IMMUNOGLOBULIN FORMULATIONS DEPLETED IN IgA

Abstract

IgA depleted IgG formulations, kits comprising these formulations and methods of making and using these formulations are provided.

Description

IMMUNOGLOBULIN FORMULATIONS DEPLETED IN IgAFIELD OF THE INVENTION

[0001] The present invention resides in the field of immunoglobulin G (IgG) products for injection (IGI, e.g., IVIG and SCIG compositions) depleted in immunoglobulin A (IgA) and methods of making those products derived from human plasma, and their use in treating diseases or conditions.BACKGROUND OF THE INVENTION

[0002] Plasma-derived blood products are used to treat not only a variety of blood disorders, but diseases of other origin. For example, immunoglobulin G (IgG) products from human plasma were first used in 1952 to treat immune deficiency. Since then, IgG preparations have found widespread use in at least three main categories of medical conditions: (1) immune deficiencies such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immune deficiencies), and acquired compromised immunity conditions (secondary immune deficiencies), featuring low antibody levels; (2) inflammatory and autoimmune diseases; and (3) acute infections.

[0003] Specifically, many people with primary immunodeficiency disorders lack antibodies needed to resist infection. In certain cases, these deficiencies can be supplemented by the infusion of purified IgG, commonly through intravenous administration (i.e., IVIG therapy). Several primary immunodeficiency disorders are commonly treated in the fashion, including X- linked Agammaglobulinemia (XLA), Common Variable Immunodeficiency (CVID), Hyper-IgM Syndrome (HIM), Severe Combined Immunodeficiency (SCID), and some IgG subclass deficiencies (Blaese and Winkelstein, J. Patient & Family Handbook for Primary Immunodeficiency Diseases. Towson, MD: Immune Deficiency Foundation; 2007).

[0004] While IgG therapy can be very effective for managing primary immunodeficiency disorders, this therapy is only a temporary replacement for antibodies that are not being produced in the body, rather than a cure for the disease. Accordingly, patients dependent upon IgGtherapy require repeated doses, typically about once a month for life. This need places a great demand on the continued production of IgG compositions. However, unlike other biologies that are produced via in vitro expression of recombinant DNA vectors, IgG is fractionated from human blood and plasma donations. Thus, IgG products cannot be increased by simply increasing the volume of production. Rather, the level of commercially available IgG is limited by the available supply of blood and plasma donations.

[0005] Several factors drive the demand for IgG products, including the acceptance of IgG treatments, the identification of additional indications for which IgG therapy is effective, and increasing patient diagnosis and IgG therapy prescription. Notably, the use of blood (z.e., erythrocytes) has declined by almost 40% but the use of IgG tripled between 2004 and 2018, with a 5-7% estimated annual increase until 2024 (Brand, A., et al. Transfusion Clinique et Biologique, 2021, 28(1), 96-122). Due in part to the increasing global demand and fluctuations in the available supply of IgG products, several countries, including Australia and England, have implemented demand management programs to protect supplies of these products for the highest demand patients during times of product shortages.

[0006] A number of IgG commercial suppliers provide a variety of Immunoglobulin for Injection (“IGI”), e.g., IVIG (intravenous immunoglobulin), products. More than a dozen IgG products are available in North America and Europe, which vary with respect to IgG concentration, infusion frequency, route of administration, and other considerations (Perez, et al., J Allergy Clin Immunol. (2017), 139: S1-S46). Compared to the older lyophilized IVIG products containing 50 mg / mL or 100 mg / mL protein in the solution after re-dissolving, current formulations provide a 100 mg / mL and 200 mg / mL ready-for-use sterile, liquid preparation of highly purified and concentrated human IgG antibodies.

