WO2013096791A1

WO2013096791A1 - Process for making high concentration protein formulations

Info

Publication number: WO2013096791A1
Application number: PCT/US2012/071284
Authority: WO
Inventors: Jenny Hsiung; Steven J. Shire; Jun Liu; Fredric John LIM; Sreedhara Alavattam
Original assignee: Genentech, Inc.; F. Hoffmann-La Roche Ag
Priority date: 2011-12-23
Filing date: 2012-12-21
Publication date: 2013-06-27
Also published as: AR089434A1

Abstract

The invention provides a process of making liquid protein formulations of high concentration even under conditions of high viscosity that might make TFF processing prohibitive. More specifically, the invention provides for a process of creating protein drug product formulations having a concentration of about 50 - 700 mg/ml, and/or a viscosity of at least 20 cP. Moreover, the invention provides an alternative manufacturing process whereby the protein formulations (e.g., polypeptide or antibody) are prepared using a bulk lyophilization and reconstitution process (BLRP). Alternatively, the invention provides for an improved process for making liquid protein formulations, which may be stored avoiding the expense and inconvenience of freezing and maintaining at -20° C. Specifically, the invention provides an alternate method to store bulk freeze dried drug substance having a concentration of around 2-200 mg/mL at temperatures greater than -20°C, more specifically, 15-25°C or 2-8°C.

Description

PROCESS FOR MAKING HIGH CONCENTRATION PROTEIN

FORMULATIONS

RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application Number 61/579,844 filed 23 December 2011, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for creating high concentration protein formulations. BACKGROUND

Current manufacturing techniques for liquid protein formulations use tangential flow filtration ("TFF"). TFF techniques are desirable to create protein formulations in that the final filtration step can also include the final excipients so that the resulting filtrate is in a format ready to use without further formulation or reconstitution steps.

However, the solution viscosity limits the application of TFF for high viscosity protein formulations. Higher viscosity is a problem because, among other things, it increases backpressure and decreases flux across the membrane. Thus, there exists a strong need to for an improved process for creating high concentration liquid protein formulations.

One solution to the manufacturing problem posed by high viscosity of high protein concentration formulations is to lower the viscosity by elevating the temperature during the TFF process to above 30°C (See US 2007/0237762 to Winter). However, not all proteins retain stability and the desired therapeutic properties at elevated temperatures. Alternatively, another disadvantage of the TFF process is that long-term storage can be cumbersome and expensive in that the drug substance must typically be frozen and maintained at sub zero temperatures (e.g., - 20° C) and in bulky stainless steel tanks. Further, the filling of partial lots can result in multiple freeze/thaw cycles, which can negatively affect stability.

Alternatively, when high concentration protein formulations are desired and viscosity is not a limitation, there are also applications where devices can be employed which are suitable to deliver very viscous formulations. When protein formulations of very high concentrations are desired (e.g., > 300 mg/ml), it may not be possible to formulate with excipients to lower the viscosity sufficiently to allow for TFF processing. Thus, there still remains a need for an improved process for creating high concentration protein formulations both when the protein drug product formulation has high viscosity as well as when there are limitations for extended storage (e.g., temperature, cost, space) of the drug substance is desired.

SUMMARY

The invention provides a process of creating liquid protein formulations of high concentration even under conditions of high viscosity that might make TFF processing prohibitive. More specifically, the invention provides for a process of creating protein drug product formulations having a concentration of about 50 - 700 mg/ml, and/or a viscosity of at least 20 cP at the TFF processing temperature while preserving product quality attributes (e.g., potency, purity, stability, SEC, IEC profiles).

Moreover, the invention provides an alternative manufacturing process whereby the protein formulations (e.g., polypeptide or antibody) are prepared using a bulk lyophilization and reconstitution process (BLRP). In summary, a bulk formulation (i.e., drug substance) is bulk freeze-dried followed by resconstitution at a lower volume, resulting in a high concentration liquid that can be filed into the into final container/closure systems. This process is an improvement over conventional TFF processes, and allows higher final product concentrations because it avoids the high viscosity and stability restrictions of the TFF/UF/DF process.

Alternatively, the invention provides for an improved process for making high

concentration liquid protein formulations, which are processed and stored at temperatures above -20° C, including a method to store bulk freeze dried drug substance formulations at

temperatures greater than 0°C. Such bulk drug substance formulations can be reconstituted to create drug product formulations having concentrations of between 2-700 mg/ml.

In one embodiment, the invention relates to a process for making a high concentration protein formulation comprising the steps of:

Preparing a protein drug substance in a formulation of optimal molar ratios of desired excipients and purity, optionally using TFF and/or UF/DF,

Bulk lyophilizing the formulation of (a),

Optionally storing the formulation of (b),

Reconstituting the lyophilized formulation of (b) or (c) to create a stable drug product formulation having a viscosity of at least 20 cP.

In one aspect, the viscosity of the drug product formulation is between 20 cP to 47,400 cP, alternatively 20 to 40000 cP, 20 to 20000 cP, 20 to 10000 cP, 20 to 1000 cP, 20 to 600 cP, 20 to 400 cP, 20 to 200 cP, 20 to 150 cP, 20 to 140 cP, 20 to 140 cP, 20 to 130 cP, 20 to 120 cP, 20 to 110 cP, 20 to 100 cP. In another aspect, the viscosity is about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 cP. In a further aspect, the concentration of protein in the drug product formulation is at least about 50 mg/ml. In a further aspect, when the protein is a full length antibody, the concentration of protein in the drug substance formulation is at least about 2-200 mg/ml, alternatively, 4-200 mg/ml, 10-200 mg/ml, 20-200, 30-150, 40-150, 45-150, 50-150) and is reconstituted to achieve a drug product formulation that is concentrated 2-20 times, alternatively 2-6 times. In a further aspect, when the protein is a polypeptide or an antibody fragment, higher concentrations (e.g., 50-700 mg/ml) of drug substance may be used. In a further aspect, the drug product is concentrated 5 times. In further aspects, the drug product is concentrated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In a further aspect, the temperature of step (c) is above -20°C. In a further aspect the temperature is between 2-25°C, alternatively 2-8° C, 8-15°C, 15-25°C, -20-0°C, 0-10°C, 1-9°C, 3-7°C, 4-6°C. 16-24°C, 17-23°C, 18-22°C, 19-2FC. In a further aspect, the temperature is -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -FC, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C. In a further aspect, step (a) is prepared using a TFF, and/or UF/DF process. In yet another aspect the drug product formulation is isotonic. In yet another aspect, the drug product formulation is hypertonic. In yet another aspect, the drug product formulation is hypotonic.

In yet another aspect, the protein is an antibody. In yet a further aspect, the antibody is monoclonal. In yet another aspect the concentration of the drug product is about 50-400 mg/ml, including 60-375, 70-350, 75-325, 80-300, 85-275, 90-250, 95-225, 100-200, 100-300, 110-290, 120-280, 130-270, 140-260, 150-250, 160-240, 170-230, 180-220, 190-210 mg/ml. In yet another aspect, the concentration of drug product is about 170-300 mg/ml, alternatively 180-300, 190-300, 200-300. In yet a further aspect the concentration of drug product is about 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 mg/ml.

In yet another aspect, the protein is a polypeptide or antibody fragment. In yet a further aspect the concentration of drug product is about about 50-700 mg/ml. In yet a further aspect, the concentration of drug product is about 100-600 mg/ml, alternatively, 120-550, 130-500, 140- 450, 150-400, 160-375, 170-350, 180-325, 190-300, 200-275, 225-250 mg/ml. In yet a further aspect, the concentration of drug product is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700 mg/ml. In another embodiment, the invention relates to a process for making a high concentration protein formulation capable of storage at temperatures above -20°C comprising the steps of:

(a) Preparing a protein drug substance in a formulation of optimal molar ratios of desired excipients and purity,

(b) Bulk lyophilizing the formulation of (a) to create a bulk cake,

(c) Storing the bulk cake of (b),

(d) Reconstituting the lyophilized formulation of (b) or (c) to create a drug product formulation.

