the stability water bath) at 2 mg/mL concentration, pH 6. Data were com of 13C5.5 formulations based on universal buffer (10 mM pared with data of various formulations (2 mg/mL protein, pH phosphate, 10 mM citrate) that has been combined with a 6) using buffers and excipients commonly used in parenteral variety of excipients that are commonly used in protein for protein formulation development. It was found that the sta mulation (e.g. 10 mg/mL mannitol, 10 mg/mL Sorbitol. 10 bility of 1D4.7 formulated in water exceeded the stability of mg/mL sucrose, 0.01% polysorbate 80, 20 mM NaCl, 200 1D4.7 formulated in established buffer systems (e.g. 20 mM mM NaCl). histidine, 20 mM glycine, 10 mM phosphate, or 10 mM 10 Sample preparation, experiment processing, sample pull citrate) and even exceeded the stability of 1D4.7 formulations and sample analysis was performed in the same way as out based on universal buffer (10 mM phosphate, 10 mM citrate) lined in the above examples. combined with a variety of excipients that are commonly used 13C5.5 formulated in water compared with formulations to stabilize protein formulations, e.g. 10 mg/mL mannitol, 10 listed above mg/mL sorbitol, 10 mg/mL sucrose, 0.01% polysorbate 80, or 15 4 freeze/thaw cycles applied 20 mM. NaCl. 30 mL PETG repository SEC, DLS and particle counting analysis were applied to 2 mg/mL, pH 6 monitor protein stability, and particle counting was per sampling at T0, T1, T2, T3, and T4 formed using a particle counting system with a 1-200 um analytics: visual inspection, SEC, DLS, subvisible particle measurement range (particle counter Model Syringe, Markus measurement Klotz GmbH, Bad Liebenzell, Germany). Experiment details FIG.34 shows 13C5.5 stability during repeated fit cycling are as follows: (-80° C./25° C.), mirrored by formation of subvisible par 1D4.7 formulated in water compared with formulations ticles >10 um. 13C5.5 was formulated in either 10 mM phos listed above phate buffer, 10 mM citrate buffer, 20 mM glycine buffer, and 4 freeze/thaw cycles applied 25 20 mM histidine buffer. 13C5.5 was also formulated in the 30 mL PETG repository, about 20 mL fill, 2 mg/mL pro formulation of invention (by dialysis) with no excipients tein, pH 6 added at all. Water for injection was also subjected to fit sampling at T0, T1 (i.e. after one fit step), T2, T3, and T4 cycling and Subvisible particle testing to evaluate a potential analytics: visual inspection, SEC, DLS, subvisible particle impact of material handling, fit, and sample pull on particle measurement 30 load (referred to as blank). FIG.33 shows 1D4.7 stability during repeated fit cycling The stability of 13C5.5 formulated in water upon f7t (-80° C./25° C.), mirrored by formation of subvisible par exceeded the stability of 13C5.5 solutions formulated in buff ticles >1L m. 1D4.7 was formulated in universal buffer (10 ers typically used in protein formulations. No instabilities of mM citrate, 10 mM phosphate) and then the following excipi 13C5.5 solutions formulated in water have been observed ent variations were tested: sorbitol (10 mg/mL), mannitol (10 35 with other analytical methodologies applied (e.g. SEC, visual mg/mL), sucrose (10 mg/mL), NaCl (100 mM), and polysor inspection, etc.) bate 80 (0.01%). 1D4.7 was also formulated in water (by FIG.35 shows 13C5.5 stability during repeated fit cycling dialysis) with no excipients added at all (“water in FIG. 33). (-80° C./25° C.), mirrored by formation of subvisible par Water for injection was also subjected to fit cycling and ticles >1 um. 13C5.5 was formulated in universal buffer (10 Subvisible particle testing to evaluate a potential impact of 40 mM citrate, 10 mM phosphate) and in universal buffer com material handling, fit, and sample pull on particle load. bined with the following excipient variations were tested: The stability of 1D4.7 formulated in water upon f7t sorbitol (10 mg/mL), mannitol (10 mg/mL), sucrose (10 exceeded the stability of 1D4.7 solutions formulated with mg/mL), NaCl (200 mM), NaCl (20 mM) and polysorbate 80 excipients typically used in protein formulations. Mannitol, (0.01%). 