[0007] More recently entering the IgG therapeutic market are IgG formulations formatted for subcutaneous administration (i.e., SCIG therapy). These formulations represent a significant advance in the overall patient experience with IgG formulations. For example, a patient or caregiver trained in subcutaneously infusing an IgG formulation, can infuse the formulation in practically any setting. This innovation frees the patient from visits to an infusion center, allows them to infuse, e.g., self-infuse, in the comfort of their own home, or anywhere of their choosing. Exemplary subcutaneously infused IgG formulations include HyQvia® [Immune GlobulinInfusion 10% (Human) with Recombinant Human Hyaluronidase], Hizentra® [Immune Globulin Subcutaneous Human 20% Liquid],

[0008] There is a subset of immunoglobulin-treated patients that may react to immunoglobulins containing higher levels of immunoglobulin A (IgA); therefore, IgG preparations carry the risk of inducing an anaphylactic reaction in IgA-sensitive patients. GAMMAGARD® S / D Immunoglobulin Intravenous (Human) [IVIG], Solvent / Detergent-Treated (Freeze-Dried Concentrate) has the lowest levels of IgA of all immunoglobulins on the market globally. GAMMAGARD® S / D was one of the first immunoglobulins marketed in the US and has been available for over 20 years for the safe and effective treatment of Primary Immunodeficiency and other ailments; approximately 3000 patients use GAMMAGARD® S / D around the world. Other pharmaceutical formulations of immunoglobulins have become available in ready-to-use solutions with more modern manufacturing methods.

[0009] Reducing the potential of adverse patient reactions to therapeutic agents is a constant and significant goal of research into these agents; however, with protein therapeutics isolated from human plasma, this goal is uniquely difficult to obtain. Isolation processes for plasma proteins begin with a highly complex mixture, human plasma, and are multi-step with subroutines within many of the major steps. Each step of the purification process and its products depend upon the conditions of one or more previous step and the amount and identity of impurities remaining in the intermediates following those steps. Accordingly, altering the process of isolating a plasma protein therapeutic from plasma to increase purity or to decrease the amount of one or more specific impurities is not a trivial process.BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides a facile new process for purifying immunoglobulin G (IgG) from human plasma, providing an IgG formulation with surprisingly low IgA content, and pharmaceutical formulations prepared by this process.

[0011] Provided herein, in various embodiments, are storage-stable IgA depleted IgG pharmaceutical formulations, kits comprising these formulations, methods of producing these formulations, and methods of using them to treat a disease in a subject by administering a therapeutically useful amount of a formulation of the invention. An exemplary storage-stableIgG pharmaceutical formulation of the present invention contains IgA at not more than about 2 pg / mL, for example, not more than about 2 pg / mL, not more than about 1.9 pg / mL, not more than about 1.8 pg / mL, not more than about 1.7 pg / mL, not more than about 1.6 pg / mL, not more than about 1.5 pg / mL, not more than about 1.4 pg / mL, not more than about 1.3 pg / mL, not more than about 1.2 pg / mL, not more than about 1.1 pg / mL, not more than about 1 pg / mL, not more than about 0.9 pg / mL, not more than about 0.8 pg / mL, not more than about 0.7 pg / mL, not more than about 0.6 pg / mL, not more than about 0.5 pg / mL, not more than about 0.4 pg / mL, not more than about 0.3 pg / mL, not more than about 0.2 pg / mL, not more than about 0.1 pg / mL, not more than about 0.01 pg / mL, or less. In exemplary embodiments, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 2 pg / mL. In another exemplary embodiment, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 1.8 pg / mL.

[0012] In various embodiments, a storage-stable IgA depleted IgG pharmaceutical formulation of the present invention contains IgA at a ratio of IgA : IgG of less than 1 : 50000 (by weight), at a ratio of less than 1 : 75000, at a ratio of less than 1 : 100000, at a ratio of less than 1 : 250000, at a ratio of less than 1 : 500000.

[0013] In various embodiments, a storage-stable IgA depleted IgG pharmaceutical formulation comprises the purified IgG in an aqueous pharmaceutically acceptable carrier. In various embodiments, the formulation has a property selected from a pH of from about 4.0 to about 5.5, from about 0.01 M to about 1.0 M glycine, and a combination thereof. In some embodiments, the formulation has a property selected from a pH of from about 4.4 to about 5.1, from about 0.1 M to about 0.4 M glycine, and a combination thereof. In exemplary embodiments, the formulation has a property selected from a pH of from about 4.6 to about 5.1, from about 0.2 M to about 0.3 M glycine, and a combination thereof. The pH value always refers to a ready to use formulation.