In another aspect, the viscosity of the drug product formulation ranges from about 20 cP to 47,400 cP, alternatively 20 to 40000 cP, 20 to 20000 cP, 20 to 10000 cP, 20 to 1000 cP, 20 to 600 cP, 20 to 400 cP, 20 to 200 cP, 20 to 150 cP, 20 to 140 cP, 20 to 140 cP, 20 to 130 cP, 20 to 120 cP, 20 to 110 cP, 20 to 100 cP. In yet another aspect, the viscosity is about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 cP. In a further aspect, the protein is a polypeptide. In a further aspect the concentration of the drug product is at least about 50 mg/ml. In a further aspect, when the protein is a full length antibody, the concentration of protein in the drug substance formulation is at least about 2-200 mg/ml, alternatively 4-200, 10-200, 20-200, 30-150, 40-150, 45-150, 50-150 mg/ml, and is reconstituted to achieve a drug product formulation that is concentrated 1 - 20 times, alternatively 2-6 times. In a further aspect, when the protein is a polypeptide or an antibody fragment, higher concentrations (e.g., 50-700 mg/ml) or drug substance may be used. In another aspect, the drug product is concentrated 5X. In further aspects, the drug product is concentrated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In a further aspect the temperature is between 2-25°C, alternatively 2-8° C, 8-15°C, 15-25°C, -20-0°C, 0-10°C, 1-9°C, 3-7°C, 4-6°C. 16-24°C, 17-23°C, 18-22°C, 19- 2FC. In a further aspect, the temperature is -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, - 2°C, -FC, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C. In yet another aspect the drug product formulation is isotonic. In yet another aspect the drug product formulation is hypertonic. In yet another aspect the drug product formulation is hypotonic. In yet another aspect, step (a) is prepared using a TFF, and/or UF/DF process. In yet a further aspect, the drug product is sterile filtered into a final container.

In yet another aspect, the protein is an antibody. In yet a further aspect, the antibody is monoclonal. In yet another aspect the concentration of drug product is about 50-400 mg/ml, including 60-375, 70-350, 75-325, 80-300, 85-275, 90-250, 95-225, 100-200, 100-300, 110-290, 120-280, 130-270, 140-260, 150-250, 160-240, 170-230, 180-220, 190-210 mg/ml. In yet another aspect, the concentration of drug product is about 170-300 mg/ml, alternatively 180-300, 190-300, 200-300. In yet a further aspect the concentration of drug product is about 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 mg/ml.

In yet another aspect, the protein is a polypeptide or antibody fragment. In yet a further aspect the concentration of drug product is about about 50-700 mg/ml. In yet a further aspect, the concentration of drug product is about 100-600 mg/ml, alternatively, 120-550, 130-500, 140- 450, 150-400, 160-375, 170-350, 180-325, 190-300, 200-275, 225-250 mg/ml. In yet a further aspect, the concentration of drug product is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700 mg/ml.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A is a schematic of the bulk lyophilization process. Figure IB is a flow chart of a comparison between the bulk lyophilization process and stability testing.

Figure 2 depicts a cartoon representation of one embodiment of the process of product recovery from resealable bulk trays. Figure 2A shows a lyophilized drug substance [20] inside a resealable bulk lyo tray container [10], further comprising a vessel wall [12], a lid [14], opening

[16] and cap [18]. Figure 2B shows the container inverted to receive reconstitution fluid [26] to create an intermediate drug substance mixture [30]. Figure 3B shows a full reconstituted drug product [40] being removed from the bulk tray [10] and poured into a container [46] to create recovered drug product [50]. Figure 2D shows the bulk tray [10] with unrecovered drug product

[60].

Figure 3 is a plot of product recovery vs. viscosity after lyophilization in Lyo-Guard

Trays. Figures 4A-1 to 4A-2 are plots of SEC results for omalizumab.

Figures 4B-1 to 4B-2 are plots of SEC results for aIL17. Figures 5A-1 to 5A-2 are plots of IEC results for omalizumab.

Figures 5B-1 to 5B-2 are plots of IEC results for aIL17. Figures 6A-6B are plots of HIC results for omalizumab.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. DEFINITIONS

By "protein" is meant a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. Thus, proteins are distinguished from "peptides" which are also amino acid sequence - based molecules that do not have such structure. Typically, a protein for use herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. As used herein, "polypeptide" is a single chain amino acid sequence with some form of secondary and/or teriary structure. As intended herein, the term "protein" includes both "polypeptide," "antibody," and "antibody fragments."

"Ultrafiltration " ("UF") refers to the use of a semi-permeable membrane of very small (0.001 and 0.1 μιη) pores to sort or concentrate molecules in a feed stream using a pressure differential across the membrane. UF membranes are sometimes classified on the basis of molecular weight cut-off (MWCO) rather than pore size.

"Tangential flow filtration " ("TFF")" or "cross flow filtration" refers to filtration in which the feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir. It is often used instead of "Direct Flow Filtration," ("DFF"), wherein the feedstream is applied perpendicular to the membrane, and in which in compression of mixture at the membrane occurs causing insufficient separation.

"Diafiltration," "(DF") refers to a filtration or fractionation process that washes smaller molecules through a semi-permeable membrane and leaves larger molecules in the retentate without changing the final concentration. It can be performed either continuously or discontinuously.

In continuous diafiltration, the DF solution is added to the sample feed reservoir at the same rate as filtrate is generated. The result is that while the volume in the sample reservoir remains constant, the smaller molecules, which permeate the membrane are washed away. DF processes sometimes use DF volumes, which refers to the volume of the solution before the DF solution is added.

In discontinuous diafiltration, the DF solution is first diluted to increase the initial volume and then concentrated as the filtrate is generated. The process is then repeated until the desired concentration of small molecules remaining in the sample reservoir is achieved.

Discontinuous diafiltration requires more filtrate volume to achieve the same degree or small molecule permeate reduction as continuous diafiltration.

"Viscosity, "broadly defined, is the measure of a fluid's resistance to flow. Multiple methodologies and terms can be used to characterize viscosity. As used herein, unless otherwise indicated, the viscosity means "absolute viscosity." As used herein, unless stated otherwise, the temperature at which the viscosities were measured is 20°C. One example instrument for use to determine the actual viscosities is an Anton Paar Physica MCR 501 rheometer (with the cone and plate fixture).

Absolute viscosity, sometime also known as "dynamic viscosity" or "the coefficient of viscosity " is the form most often employed to describe fluids. Generally, absolute viscosity is is a measure of a fluid's internal resistance. More precisely, absolute viscosity is the tangential force per unit area of two parallel plans at unit distance apart when the space between them is filled with a fluid and one plane moves with unit velocity in its one plane relative to the other. It is expressed using the units of centipoise (cP) or milliPascal seconds (mPaS).

Kinematic viscosity is the measure of a fluid's resistance to flow under the influence of gravity. Kinematic viscosity is commonly measured in stokes (St) or centistokes (cSt). When determined using glass capillary viscometry, units may be expressed in mm 2 /s, wherein 1 mm 2 /s = 1 cSt. Kinematic viscosity of a fluid may be determined by dividing absolute viscosity by the density.

Bulk lyophilization and reconstitution process ("BLRP") refers to a process in which a drug substance is lyophilized and reconstituted in bulk, as opposed to lyophilization followed by reconstitution in vials.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.

Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG₂, IgG₃, IgG₄, IgAi, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophy

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of the methods for assessment of antibody purity, see, e.g., Flatman et al, J. Chromatogr. B 848:79-87 (2007).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier

"monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. "Drug substance" ("DS") means an active ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body, but does not include intermediates use in the synthesis of such ingredient.

"Drug product" ("DP") means a finished dosage form, for example, tablet, capsule, or solution, that contains a drug substance, generally, but not necessarily, in association with one or more other ingredients.

Drug "potency" is a measure of drug activity expressed by the amount required to produce a pharmacological effect of a given intensity. For example, a highly potent drug achieves a large effect at low concentration, while a drug with lower potency achieves a small effect at low concentration. Potency can also be expressed as a combined measure of affinity and efficacy. Affinity refers to the ability of a drug to bind to a target molecule, or interfere with the target binding to a natural binding partner. Efficacy is the relationship of target occupancy of the drug and the pharmacological effect at the molecular, cellular, tissue or system level.

The potency of a therapeutic anti-IgE antibody may be determined by measuring the ability of the therapeutic anti-IgE antibody to bind to IgE in competition with the high affinity receptor (FcsRI) as compared to a reference control. Typical assay methods include

immunoassays, such as ELISA, ECLA, and the like that include a capture agent bound to an assay surface to capture and immobilize the desired target molecule. Captured target molecules are detected with a detection agent that binds the target molecule and provides a detection label for quantification.

In some embodiments, the potency of a therapeutic anti-IgE antibody is determined by an inhibition ELISA as shown in Figure 3 of WO2011/056606. Increasing concentrations of an anti-IgE antibody antibody is incubated with labeled IgE. The mixture is added to a plate containing an immobilized FcsRIa polypeptide as a capture agent. The anti-IgE antibody that binds labeled IgE effectively inhibits the binding of the labeled IgE to the capture agent, reducing the detectable signal. Thus, an anti-IgE potency of the sample is inversely correlated with the signal detected.