13C5.5 was also formulated in water (by dialysis) Sucrose, and Sorbitol are known to act as lyoprotectant and/or 45 with no excipients added at all for comparison (pure water). cryoprotectant, and polysorbate 80 is a non-ionic excipient Water for injection was also subjected to fit cycling and prevalently known to increase physical stability of proteins Subvisible particle testing to evaluate a potential impact of upon exposure to hydrophobic-hydrophilic interfaces such as material handling, fit, and sample pull on particle load. air-water and ice-water, respectively. The stability of 13C5.5 formulated in water upon f7t In Summary, 1D4.7 solutions formulated in water appeared 50 exceeded the stability of 13C5.5 solutions formulated with to be surprisingly stable when analyzed with various analyti excipients typically used in protein formulations. Mannitol, cal methodologies typically applied to monitor stability of Sucrose, and Sorbitol are known to act as lyoprotectant and/or pharmaceutical proteins upon freeze-thaw processing (e.g. cryoprotectant, and polysorbate 80 is a non-ionic excipient SEC, visual inspection, dynamic light scattering, and espe prevalently known to increase physical stability of proteins cially light obscuration). 55 upon exposure to hydrophobic-hydrophilic interfaces such as air-water and ice-water, respectively. The low number of sub Example 27 visible particles in 13C5.5 samples formulated into the for mulation of invention was found to be at Surprisingly low Freeze/Thaw Stability of Low-Ionic 13C5.5 levels, demonstrating the high safety and stability potential of Solutions 60 Such formulations. No instabilities of 13C5.5 solutions formulated in water 13C5.5 (an anti IL-13 IgG1) formulated in water was dem have been observed with other analytical methodologies onstrated to be stable during repeated freeze? thaw processing applied, (e.g. SEC, visual inspection, etc.). (-80°C./25°C. water bath) at 2 mg/mL concentration, pH 6. DLS analysis of 13C5.5 solutions after fit procedures was Data were compared with other formulations (2 mg/mL pro 65 performed as described above. Results from the DLS analysis tein, pH 6), and it was found that the stability of 13C5.5 showed that an 13C5.5 Solution with 0.01% Tween-80 con formulated in water exceeded the stability of 13C5.5 formu tained significant high molecular weight (HMW) aggregate US 8,883,146 B2 117 118 forms after only 1 fit step, whereas 13C5.5 in water contained anhydrous sodium sulfate were dissolved in approx. no HMW aggregate forms, even after 3 ft steps applied. 3300 mL Milli-Q water, the pH was adjusted to 7.0 using In summary, 13C5.5 solutions formulated in water 1Mphosphoric acid, filled to a volume of 3500 mL with appeared to be surprisingly stable when analyzed with vari Milli-Q water, and the solution was filtered through a ous analytical methodologies typically applied to monitor membrane filter). SEC column, TSK gel G3000SW (cat. stability of pharmaceutical proteins upon freeze-thaw pro no. 08541) 7.8 mmx30 cm, 5um combined with a TSK cessing (e.g. SEC, visual inspection, dynamic light scatter gel guard (cat. no. 08543) 6.0 mmx4.0 cm, 7 um. Flow ing, and especially light obscuration). 0.3 mL/min, injection volume 20LL (equivalent to 20 Jug sample), column temperature room temperature, Example 28 10 autosampler temp. 2 to 8° C., run time 50 minutes, gradient isocratic, piston rinsing with 10% isopropyl Freeze/Thaw Stability of Low-Ionic 7C6 Solutions alcohol, detection via UV absorbance, diode array detector: wavelength 214 nm, peak width:0.1 min, band 7C6 (an anti amyloid beta IgG1) formulated in water was width:8 nm, reference wavelength 360 nm, band width demonstrated to be stable during repeated freeze/thaw pro 15 100 nm cessing (-80° C./30°C. water bath) at 2 mg/mL concentra FIG. 36 shows 7C6 stability during repeated fit cycling tion, pH 6. Data were compared with other formulations (2 (-80° C./25° C.), mirrored by formation of subvisible par mg/mL protein, pH 6), and it was found that the stability of ticles >1 um. The stability of7C6 formulated in water uponft 7C6 formulated in water exceeded the stability of 7C6 for formany formulations exceeded the stability of 7C6 solutions mulated in buffer systems often used in parenteral protein formulated in buffers typically used in protein formulations. formulations and even exceeded the stability of 7C6 formu No instabilities of 7C6 solutions formulated in water have lations based on universal buffer (10 mM phosphate, 10 mM been observed with other analytical methodologies applied citrate) that has been combined with a variety of excipients (e.g. SEC, visual inspection, dynamic light scattering) that are commonly used in protein formulation. Surprisingly, the stability of 7C6 formulated