[0014] In various embodiments, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) contains at least about 5% (w / v) to about 25% (w / v) IgG and / or total protein, for example, about 5% (w / v) to about 20% (w / v), about 5% (w / v) to about 15% (w / v), about 5% (w / v) to about 10% (w / v), about 5% (w / v) to about 15% (w / v), about 7% (w / v) to about 13% (w / v), about 9% (w / v) to about 11% (w / v). In some embodiments, IgGand / or total protein is about 9% (w / v), about 10% (w / v), about 11% (w / v), about 12% (w / v), about 13% (w / v), about 14% (w / v), about 15% (w / v), about 16% (w / v), about 17% (w / v), about 18% (w / v), about 19% (w / v), or about 20% (w / v). In exemplary embodiments, IgG and / or total protein is about 10% (w / v). In exemplary embodiments, IgG and / or total protein is from about 9% (w / v) to about 11% (w / v). In exemplary embodiments, IgG and / or total protein is about 20% (w / v). In exemplary embodiments, IgG and / or total protein is from about 19% (w / v) to about 21% (w / v). In another exemplary embodiments, IgG and / or total protein is about 10% (w / v). In another exemplary embodiments, IgG and / or total protein is from about 9% (w / v) to about 11% (w / v). In another exemplary embodiments, IgG and / or total protein is about 5% (w / v). In various embodiments, when the formulation is intended for IV administration, it is formatted as a solution of about 10% IgG and / or total protein. In various embodiments, when the formulation is intended for subcutaneous or intramuscular administration, it is formatted as a solution of at least about 20% IgG and / or total protein.

[0015] In various embodiments, the present invention provides methods of forming a storagestable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) by (a) performing an anion exchange step on an anion exchange precursor solution comprising the IgG, the step comprising: (i) submitting the anion exchange precursor solution to anion exchange chromatography in a single pass through a first portion of an anion exchange medium contained in a first column and collecting a first anion exchange flowthrough from the first column.

[0016] In various embodiments of the methods described above, the step further comprises (ii), following (i), washing the anion exchange medium with a wash buffer, wherein the washing uses from about 0.25 column volumes to about 5 column volumes of the wash buffer, for example, about 0.25 to about 4 column volumes, about 0.25 to about 3 column volumes, about 0.25 to about 2 column volumes, about 0.25 to about 1 column volumes, about 0.50 to about 5 column volumes, about 0.75 to about 5 column volumes, about 1 to about 5 column volumes, about 2 to about 5 column volumes, or about 3 to about 5 column volumes. In some embodiments the washing uses about 0.25 to about 3 column volumes, about 0.25 to about 2 column volumes, or about 0.25 to about 1 column volumes.

[0017] In various embodiments of the methods described above, storage-stable IgA depleted IgG pharmaceutical formulations (e.g., not more than about 2 pg / mL of IgA) are formed using the method of the present invention.

[0018] In various embodiments, a storage-stable IgA depleted IgG formulation is formatted for injection (IGI), e.g., intravenous immunoglobulin (IVIG) administration. In various embodiments, the IGI formulation is formatted for subcutaneous immunoglobulin (SCIG) administration. In exemplary embodiments, the formulation is useful to treat immunoglobulin- treatable diseases and conditions, particularly in IgA-sensitive patients in need thereof.

[0019] Other embodiments, objects, and advantages of the invention will be apparent from the detailed description following.DETAILED DESCRIPTION OF THE INVENTIONI. Introduction

[0020] Given the broad use of therapeutic plasma-derived blood protein compositions, such as immune globulin compositions, blood coagulation factors, coagulation factor inhibitors, and proteins of the complement system, ensuring the safety of these compositions and maximizing their tolerability is of paramount importance. A subset of immunoglobulin-treated patients reacts to immunoglobulins containing higher levels of immunoglobulin A (IgA); therefore, IgG preparations carry the risk of inducing an anaphylactic reaction in IgA-sensitive patients. GAMMAGARD® S / D Immunoglobulin Intravenous (Human) [IVIG], Solvent / Detergent- Treated (Freeze-Dried Concentrate) has the lowest levels of IgA of all immunoglobulins on the market globally. Given the perennially motivation to devise safer and more efficacious therapeutics, a need remains in the art for ready-to-use solutions comprising immunoglobulin compositions depleted in IgA and methods of manufacturing those compositions.