A "stable" formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991), and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40° C for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8° C, generally the formulation should be stable at 30° C. or 40° C for at least 1 month and/or stable at 2-8° C for at least 2 years. Where the formulation is to be stored at 30° C, generally the formulation should be stable for at least 2 years at 30° C. and/or stable at 40° C. for at least 6 months. For example, the extent of aggregation following lyophilization and storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one wherein less than about 10% and preferably less than about 5% of the protein are present as an aggregate in the formulation. In other embodiments, any increase in aggregate formation following lyophilization and storage of the lyophilized formulation can be determined. For example, a "stable" lyophilized formulation may be one wherein the increase in aggregate in the lyophilized formulation is less than about 5% and preferably less than about 3%, when the lyophilized formulation is stored at 2-8° C (alternatively 15-30°C) for at least one year. In other

embodiments, stability of the protein formulation may be measured using a biological activity assay.

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted

formulation. The reconstituted formulation is suitable for administration (e.g. parenteral administration) to a patient to be treated with the protein of interest and, in certain embodiments of the invention, may be one which is suitable for subcutaneous administration.

An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure below that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. The formulations of the present invention are either isotonic or hypertonic as a result of the addition of salt and/or buffer. For IV

administration, the formulation may also be hypotonic.

A " lyoprotectant" is a molecule which, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage. Exemplary lyoprotectants include sugars and their corresponding sugar alchohols; an amino acid such as monosodium glutamate, histidine or arginine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g. glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronics®; and combinations thereof. Additional exemplary lyoprotectants include glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose.

Examples of reducing sugars include glucose, maltose, lactose, maltulose, iso-maltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols. Preferred sugar alcohols are monoglycosides, especially those compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glycosidic side group can be either glucosidic or galactosidic. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and iso-maltulose. Example lyoprotectants are the non-reducing sugars trehalose or sucrose and arginine salts.

The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotecting amount" which means that, following lyophilization of the protein in the presence of the lyoprotecting amount of the lyoprotectant, the protein essentially retains its physical and chemical stability and integrity upon lyophilization and storage.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject., A

pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

A "pharmaceutically acceptable acid" includes inorganic and organic acids which are non toxic at the concentration and manner in which they are formulated. For example, suitable inorganic acids include hydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric, sulfonic, sulfuric, sulfanilic, phosphoric, carbonic, etc. Suitable organic acids include straight and branched-chain alkyl, aromatic, cyclic, cyloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono, di-and tri-carboxylic, including for example, formic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, t-butyl acetic, anthranilic, propanoic, 2- hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic, cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic, benzoic, 3-(4-hydroxybenzoyl) benzoic, 2- acetoxy-benzoic, ascorbic, cinnamic, lauryl sulfuric, stearic, muconic, mandelic, succinic, embonic, fumaric, malic, maleic, hydroxymaleic, malonic, lactic, citric, tartaric, glycolic, glyconic, gluconic, pyruvic, glyoxalic, oxalic, mesylic, succinic, salicylic, phthalic, palmoic, palmeic, thiocyanic, methanesulphonic, ethanesulphonic, 1 ,2-ethanedisulfonic, 2- hydroxyethanesulfonic, benzenesulphonic, 4-chorobenzenesulfonic, napthalene-2-sulphonic, p- toluenesulphonic, camphorsulphonic, 4-methylbicyclo[2.2.2]-oct-2-ene- 1 -carboxylic, glucoheptonic, 4,4'-methylenebis-3-(hydroxy-2-ene-l-carboxylic acid), hydroxynapthoic. " ' Pharmaceutically-acceptable bases" include inorganic and organic bases were are nontoxic at the concentration and manner in which they are formulated. For example, suitable bases include those formed from inorganic base forming metals such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N- methylglucamine, morpholine, piperidine and organic nontoxic bases including, primary, secondary and tertiary amine, substituted amines, cyclic amines and basic ion exchange resins, [e.g., N(R') 4+ (where R' is independently H or C 1-4 alkyl, e.g., ammonium, Tris)], for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.

Additional pharmaceutically acceptable acids and bases useable with the present invention include those which are derived from the amino acids, for example, histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

"Pharmaceutically acceptable" buffers and salts include those derived from both acid and base addition salts of the above indicated acids and bases. Specific buffers and or salts include histidine, succinate and acetate.

A "preservative" is a compound which can be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential preservatives include

octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

A "bulking agent" is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake {e.g. facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The liquid

formulations of the present invention obtained by reconstitution of a lyophilized formulation may contain such bulking agents. As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

"Anti-IgE antibody" includes any antibody that binds specifically to IgE in a manner so as to not induce cross-linking when IgE is bound to the high affinity receptor on mast cells and basophils. Exemplary antibodies include the antibodies of the invention as well as omalizumab, rhuMabE25 (omalizumab, E25, XOLAIR®), E26, E27, as well as CGP-5101 (Hu-901) and the HA antibody. The amino acid sequences of the heavy and light chain variable domains of the humanized anti-IgE antibodies E25, E26 and E27 are disclosed, for example in U.S.P. 6,172,213 and WO99/01556. The CGP-5101 (Hu-901) antibody is described in Corne et al, (1997), J. Clin. Invest. 99(5): 879-887, WO 92/17207 and ATCC Dep. Nos. BRL-10706, BRL-11130, BPvL-11131, BPvL-11132 and BRL-11133. The HA antibody is described in USSN 60/444,229, WO2004/070011 and WO2004/070010. An anti-IgE antibody specific for the Ml ' segment of the extracellular portion of membrane form of IgE (RG7449) is disclosed in U.S. 8,071,097.

II. PROTEINS USABLE WITH THE INVENTION

Many distinct protein drug product formulations can be made using the process of the current invention. It is especially suited to make drug product formulations of high

concentration even under conditions of high viscosity that might make TFF processing prohibitive. It is also suited as a means for long term storage of bulk freeze-dried drug substances at temperatures above -20°C. Examples of drug substances which can be formulated using the current process are recited below.

In one embodiment, the invention describes an improvement in the TFF process. While alternative means exist [e.g., (i) elevated tempeatures, (ii) membrane pore size, (iii) larger pump size] may be employed to circumvent the problem of high ambient temperature viscosities using the TFF process, these alternatives create new problems in drug formulations. For example, elevated temperatures can accelerate degregation pathways (e.g., isomerization, amidation, denaturation, aggregation, etc.) resulting in decreased potency. Larger membrane sizes can decrease purity, while larger pump size requires a purification system capable of handling larger back pressures. Hence the advantage of an ambient temperature (e.g. 20°C) production of high viscosity drug products is significant.

A. Exemplary Antibodies and Polypeptides.

The process of the present invention may be employed on any antibody which has elevated viscosity when formulated at high concentrations. One example antibody is an anti-IgE antibody.

Additional antibodies which may be formulatd using the process of the present invention include: Abciximab (ReoPro®) (a-glycloprotein Ilb/IIIa), adalimumab (Humira®)(a-TNF-a), alemtuzumab (Campath®)(a-CD52), basiliximab (Simulect®, a-CD25/IL-2Ra), belimumab (Benlysta®, a-BAFF), bavacizumab (Avastin®, a-VEGF), brentuximab vedotin (Adcetris®, a- CD30, SGN-35/cAC10-vcMMAE), canakinumab (Illaris®, a-IL-Ι β), cetuximab (Erbitux®, <x- EGFR), certolizumab pegol (Cimzia®, CDP870, α-TNF-a+Peg), daclizumab (Zenapax®, a- CD25/IL-2R), denosumab (Prolia®, Xgeva®, a-RANKL), eculizumab (Soliris®, a-complement C5), efalizumab (Raptiva®, a-CDl la), gemtuxumab (Mylotarg®, a-CD33), golimumab