in water upon The following solution compositions were evaluated for fit exceeded the stability of 7C6 solutions formulated with their potential to maintain 7C6 physical stability during 25 excipients typically used in protein formulations. Mannitol. freeze/thaw experiments: sucrose, and sorbitol are known to act as lyoprotectant and/or Phosphate buffer, 15 mM cryoprotectant, and polysorbate 80 is a non-ionic excipient Citrate buffer, 15 mM prevalently known to increase physical stability of proteins Succinate buffer, 15 mM upon exposure to hydrophobic-hydrophilic interfaces such as Histidine buffer, 15 mM 30 air-water and ice-water, respectively. The low number of sub Arginine buffer, 15 mM visible particles in 7C6 samples formulated into the formu Low ionic protein formulation, no excipients added lation of invention was found to be at Surprisingly low levels, Universal buffer, sorbitol (10 mg/mL) demonstrating the high safety and stability potential of Such Universal buffer, mannitol (10 mg/mL) formulations. Universal buffer, sucrose (10 mg/mL) In summary,7C6 solutions formulated in water appeared to Universal buffer, trehalose (10 mg/mL) 35 be surprisingly stable when analyzed with various analytical Universal buffer, 0.01% (w/w) polysorbate 80 methodologies typically applied to monitor stability of phar Sample preparation, experiment processing, sample pull maceutical proteins upon freeze-thaw processing (e.g. SEC. and sample analysis was performed in very similar way as visual inspection, dynamic light scattering, and especially outlined in Examples 26 and 27. light obscuration). 7C6 formulated in water compared with formulations 40 listed above Example 29 4 freeze/thaw cycles applied 30 mL PETG repository, approx. 20 mL fill Preparation of Jó95 Formulated in Water and 2 mg/mL, pH 6 Stability Studies Thereof sampling at T0, T1, T2, T3, and T4 Analytics: AB-antibody stability was assessed by the fol 45 lowing methods: Materials and Methods Visual inspection of the protein solution was performed in 427.1 g (80 mg/mL) of Jó95 were diluted to 40 mg/mL and polypropylene round-bottom tubes wherein the samples diafiltered using purified water. After a 5-fold volume were filled for light obscuration measurements. It was exchange with purified water (theoretical excipients reduc carefully inspected against both a black and a white 50 tion, 99.3%), the protein solution was ultrafiltered to target background for signs indicating protein physical insta concentration of 100 mg/mL. pH, osmolality, density, Visual bility such as haziness, turbidity and particle formation. inspection and protein concentration measurements (OD280) Dynamic light scattering (eZetasizer Nano ZS, Malvern were performed to monitor the status of the protein after Instruments, AI9494; equipped with Hellma precision DF/UF processing. cells, suprasil quartz, type 105.251-QS, light path 3 mm, 55 After DF/UF processing, the protein solution was sterile center Z8.5 mm, at least 60 uL sample fill, protein filtered (0.22 Lum Sterivex GV membrane filter) into a 60 mL sample remaining from light obscuration measurements PETG bottle (Nalgene) and subsequently storedat-80°C. for in PP round-bottom tubes were used for DLS measure 3 months. ments). Automated measurements (1 measurement per sample) were performed. After thawing at 37° C., the solution was sterile filtered Light obscuration analysis. 3.5 mL of sample were filled in 60 (0.22 um Sterivex GV membrane filter) and filled into sterile 5 mL round-bottom tube under laminar air flow condi BD Hypak Physiolis SCFTM1 mL long syringes 29G, /2 inch, tions, measurement was performed in n=3 mode (0.8 mL 5-bevel, RNSTPE and closed with sterile BD Hypak SCFTM per single measurement) after an initial 0.8 mL rinse. 1 ml W4023/50 Flur Dalkyo stoppers. The filling volume was Size-exclusion chromatography, combined with UV24/ 1.000 mL per syringe. UVso and multi-angle light scattering. Mobile phase: 65 After filling, the syringes were stored at 2-8°C. and 40°C., 100 mM Na2HPO4/200 mM NaSO, pH 7.0 (49.68 g respectively, and analyzed as indicated in the sample pull anhydrous disodium hydrogen phosphate and 99.44 g scheme depicted below. US 8,883,146 B2 119 120 J695 Drug Substance (extinction coefficient at 280 nm: e—absorption coefficient 1.42 mL/mg cm): Drug Substance, pH 6.0, did not con c—concentration tain polysorbate 80. Sartorius Sartocon Slice diafiltration system, equipped d—length