(Simponi®, a-TNF-a), ibritumomab tiuxetan (Zevalin, a-CD20+radioisotope), infliximab (Remicade®, a-TNF-a), ipilimumab (Yervoy®, MDX- 101 , a-CTLA-4), muromonab-CD3 (Orthoclone OKT3®), a-CD3), natalizumab (Tysabri®, a-alpha-4 integrin), ofatumumab (Arzerra®, a-CD20), palivizumab (Synagis®, a-RSV-F), panitumumab (Vectibix®, a-EGFR), ranibizumab (Lucentis®, a-VEGF Fab), rituximab (Rituxan®/MabThera®, a-CD20), tocilizumab/atlizumab (Actemra®/ RoActemra®, a-IL-6R), trastuzumab (Herceptin®, anti- ErbB2), ID1 1 (a-TGF-β), a-PSA (prostate specific antigen), AVX-205 (a-gluten Ab), CSL-324 (a-GCSF), CSL-360, CSL-362 (a-CD123/IL-3Ra), CT-01 1 (BAT-1 , a-PD-1), elotuzumab (HuLuc63; PDL-063; BMS-901608; a-CS l/CD2), epratuzumab (205923-57-5, AMG-412, IMMU-103, LymphoCide®, a-CD22), lebrikizumab (a-IL-13), MK-6105 (a-IL-13R), labetuzumab-SN-38 (IMMU-130; hMN-14; hMN-14-CL2-SN-38; hMN-14-CL2A-SN-38), mavrilimumab (CAM-3001, a-GM-CSFR), MGAH-22 (a-ErbB2), milatuzumab, milatuzumab- doxorubicin (IMMU-110, a-CD74-Dox), MOR-103 (MOR-04357, a-GMSCF), MOR-202 (MOR-03087, a-CD38), MOR-208, (XENP-5574, XENP-5603, a-CD19), PF-3446962 (a- activin receptor like kinase -1/ALK-l), RG-7356 (ARH460-16-2, RO-5429063, a-CD44), siltuximab (cCLB8, CNTO-328, a-IL6), U3-1287 (AMG-888, a-Her-3), veltuzumab (IMMU- 106, a-CD20), clivatuzumab tetraxetan-[Y-90] (IMMU-107, PAM-4, a-MUCl+radio Y90), 131 [I]-chTNT-l/B (TNT-1, Vivatuxin^®, Cotara^®, Peregrin^®, a-Histone 1), ALD-518 (BMS- 945429, a-IL6), AME-133 (LY-2469298, AME-133v, a-CD20), a-CD22, a-IL-31, bavituximab (3G4, 9D2, Tavacin®, a-phosphatidylserine), a-CD19, IST-1458 (IMC-071, MDX-1458, a- lamba myleloma antigen (LMA)), L-DOS47 (adenocarcinoma Ag + urease), LFA-102

(a-prolactin receptor (PRLR)), [11 l]In-capromab pendetide (OcoScint PR-356, ProstaScint®, CYT-356, a-PMSA), [11 l]In-imciromab pentetate (miframonab-Fab, Myoscint®), 11 lln- satumomab pendetide (Cyt- 103 -indium 111, OncoScint CR/OV3^®), 99mTc-besilesomab

(Scintimun^®, [99m]Tc-a-carcinoembryonic antigen (CEA)), 99mTc-sulesomab (ImmuRAID- MN3-Tc-99m, MyeloScan®, Leukoscan®, [99m]-a-NCA90), arcitumonab (ImmunRAID-CEA- Tc99m, IMMU-4, [Tc99m)-a-CEA], biciromab (Fibriscint®), cantumaxomab (Removab®, a- CD3/EpCAm bsp), nimotuzumab (OSAG-101, TherCIM^®, Vecthix®, BioMab®, a-EGFR), raxibacumab (ABthrax^®, anti-anthrax protective antigen), votumumab (Tc-99M-votumumab, HumaSPECT®, OncoSPECT-Y90^®), Tetagam^® (a-clostridium tenani), ustekinumab (Stelara®, CNTO-1275, a-p40 (subunit of IL-12R and IL-23R), mogamulizumab (KW-0761, Potelligent®, a-CCR-4), CGEN-243, CGEN-973A, CGEN-973B, H5N1 (iBioLaunch®, anti-H5Nl influenza), HX-02 (Humanyx®, anti-TNF-a synthesis inhibitor), IC-14, (a-CD14),

MabionCD20® (anti-CD20), motavizumab (MEDI-524, Numax^®, a-RSV), motavizumabYTE (a-RSV), MP-196 (a-IL23), PAT-LM1 (OmcoMab®, Acceptys®, a-Nono/nmt55 Ag), supraagonist monoclonal (agonist T-cell glycoprotein a-CD-28), TOL-102, APN-311 (a-GD2- GMCSF/a-GD-2), bapineuzumab (AAB-001, a-amyloid beta), briakinumab (ABT-874, J-695, a-IL-12R/IL-23R), conbercept (Fumintide®, KH-902, VEGFR-Fc), dalotuzumab (F-50035, MK-0646, h7C10, anti-IGFl-R), elotuzumab (BMS-901608, PDL-063, HuLuc63, a-CD2 (CSl glycoprotein), EMD-525797 (17E6, a-CD51), epratuzumab (AMG-412, IMMU-103,

LymphoCide®, a-CD22), farletuzamab (MORAb-003, a-folate receptor alpha), ganitumab (AMG-479, α-IGF-lR), girentuximab (WX-G250, Rencarex®, anti-G250 (tumor antigen carbonic anhydrase IX), [1241] -girentuximab (CA9-SCAN^®, REDECTANE^®), inolimonab (BT- 563, B-B10, Leukotac^®, a-CD25(IL-2Ra), inotuzumab ozogamicin (CMC-544, PF-5208773, <x- CD22-calicheamicin), ixekizumab (LY-2439821, a-IL-17), MK-3415A (MDX-066/MK- 3415/GS-CDAl + MDX-1388 (MBL-CDBl/MK-6072, a-enteroxin A and B Ag of Clostridium difficile), necitumumab (IMC-11F8, a-EGFR), oportuzumab monatox (VB4-845, Viventia^®, anti-EpCAM-Pseudomonas exotoxin, ScFv fragment), oregovomab (B43.13, ViRexx®, AltaRex®, OvaRex®, a-CA125), pagibaximab (HU96-110, BSYX-Al lO, anti-lipoteichoic acid (LTA)), racotumomab (1E10, a-idiotypic P3 protein), ramucirumab (IMC-1121B, LY-3009806, a-VEGFR2), reslizumab (CTX-55700, SCH-55700, CDP-835, DCP-835, CEP-38072,

Ception®, a-IL-5), RIGS CC49 ([125I]-CC49, Neo2-17, Neoprobe®, RIGScan CR49®), sarilumab (REGN-88, SAR-153191, a-IL-6R), secukinumab (AIN-457, a-IL17A), siltuximab (cCLB8, CNTO-328, a-IL-6R), solanezumab (LY-2062430, a-amyloid beta), itolizumab (h-Tl, T-lh-mAb, a-CD6), teplizumab (hOKT3 gamma 1 (Ala- Ala), MGA-031, a-CD3), trastuzumab emtansine (trastuzumab-DMl, trastuzumab-mertansine, a-Erbb2 + mertansine), vedolizumab (MLN-02, LDP-02, a-alph4/beta-7 integrin), zanolimumab (HuMax-CD4, MDX-CD4, a-CD4).

Additional examples of antibodies include: Anti-CD22-vcMMAE, anti-EGFL7

(RG7414), anti-FGFR3 (RG7444), anti-HER3/EGFR (RG7597), anti-Factor-D (RG7417), anti- LT-alpha (RG7416), anti-beta-7 integrin (RG7413), rontalizumab (RG7415, anti-INF-a), anti- amyloid beta, anti-oxLDL (RG7418), anti-PD-Ll, [MDX-1105], anti-PD-1 [MDX-1106, CT- 011, Merck 43745, AMP-224], lebrikizumab (a-IL13), RG4934 (a-IL-17), anti-P-selectin (RG1512), gantenerumab (a-amyloid beta), RG7412 (a-amyloid beta), ocrelizumab (RG1594, a- CD20), RG3638 (MetMab/anti-cMet), RG7159 (a-CD20), RG7160 (a-EFGR), RG7212 (a- TWEAK), RG7334 (anti-PLGF), RG7356 (anti-CD44), RG7593 (anti-CD22), RG7686 (anti- glypican).

Alternatively, the process of the present invention can be employed with polypeptides. Suitable polypeptides are: polypeptides encompassed within the definition herein include mammalian proteins, such as, e.g., growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; a- 1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor ΓΧ, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA, e.g., Activase®, TNKase®, Retevase®); bombazine; thrombin; tumor necrosis factor-a and -β; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; an integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT -4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-β, including TGF-βΙ, TGF^2, TGF^3, TGF^4, or TGF^5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;

erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -β, and -γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;

superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); a viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the above-listed polypeptides.

B. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. 1. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al, Nature 332:323-329 (1988); Queen et al, Proc. Nat 'lAcad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489- 498 (1991) (describing "resurfacing"); Dall'Acqua et al, Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68 (2005) and Klimka et al, Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al, J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. 5zo/. Chem. 271 :22611-22618 (1996)).

2. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing

VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al,

Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol., 147: 86 (1991).) Human antibodies generated via. human B-cell hyb.rido.ma technology are also described in Li et al., Proc, Natl, Acad, Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human- human hybridomas). Human hybridoma technology (Trioma technology) is also described in VoUmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and VoUmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

3. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al, Nature 348:552-554; Clackson et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and

Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol. 340(5): 1073- 1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.

Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574,

2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

4. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731 , 168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al, Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al, J. Immunol, 148(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl Acad. Sci. USA, 90:6444- 6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al, J. Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J.

Immunol 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to a specific antigen as well as another, different antigen (see, US 2008/0069820, for example). 5. Protein Variants

In certain embodiments, amino acid sequence variants of the protesin herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the protein. Amino acid sequence variants of a protein may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the protein. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding, binding partner interaction. a) Substitution, Insertion, and Deletion Variants

In certain embodiments, protein variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs, FRs and binding sites or pockets. Conservative substitutions are shown in Table 1 under the heading of

"conservative substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a protein of interest and the products screened for a desired activity, e.g., retained/improved antigen/partner binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1

Original Exemplary Preferred

Residue Substitutions Substitutions

Met (M) Leu; Phe; He Leu

Phe (F) Trp; Leu; Val; He; Ala; Tyr Tyr

Pro (P) Ala Ala

Ser (S) Thr Thr

Thr (T) Val; Ser Ser

Trp (W) Tyr; Phe Tyr

Tyr (Y) Trp; Phe; Thr; Ser Phe

Val (V) He; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of a protein that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the protein with binding partner is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.

Alternatively, or additionally, a crystal structure of an protein-binding partner complex to identify contact points. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or polypeptide with an N-terminal methionyl residue. Other insertional variants of the antibody or polypeptide molecule include the fusion to the N- or C- terminus of the molecule to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life.

b) Glycosylation variants

In certain embodiments, a protein provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to a protein may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the protein comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The

oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in a protein of the invention may be made in order to create protein variants with certain improved properties.

In one embodiment, protein variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such protein may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch.

Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function.

Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et al); and US 2005/0123546 (Umana et al). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). c) Fc region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence {e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification {e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see

Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96^® nonradioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat 'l Acad. Sci. USA

95 :652-656 (1998). C 1 q binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int 'l. Immunol.

18(12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J.

Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)), are described in

US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 31 1 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371 ,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 ; and WO 94/29351 concerning other examples of Fc region variants. d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g.,

"thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521 ,541. e) Protein Derivatives

In certain embodiments, an protein provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the protein include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol,

carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3-dioxolane, poly-l ,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either

homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)poly ethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the protein may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the protein to be improved, whether the protein derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an protein and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the

nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the protein-nonproteinaceous moiety are killed.

C. Recombinant Methods and Compositions

Antibodies and polypeptides may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding a protein described herein is provided. Such nucleic acid may encode such polypeptide or an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises {e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an protein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the protein or polypeptide, as provided above, under conditions suitable for expression of the protein or polypeptide, and optionally recovering the protein from the host cell (or host cell culture medium).

For recombinant production of a protein, nucleic acid encoding a protein, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of protein-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies or polypeptides may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody or polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for protein-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody or polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated protein are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177,

6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor

(MMT 060562); TRI cells, as described, e.g., in Mather et al, Annals N Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR^" CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255- 268 (2003).

D. Preparation of Lyophilized Formulations

Under the process of the current invention, in a particular embodiment, the proteins are lyophilized and then reconstituted to produce the reduced-viscosity stable liquid formulations of the invention. In this particular embodiment, after preparation of the protein of interest as described above, a "pre-lyophilized formulation" is produced. The amount of protein present in the pre-lyophilized formulation is determined taking into account the desired dose volumes, mode(s) of administration etc. For example, the starting concentration of an intact protein can be from about 2 mg/ml to about 100 mg/ml, specifically from about 5 mg/ml to about 40 mg/ml and most preferably from about 20-80 mg/ml. The protein to be formulated is generally present in solution. For example, in the elevated ionic strength reduced viscosity formulations of the invention, the protein may be present in a pH-buffered solution at a pH from about 4-8, and preferably from about 5-7. The buffer concentration can be from about 1 mM to about 20 mM, alternatively from about 3 mM to about 15 mM, depending, for example, on the buffer and the desired tonicity of the formulation (e.g. of the reconstituted formulation). Exemplary buffers and/or salts are those which are pharmaceutically acceptable and may be created from suitable acids, bases and salts thereof, such as those which are defined under "pharmaceutically acceptable" acids, bases or buffers.

In one embodiment, a lyoprotectant is added to the pre-lyophilized formulation. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that, upon reconstitution, the resulting formulation will be isotonic. However, hypertonic reconstituted formulations may also be suitable. In addition, the amount of lyoprotectant must not be too low such that an unacceptable amount of degradation/aggregation of the protein occurs upon lyophilization. However, exemplary lyoprotectant concentrations in the pre-lyophilized formulation are from about 10 mM to about 500 mM, alternatively from about 30 mM to about 300 mM, alternatively from about 50 mM to about 100 mM. Exemplery lyoprotectants include sugars and sugar alcohols such as sucrose, mannose, trehalose, glucose, sorbitol, mannitol, arginine and salts of the foregoing. However, under particular circumstances, certain lyoprotectants may also contribute to an increase in viscosity of the formulation. As such, care should be taken so as to select particular lyoprotectants which minimize or neutralize this effect. Additional lyoprotectants are described above under the definition of ^' " 'lyoprotectants" . The ratio of protein to lyoprotectant can vary for each particular protein or protein and lyoprotectant combination. In the case of an protein as the protein of choice and a sugar (e.g., sucrose or trehalose) as the lyoprotectant for generating an isotonic reconstituted formulation with a high protein concentration, the molar ratio of lyoprotectant to protein may be from about 10 to about 1500 moles lyoprotectant to 1 mole protein, and preferably from about 200 to about 1000 moles of lyoprotectant to 1 mole protein, for example from about 200 to about 600 moles of lyoprotectant to 1 mole protein.

In a preferred embodiment, it may be desirable to add a surfactant to the pre-lyophilized formulation. Alternatively, or in addition, the surfactant may be added to the lyophilized formulation and/or the reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g. polysorbates 20 or 80); polyoxamers (e.g. poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleylmyristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or

isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyldimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUA® series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc). The amount of surfactant added is such that it reduces particulate formation of the reconstituted protein and minimizes the formation of particulates after reconstitution. For example, the surfactant may be present in the pre-lyophilized formulation in an amount from about 0.001- 0.5%, alternatively from about 0.005-0.05%.

A mixture of the lyoprotectant (such as sucrose or trehalose) and a bulking agent (e.g. mannitol or glycine) may be used in the preparation of the pre-lyophilization formulation. The bulking agent may allow for the production of a uniform lyophilized cake without excessive pockets therein etc. Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the pre-lyophilized formulation (and/or the lyophilized formulation and/or the reconstituted formulation) provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; preservatives; co- solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium. The formulation herein may also contain more than one protein as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect the other protein. Such proteins are suitably present in combination in amounts that are effective for the purpose intended.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to, or following, lyophilization and reconstitution. Alternatively, sterility of the entire mixture may be accomplished by autoclaving the ingredients, except for protein, at about 120° C. for about 30 minutes, for example.

After the protein, optional lyoprotectant and other optional components are mixed together, the formulation is lyophilized. Many different freeze-dryers are available for this purpose such as Hull50® (Hull, USA) or GT20® (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about -30 to 25° C. (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs). Optionally, a secondary drying stage may also be performed depending upon the desired residual moisture level in the product. The temperature at which the secondary drying is carried out ranges from about 0 - 40° C, depending primarily on the type and size of container and the type of protein employed. For example, the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30° C (e.g., about 20° C). The time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours). The pressure may be the same as that employed during the primary drying step. Freeze- drying conditions can be varied depending on the formulation and bulk container size. E. Reconstitution of a Lyophilized Formulation

Prior to administration to the patient, the lyophilized formulation is reconstituted with a pharmaceutically acceptable diluent such that the protein concentration in the reconstituted formulation is at least about 50 mg/ml, for example from about 50 mg/ml to about 700 mg/ml, alternatively from about 80 mg/ml to about 300 mg/ml, alternatively from about 90 mg/ml to about 150 mg/ml. When full length antibodies are formulated using the current process, suitable results may be not be achieved with concentrations above 500 mg/ml, with optimal resuls at or below 400 mg/ml. Such high protein concentrations in the reconstituted formulation are considered to be particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower concentrations of the protein in the reconstituted formulation may be desired (for example from about 2-200 mg/ml, or from about 10-40 mg/ml protein in the reconstituted formulation). In certain embodiments, the protein concentration in the reconstituted formulation is

significantly higher than that in the pre-lyophilized formulation. For example, the protein concentration in the reconstituted formulation may be about 1-20 times, alternatively 3-10 times, alternatively 2-6 times (e. g. at least two, three, four, five or six fold, or fractions thereof) that of the pre-lyophilized formulation.