of cuvette that the light has to pass with Ultrasert PES membrane cassettes (50 kDa cutoff). E—absorbance The Sartocon Slice system was operated in continuous Io initial light intensity mode at ambient temperature according to Sartorius I—light intensity after passing through sample Operating Instructions. pH electrodes PerkinElmer UV/Vis spectrophotometer, Lambda 25, was & 695 = 1.42 used for protein concentration measurements (280 nm 10 wavelength). Disposable UV cuvettes, 1.5 mL, semi Sample Pull Scheme micro, were used for the concentration measurements. Samples of the prepared solutions were stored at the tem 0.22 um filtered purified water was used as DF/UF medium. peratures listed below and pulled (x) at the indicated time 15 points after study start (Table 63). Test parameters and meth Anton Paar Density Meter DMA 4100 was used for density ods are described in Table 64. measurements A Knauer Osmometer Type ML, was used for osmolality measurements (calibrated with 400 mOsmol/kg NaCl TABLE 63 calibration solution, Art. No. Y 1241, Herbert Knauer GmbH, Berlin, Germany). Temp. TO 1m 3m 6m Analytical Methods 50 C. X X X X J695, SEC analysis: Superdex 200 column (Pharmacia). 40° C. X X X Mobile phase 92 mM di-sodium hydrogen phosphate, 211 mM sodium sulfate, pH 7.0, 0.75 mL/min flow rate, ambient temperature, detection UV 214 nm. Each sample was diluted to 2.0 mg/mL with mobile phase, 25 TABLE 64 sample injection load 20 Jug (duplicate injection). J695, IEC analysis: Dionex, Propac WCX-10 column Test parameter Test method along with a corresponding guard column Separation Appearance Visual inspection conditions: mobile phase A: 20 mM di-sodium hydrogen Visible particles analogous DAC (EA 4.43) phosphate and 20 mM sodium acetate, pH 7.0; mobile 30 Sub-visible particles analogous phase B 20 mM di-sodium hydrogen phosphate, 400 Ph. Eur?USPEA 444 mM Sodium chloride, pH 5.0. 1.0 mL/min flow rate, Clarity Ph. Eur. (EA 4.42) ambient temperature. Each sample was diluted to 1.0 Color (visual) Ph. Eur. (EA 4.50) mg/mL with Milli-Q water, sample injection load 100 ug pH Ph. Eur. (EA 4.24) (duplicate injection). Size exclusion HPLC See above 35 Cation exchange HPLC See above J695, SDS-PAGE analysis: Novex acryl amide slab gels SDS-PAGE See above (8-16% for non-reducing conditions, 12% for reducing PCS See above conditions, Invitrogen), Coomassie Staining (Invitro gen). Separation under reducing (B-mercaptoethanol) and non-reducing conditions using Tris-Glycine buffer DF/UF Processing of Jó95 made of 10x stock Solution (Invitrogen). 40 Table 65 provides the JG95 status after diafiltration. J695, quantitation of buffer components: Mannitol: separation per ReproGel Ca column (Dr. Maisch, Germany) and RI detection, mobile phase: TABLE 65 deionized water, 0.6 mL/min flow rate, 20 uL sample Protein injection. Quantitation was performed using external Concentration Osmolality calibration standard curve. 45 Sample mg/mL pH mosmol/kg Visual Inspection Histidine and Methionine: fluorescence labelling of the after DFAUF 107 6.4 10 Slightly opalescent, amino acids with OPA (ortho-phthalic aldehyde) and slightly yellow essen HPLC separation per ReproSil ODS-3 column (Dr. tially Maisch, Germany) and fluorescence detection at 420 free from visible par nm (extinction at 330 nm), mobile phase A: 70% citric 50 ticles acid (10.51 g/L) buffer, pH 6.5, 30% methanol, mobile phase B: methanol, 1.0 mL/min flow rate, 20 LL sample injection. Quantitation was performed After DF/UF the concentrations of the originating buffer using external calibration standard curve. components were quantitatively monitored to assess the DF J695, PCS analysis: was performed undiluted at 100 effectiveness. All results were found to be below the practical mg/mL in single-use plastic cuvettes at 25°C. using a A 55 detection limits (see Table 66) of the corresponding analytical Malvern Instruments Zetasizer nano ZS at 173° angle assuming solution viscosity of 4.3875 mPas, refractive methods (HPLC with RI for Mannitol and fluorescence detec index of the protein of 1.450 and refractive index of the tion for the methionine and histidine after OPA labeling, buffer solution of 1.335. The averaged results of 20 respectively). scans, 20 seconds each, are reported. 60 Calculation of the Protein Concentration TABLE 66 Calculation Formula: Methionine Histidine Mannitol Sample mg/mL mg/mL) mg/mL before DFUF O.669 O.S86 18.36 65 after DFUF <0.13 <0.14 <3.20