Reconstitution generally takes place at ambient temperature (15-30°C) to ensure complete hydration, although other temperatures may be employed as desired (such as 2-8°C). Elevated temperatures may be used to aid in recovery of the reconstituted material. The time required for reconstitution will depend, e.g. , on the type of diluent, amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives. The amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the protein and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, the reconstituted formulation has less than 6000 particles per vial which are < 10 μιη in size. III. EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

EXAMPLE 1

Introduction: The objective of this study is to prepare a high concentration liquid monoclonal protein formulation using a pure TFF with BLRP, and to compare the stability of the resulting drug product produced by both processes (Figure IB). This BLRP process enables the attainment of higher concentrations than conventional pure TFF procesesses because it is not subject to the constraint of high viscosity drug substance preparations.

1. Material and Methods 1.1. Monoclonal Antibodies

E25 (omalizumab, lot: Ml 1/Ml 1-RD136) stored at -70°C, and anti-IL17 Ab v.2 in 5 mM His HCl, 23mM ArgHCl, 8 mM Sucrose at pH 6.0 were used as model molecules. Both of these mAbs have the same human Fc IgGl construct and differ only in their complementarity determining regions and antigen binding specificity. 1.2 Optimization of formulation for BLRP

Several formulations with different amounts of sucrose and arginine-HCl were prepared at omalizumab (Xolair, E25) concentrations of 40 and 80 mg/mL using UF/DF technology, and are shown in Table 1. These formulations were freeze-dried using the lyo cyscle shown in Table 2. These formulations were reconstituted to final concentrations of - 120 or 200 mg/mL and final viscosities and reconstitution times are also shown in Table 1. Formulation 8 which attained a final concentration of 200 mg/mL at 50 cP was chosen for the comparative studies discussed in the following subsections. Table 1 Screening of Omalizumab formulations for Process and Stability Studies*

*Current commercial formulation for Omalizumab is formulation 4. Formulation 8 was chosen for process and

stability studies

1.3 Preparation of high-concentration liquid MAb using diafiltration/ ultrafiltration.

A high concentration liquid formulation for omalizumab was prepared on a small scale by concentration to >200mg/mL using Centriprep^® centrifugal filter units (Centriprep

Centrifugal Filter Units (Millipore/Product #4305). The resulting concentrate was then dialyzed using a Slide-A-Lyzer dialysis cassette (Thermo Scientific/Product #66830) vs. 25 mM His HCl, 115 mM ArgHCl, 40mM Sucrose, pH 6.0, and the protein concentration confirmed by UV spectrophotometry. The PS20 concentration was measured by chromatography with an evaporative light scattering detector ("ELSD"). Based on the measured concentration of PS20, the necessary amount of PS20 to achieve a final 0.05% PS20 concentration was spiked into the 200 mg/mL omalizumab preparation. The resulting viscosity was 70 cP.

a-IL17 Ab was concentrated to > 200mg/mL using Centriprep^® centrifugal filters (Centriprep^® Centrifugal Filter Units (Millipore/Product #4305) and final concentration at 200 mg/mL obtained by dilution with 25mM His HC1, 115mM ArgHCl, 40mM Sucrose, at pH 6.0, which was confirmed by UV absorption spectrophotometry. The final concentration of 0.05% PS20 was obtained by addition of PS20. The resulting viscosity was 240 cP.

1.4 Preparation of high-concentration liquid MAb Formulation using BLRP The omalizumab formulation (#8 in Table 1) at 40 mg/mL (5 mM Histidine-HCl buffer,

23 mM ArgHCl, 8 mM Sucrose, and 0.01% PS20 at pH 6) was prepared by dialysis (Spectra/Por Dialysis Membrane, 10-12 kDa MWCO, 29.0 mm) and concentration confirmed by UV absorption spectrophotometry. The a-IL17 formulation was prepared by first adding sufficient PS20 to yield a final concentration at 0.01% PS20 followed by dilution with 5mM His HC1, 23mM ArgHCl, 8mM Sucrose, 0.01% PS20, pH 6.0 buffer. The final protein concentration was confirmed by UV absorption spectrophotometry.

The prepared solutions were loaded into separate Lyoguard^® single-use autoclavable containers (W. L. Gore & Associates, Newark, DE) constructed of polypropylene and expanded polytetrafluoroethylene (ePTFE) at a fill volume of 20 mL.

1.5 Freeze-drying of Lyoguard^® Trays

The Lyoguard containers were loaded into the freeze dryer and lyophilized according to the lyophilization cycle shown in Table 2. The samples were first held at 5°C for 1 hour to equilibrate before the freezing ramp was initiated. The lyophilizer shelves were then cooled to the -50°C freezing temperature over 1.5 hours. The shelf temperature was held at -50°C for no less than 6 hours to equilibrate the temperature of the frozen samples. The chamber pressure was then reduced to 150 mTorr and shelf temperatures were warmed to 0°C over 6 hours to initiate primary drying. Drying completion was determined based on the Pirani measurement. The shelf temperature was then raised to 25°C and held for 16 hours for secondary drying. At the end of the drying step, the chamber was aerated to atmospheric pressure with sterile filtered nitrogen. Upon unloading, the trays containing freeze-dried Drug Substance (DS) were immediately transferred and sealed in separate Mylar bags and stored at 2-8°C. The average residual moisture for this freeze-dried DS was 1.0%.

Table 2. Lyophilization Cycle for omalizumab and anti-IL17 Ab

Shelf Temperature Chamber Pressure

Step Operation (°C) (mTorr) Duration (hours)

1 Sample loading 5 Ambient As required

2 5°C hold 5 Ambient 1

3 Freeze ramp 5 to -50 Ambient 1.5

4 Freeze hold -50 Ambient > 6

5 Primary drying ramp -50 to 0 150 6

6 Primary drying hold 0 150 As required*

7 Secondary drying ramp 0 to 25 150 3

8 Secondary drying hold 25 150 16

^Depending on the load volume, primary drying time may vary. When the pirani level dropped and stabilized, indicating that drying was complete, the cycle was manually advanced to the secondary drying ramp step.

Note: At the end of the lyophilization cycle, the chamber was aerated to atmospheric pressure with sterile filtered nitrogen. Upon unloading, sample trays were immediately transferred to separate Mylar bags and heat-sealed for storage.

1.6 Evaluation of High Concentration Liquid Drug Product Recovery from Tray

Omalizumab (marketed drug substance ("DS") formulated in 40 mg/mL protein, 5 mM Histidine-HCl, 85 mM Sucrose, 0.01% PS20, pH 6) was used to assess product recovery from the Lyo tray over a wide range of viscosity. 2 L of omalizumab DS was filled into a 2-L

Lyoguard® tray and lyophilized using the lyophilization cycle shown in Table 2. After lyophilization, approximately 500 mL of purified water was added to the tray from the fill port. Reconstitution mixing at room temperature was performed at 10 rpm on a wave shaker until all solids were dissolved.

A hole 1 cm in diameter was manually drilled at the bottom corner of the tray. To drain the reconstitued product into a receiving container, the tray was held vertically at a 45 degrees for 15 minutes (see Figure 2). To measure the %> recovery by weight, the tray was weighed before and after draining. This process was repeated with product diluted with formulation buffer to ascertain recovery yield as a function of viscosity. Product with viscosity between 30 and 600 cP was tested. To ensure consistency at every viscosity point, the tray was inverted gently a few times to make sure all sides were coated with product before draining. All viscosity measurements were made using an Anton Paar Physica MCR 501 rheometer with the CP50 0.5 cone and plate configuration. The CP50-0.5 geometry has a diameter of 50 mm with a cone angle of 0.490° and tip truncation at 48 μιη. Under ideal conditions, the shear rate is constant in the cone and plate system. Measurements were performed at 20°C using the temperature controller in the rheometer (Peltier plate with circulating fluid from a water bath). Approximately 300 of sample was loaded onto the plate for measurement. The instrument controls the rotation speed and thereby the shear rate which was fixed at 100 s-1. All data were collected and analyzed using the Anton Paar Rheoplus/32 v3.40 software.

1.7 Stability Testing

Each lyophilized tray was reconstituted, either immediately or at the time points for analytical testing, with 3.2 mL of purified water to a final concentration of 200 mg/mL. The immediately reconstituted samples (DP Liquid) were stored as liquid at 40°C; whereas, the samples reconstituted at the time points (DS Solid) were kept at 2-8°C (as solid) until analytical testing (Overall process and stability testing shown schematically in Figure IB). After the samples were reconstituted within a few hours on a wave or platform shaker at a gentle speed (~ 10 rpm), the liquid DP samples were transferred to sterile glass vials. The following analytical assays were performed at 0, 1, 2 and 3 month time points, in order to compare the stability trends of the high concentration formulation prepared by

Dialysis/Ultrafiltration (Control) and the BLRP samples:

1.7.1 Clarity- Appearance-Color (CAC):

CAC is used to determine the clarity, color and appearance of the samples. The glass vials containing the samples are inverted slowly for a few times. The samples are then observed first in front of a black background for any particulates and/or opalescence, and then in front of a white background, for any color.

1.7.2 Size Exclusion Chromatography (SEC) of omalizumab (E25) and a-IL17 v.2

SEC is used to determine the percent high molecular weight species (HMWS), monomer and low molecular weight species (LMWS) based on size. E25 samples were diluted to 1 mg/niL with 25 niM Histidine-HCl, 115 niM Arginine-HCl, 40 niM Sucrose, and 0.05% PS20 buffer at pH 6.0. A Tosoh Bioscience TSKgel G3000SWxl column (7.8 x 300 mm) was equilibrated at 0.4 mL/min and at 22°C with Solvent A (0.2M tripotassium phosphate (K₃PO₄), 0.25 M potassium chloride (KC1), pH 6.2). Following injection of approximately 20 μg of E25, Solvent A was run through the column for 35 minutes. Chromatography was carried out using an Agilent 1100 HPLC instrument, and the column effluent was monitored at 280 nm.

A slightly different SEC method was employed for a-IL17. The a-IL17 samples were diluted to 1 mg/mL with 25 mM Histidine-HCl, 115 mM Arginine-HCl, 40 mM Sucrose, and 0.05% PS20 buffer at pH 6.0. A Tosoh Bioscience TSKgel G3000SWxl column (7.8x300mm) was equilibrated at 0.5 mL/min and at 22°C with Solvent A. Following injection of approximately 50 μg of aIL17, Solvent A was run through the column for 35 minutes. Chromatography was carried out using an Agilent 1100 HPLC instrument, and the column effluent was monitored at 280 nm.

Ion Exchange Chromatography (IEC) of E25 and a-IL17 v.2

IEC is used to measure the charge variants of the MAbs. Partial removal of C-terminal lysine residues from the heavy chain during cell culture is a common cause of charge heterogeneity in monoclonal antibodies. The remaining lysine residues are typically removed before IEC by treating the mAb with carboxypeptidase B (CpB). E25 samples were diluted to 1 mg/mL with a buffer containing 20 mM MES (2-[N-Morpholino] ethanesulfonic acid), pH 6.1 and then treated with carboxypeptidase B (CpB DFP treated, Roche) at a 1 : 100 (w:w) ratio. The samples were incubated at 37°C for 20 minutes prior to ion exchange analysis. A Dionex ProPac™ Weak Cation Exchange (WCX) column (4 x 250 mm) was equilibrated at 0.8 mL/minute and 48°C with 50% solvent A (20 mM MES, pH 6.1) and 50% solvent B (solvent A containing 100 mM sodium chloride). Following injection of approximately 50 μg of CpB treated E25, a linear gradient increasing from 50% to 80% solvent B in 50 minutes, then from 80% to 100% solvent B in 10 minutes, was initiated. After regeneration by washing at 100% of solvent B for 5 minutes the column was re-equilibrated with 50% solvent B for 20 minutes. Chromatography was carried out using an Agilent 1100 HPLC instrument, and the column effluent was monitored at 280 nm. The IEC assay of a-IL17 does not require CpB treatment, since virtually all the C- terminal lysine residues are cleaved during cell culture processing. The a-IL17 samples were diluted to 1 mg/mL with a buffer containing 20 mM MES (2-[N-Morpholino] ethanesulfonic acid), pH 6.5. A Dionex ProPac™ WeakCation Exchange (WCX) column (4 x 250 mm) was equilibrated at 0.5 mL/minute and 30°C with 100% solvent A (20 mM MES, pH 6.5).

Following injection of approximately 30 μg of a-IL17, a linear gradient increasing from 0% to 100% solvent B (solvent A containing 100 mM sodium chloride) in 50 minutes, was initiated. The column was regenerated by washing at 100% of solvent B for 3 minutes, and then re-equilibrated with 100% solvent A for 17 minutes. Chromatography was carried out using an Agilent 1100 HPLC instrument, and the column effluent was monitored at 280 nm.

1.7.4 Hydrophobic Interaction Chromatography (HIC) of Papain Digested E25

HIC is used to monitor the isomerization products of the MAbs. Selective protease

(papain or pepsin) digestion followed by separation of the fragments has been employed to characterize the source of heterogeneity in a mAb (Cacia et al. , Biochemistry 1996, 35, 1897- 1903). Papain digestion followed by HIC analysis is instrumental in characterizing Asp isomerization for E25 and has since been used routinely. E25 samples were diluted to 1 mg/mL with a solution containing 1.0 M Tris-HCl, 40 mM EDTA, pH 7.4 and with lOmM cysteine. Papain was added at a 1 : 100 (w/w) enzyme: substrate ratio and incubated for 2 hours at 37°C. Antipain was added at 40: 1 (w/w) papain/ antipain ratio to quench the reaction. The resulting digest was analyzed with a TSK Phenyl 5PW column (7.5 x 75 mm) using an 1100 Agilent HPLC system. The column was equilibrated with 75% of solvent A containing 2 M ammonium sulfate solution, 20 mM TRIS, pH 7.5 and 25% solvent B containing 20 mM TRIS, pH 7.5. The column temperature was 40°C and the flow rate was 1 mL/min. Following injection of about 30 μg of papain digested E25, solvent B remained at 25% for 1 minute, followed by a two-step linear gradient from 25% to 40% solvent B in 3 minutes and then from 40% to 80% solvent B in 40 minutes. Solvent B was then increased to 100% for 2 minutes to regenerate the column, followed by re-equilibration at 25% solvent B. The column effluent was monitored at 214 nm.

1.7.5 Gravimetric Spectrophotometric Scan

Gravimetric spectrophotometric scan is used to determine the concentration of the MAb solutions. On a balance, first a falcon tube was tared and then 25 of undiluted high concentration protein solution was transferred into the falcon tube. The corresponding mass of the undiluted protein solution was measured and recorded. Then this protein solution was diluted with the formulation buffer (25 mM His-HCl, 115 mM Arginine-HCl, 40 mM Sucrose and 0.05% PS20 buffer at pH 6.0) ~ 400 fold. The mass of the diluted protein solution was then measured and recorded. The steps until this point were repeated one more time, so that there would be 2 data points for each sample. Then, using UV Spec Scan, the absorbance of the diluted solutions at 278 nm, 279 nm, 280 nm and 320 nm were determined. The maximum absorbance and the absorbance at 320 nm were recorded. The dilution, mass and the absorbance data were used to calculate the concentrations of the original solutions using absorptivity values of 1.60 mL mg^-1 cm^"1 at Amax -279 nm and for E25 and 1.49mL mg^-1 cm^_1at Amax -278 nm for a-IL17. 2. Results

2.1 High Concentration Liquid Drug Product Recovery from Tray

A product recovery versus viscosity plot was generated (Figure 3). Based on this prelimary study, an approximately 98% product yield could be achieved for a liquid Drug Product up to 100 cP. The tray recovery process could be further optimized in the future by customizing the tray and port configuration.

2.2 Stability of UF/DF liquid formulations (DP control-no lyo) vs. high concentration MAb produced by BLRP (DP Liquid).

A schematic of the overall stability study design is shown in Figure 1. Overall the stability assessments for omalizumab and aIL17 showed comparable behavior for both high concentration DP preparations using UF/DF or BLRP. For both MAbs, DP liquid prepared by BLRP and control prepared by UF/DF showed similar yellowing with storage at 40°C. SEC (Figure 4A1-2 for omalizumab and Figure 4B1-2 for aIL17), IEC (Figure 5A1-2 for omalizumab and Figure 5B1-2 for aIL17, and HIC (Figures 6A-B for omalizumab) showed that the degradation rates for DP Liquid prepared by BLRP and control prepared by UF/DF were very similar. Lastly, Gravimetric Spec Scan results showed that all the final concentrations obtained were more or less around 200 mg/mL regardless of the process (Tables 3 and 4). In addition, stability testing showed that for omalizumab, there was not any significant degradation in DS solid samples stored in the Lyoguard^® containers at 5°C. Table 3 Gravimetric Spec Scan Determined Concentrations for omalizumab

Table 4 Gravimetric Spec Scan Determined Concentrations for aIL17

3. Conclusions

All the analytical testing performed for E25 (omalizumab, Xolair®) and for a-IL17 v.2 proved and supported the notion that the BLRP technology is feasible to attain at least a 200 mg/mL liquid formulation for at least 2 MAbs, and that this new technology can serve as an alternative to the current TFF system for the manufacturing of high concentration MAb drug products for SC administration. In addition, this technology can also be used to store DS at temperatures above -20°C (e.g., 2-8°, 15-25°C), no longer necessitating the use of expensive stainless steel freeze-tanks or expensive storage under frozen conditions.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

The BLRP process of the current invention may be used to generate higher

concentrations (300-400 mg/mL) monoclonal antibody formulations.

EXAMPLE 2

Introduction:

During reconstitution of large-volume trays, a higher protein concentration can be achieved by reconstituting into a lower liquid volume. However, reducing the reconstitution volume may lead to increased viscosity. This study explored the ability to not only prepare formulations of very high viscosity, but also the measure the effect of reconstituting such solutions with increasing amounts of arginine to determine the effect on viscosity.

This experiment evaluates two different molecules [e.g., omalizumab (E25) and trastuzumab (HER2)], at different viscosities and elevated protein concentration to lyophilize and reconstitute to a higher protein concentrations. The reconstitution occurs with varying amounts of arginine and measures the effect on viscosity. Before lyophilization, the proteins will be dialyzed into the same buffer system. The molecules are lyophilized (separately) into 5cc mini-trays for reconstitution, and viscosities were measured using an Anton Paar Physica MCR 501 rheometer (with the cone and plate fixture). Protein concentrations and SE-HPLC were also measured to assess purity/stability.

The following buffer solutions where prepared:

(1) 10% Polysorbate 20 (w/v) stock. Measure 1 g of 100% PS20 (GNE lot 931763) into a 15mL sterile conical tube. Q.S. to 10 mL with ultra-pure water. Mix well. (2) 10 mM His-HCl, pH 6.0. Measure 2.275 g His-HCl- 1H20 and 1.419 g Histidine, free base. QS to 2 L with ultra-pure water. Mix well. Sterile filter.

(3) 10 mM His-HCl, 40 mM sucrose, pH 6.0. Measure 2.275 g His-HCl- 1H20, 1.419 g Histidine, free base, 27.384 g Sucrose. QS to 2 L with ultra-pure water. Mix well. Sterile filter.

(4) 10 mM His-HCl, 40 mM sucrose, 0.01% PS20, pH 6.0. Measure 0.569 g His- HCl- 1H20, 0.355 g Histidine, free base, 6.846 g Sucrose (GNE lot 870309) and 0.5 mL 10% PS20 stock. QS to 0.5 L with ultra-pure water. Mix well. Sterile filter.

(5) 2 M Arginine-HCl . Measure 42.134 g Arg-HCl (Molecular Weight: 210.67). QS to 0.1 L with ultra-pure water. Mix well. Sterile filter.

Prior to lyophiliztion, the following protocols were used to prepare the therapeutic molecules:

1) Omalizumab (E25):

Starting: - 40 mg/mL in 10 mM His-HCl, 85 mM sucrose, 0.01% PS20, pH 6.0.

Ending: - 80 mg/mL in 10 mM His-HCl, 40 mM sucrose, 0.01% PS20, pH 6.0

Dilute sucrose to 40 mM with lOmM His-HCl, pH 6.0. Add 250 mL 40 mg/mL E25 in 10 mM His-HCl, 85 mM sucrose, 0.01% PS20, pH 6.0. Add 281.5 mL of buffer lOmM His- HCl, pH 6.0.

To give: ~ 531.5 mL of 16 mg/mL E25 in 10 mM His-HCl, 40 mM sucrose, PS20, pH 6.0

Concentrate protein using Millipore benchtop TFF system to 80 mg/mL. Filter sterile. Determine PS20 levels by ELSD and adjust as necessary to give 0.01%. Measure to confirm: protein concentration (Grav UV-Vis), PS20 content (ELSD), pH,

SEC, osmolality, viscosity

2) Herceptin

Starting: 25 mg/mL in 5 mM His-HCl, 60 mM trehalose, 0.01% PS20, pH 6.0

Ending: - 80 mg/mL in 10 mM His-HCl, 40 mM sucrose, 0.01% PS20, pH 6.0 Buffer exchange using Millipore benchtop TFF system from original formulation into 10 mM His-HCl, pH 6.0. Perform a 6 to 8x volume buffer exchange. Confirm removal of trehalose by measuring osmolality. Perform a second buffer exchange using Millipore benchtop TFF system into 10 mM His-HCl, 40 mM sucrose, pH 6.0. Perform a third buffer exchange to 6 to 8x volume buffer exchange. After the third buffer exchange, concentrate protein using Millipore benchtop TFF system to 80 mg/mL. Determine PS20 levels by ELSD and spike in amount needed to have 0.01%. Filter sterile.

Measure to confirm: protein concentration (Grav UV-Vis), PS20 content (ELSD), pH, SEC, osmolality, viscosity.

Reconstitution of Lyophilized Samples:

Arginine-HCl concentrations: Solutions of various concentrations (50, 100, 150, 200,

400 mM) were prepared using a 2M stock solution.

During reconstitution, in order to accommodate the liquid volume of the lyo cake (- 0.35 mL) a slightly higher concentration of Arg-HCl than target level was added. The liquid volume was constant, while reconstitution time (time on rotisserie mixer) was constant for all samples within a protein concentration series. All samples rotated on their side, so liquid was not flipping from stopper to glass bottom. The reconstitutions were done at room temperature (20°C).

Table 5: 260 mg/ml E25

Table 6: 325 mg/ml E25

Table 7: 400 mg/ml E25

Table 8: 275 mg/mL HER2

Table 9: 350 mg/mL HER2

Table 10: 400 mg/ml E25

Claims

WHAT IS CLAIMED IS:

1. A process for making a high concentration protein formulation comprising the steps of:

(a) preparing a protein drug substance in a formulation of optimal molar ratios of desired excipients and purity, optionally using TFF and/or UF/DF,

(b) bulk lyophilizing the formulation of (a),

(c) optionally storing the formulation of (b),

(d) reconstituting the lyophilized formulation of (b) or (c) to create a drug product formulation having a viscosity of at least 20 cP.

2. The process of Claim 1, wherein the concentration of drug substance is about 2 - 200 mg/ml.

3. The process of Claim 2, wherein the concentration of drug substance is about 50-150 mg/ml

4. The process of either Claim 2-3, wherein the drug substance is concentrated to drug production by a factor of 2-20 times.

5. The process of any of Claims 1-4, wherein the protein is an antibody.

6. The process of Claim 5, wherein the antibody is monoclonal.

7. The process of any of Claims 1-4, wherein the protein is a polypeptide.

8. The process of Claim 1 , wherein the viscosity of the drug product is about 20 to 47,400 cP.

9. The process of any of Claims 1-8, wherein the concentration of drug product is at least about 50 mg/ml.

10. The process of Claim 9 wherein the concentration of drug product ranges from about 100 mg/ml to 700 mg/ml.

11. The process of any of Claims 1-10, wherein the drug product is isotonic.

12. The proceses of any of Claims 1-10, wherein the drug product is hypertonic.

13. The process of any of Claims 1-10, wherein the drug product is hypotonic.

14. The process of Claim 1, wherein step (c) is occurs at a temperature of 2 - 8 °C.

15. The process of Claim 1, wherein step (c) is occurs at a temperature of 15-25 °C.

16. The process of Claim 1, wherein the drug substance is prepared using tangential flow filtrations and/or ultrafiltration/diafilration.

17. A process for making a high concentration protein formulation capable of storage at temperatures above -20°C comprising the steps of:

(b) bulk lyophilizing the formulation of (a) to create a bulk cake,

(c) storing the bulk cake of (b) ,

18. The process of Claim 17, wherein the concentration of drug substance is about 2 - 200 mg/ml.

19. The process of Claim 18, wherein the concentration of drug substance is about 50-200 mg/ml.

20. The process of either Claim 18-19, wherein the drug substance is concentrated to drug product by a factor of 1 - 20 times.

21. The process of any of Claims 17-20, wherein the protein is an antibody.

22. The process of Claim 21 , wherein the antibody is monoclonal.

23. The process of any of Claims 17-20, wherein the protein is a polypeptide.

24. The process of Claim 17, wherein the viscosity of the drug product about 20-47,400 cP.

25. The process of any of Claims 17-24, wherein the concentration of drug product is at least about 50 mg/ml.

26. The process of Claim 23 wherein the concentration of drug product ranges from about 50 mg/ml to 700 mg/ml.

27. The process of any of Claims 17-26, wherein the drug product is isotonic.

28. The process of any of Claims 17-26, wherein the drug product is hypertonic.

29. The process of any of Claims 17-26, wherein the drug product is hypotonic.

30. The process of Claim 17 wherein the temperature of the storage step (c) occurs at 2-8 °C.

31. The process of Claim 17, wherein the temperature of the storage step (c) occurs at 15-25

°C