[0021] Reducing the potential of adverse patient reactions to therapeutic agents is a constant and significant goal of research into these agents; however, with protein therapeutics isolated from human plasma, this goal can be uniquely difficult to obtain. Isolation processes for plasma proteins begin with a highly complex mixture, human plasma, and are multi-step with subroutines within many of the major steps. Each step of the purification process and itsproducts depend upon the conditions and products of one or more previous step and the amount and identity of impurities remaining in the intermediates following those steps. Accordingly, altering the process of isolating a plasma protein therapeutic from plasma to increase purity or to decrease the amount of one or more specific impurities is not a trivial process accompanied by a certain outcome.

[0022] Quite surprisingly, the present invention provides a process for purifying immunoglobulin G (IgG) from human plasma, which results in an IgG formulation having extremely low immunoglobulin A (IgA) content.

[0023] Provided herein, in various embodiments, are storage-stable IgA depleted IgG pharmaceutical formulations, kits comprising these formulations, methods of producing these formulations, and methods of using them to treat a disease in a subject by administering a therapeutically useful amount of a formulation of the invention. An exemplary storage-stable IgG pharmaceutical formulation of the present invention contains IgA at not more than about 2 pg / mL, for example, not more than about 2 pg / mL, not more than about 1.9 pg / mL, not more than about 1.8 pg / mL, not more than about 1.7 pg / mL, not more than about 1.6 pg / mL, not more than about 1.5 pg / mL, not more than about 1.4 pg / mL, not more than about 1.3 pg / mL, not more than about 1.2 pg / mL, not more than about 1.1 pg / mL, not more than about 1 pg / mL, not more than about 0.9 pg / mL, not more than about 0.8 pg / mL, not more than about 0.7 pg / mL, not more than about 0.6 pg / mL, not more than about 0.5 pg / mL, not more than about 0.4 pg / mL, not more than about 0.3 pg / mL, not more than about 0.2 pg / mL, not more than about 0.1 pg / mL, not more than about 0.01 pg / mL, or less. In exemplary embodiments, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 2 pg / mL. In an exemplary embodiment, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 1.8 pg / mL.

[0024] In various embodiments, a storage-stable IgA depleted IgG pharmaceutical formulation comprises the purified IgG in an aqueous pharmaceutically acceptable carrier. In various embodiments, the formulation has a property selected from a pH of from about 4.0 to about 5.5, from about 0.01 M to about 1.0 M glycine, and a combination thereof. In some embodiments, the formulation has a property selected from a pH of from about 4.4 to about 5.1, from about 0.1 M to about 0.4 M glycine, and a combination thereof. In exemplary embodiments, theformulation has a property selected from a pH of from about 4.6 to about 4.1, from about 0.2 M to about 0.3 M glycine, and a combination thereof. The pH values stated above always refer to a ready to use formulation.

[0025] In various embodiments, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) contains at least about 5% (w / v) to about 25% (w / v) IgG and / or total protein, for example, about 5% (w / v) to about 20% (w / v), about 5% (w / v) to about 15% (w / v), about 5% (w / v) to about 10% (w / v), about 5% (w / v) to about 15% (w / v), about 7% (w / v) to about 13% (w / v), about 9% (w / v) to about 11% (w / v). In some embodiments, IgG and / or total protein is about 9% (w / v), about 10% (w / v), about 11% (w / v), about 12% (w / v), about 13% (w / v), about 14% (w / v), about 15% (w / v), about 16% (w / v), about 17% (w / v), about 18% (w / v), about 19% (w / v), or about 20% (w / v). In exemplary embodiments, IgG and / or total protein is about 10% (w / v). In exemplary embodiments, IgG and / or total protein is from about 9% (w / v) to about 11% (w / v) (storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA). In exemplary embodiments, IgG and / or total protein is about 20% (w / v). In exemplary embodiments, IgG and / or total protein is from about 19% (w / v) to about 21% (w / v). In exemplary embodiments, IgG and / or total protein is about 10% (w / v) comprised within the storage-stable IgA depleted IgG pharmaceutical formulation having not more than about 2 pg / mL of IgA.

[0026] In various embodiments, the present invention provides methods of forming a storagestable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) by (a) performing an anion exchange step on an anion exchange precursor solution comprising the IgG, the step comprising: (i) submitting the anion exchange precursor solution to anion exchange chromatography in a single pass through a first portion of an anion exchange medium contained in a first column and collecting a first anion exchange flowthrough from the first column.

[0027] In various embodiments of the methods described above, the step further comprises (ii), following (i), washing the anion exchange medium with a wash buffer, wherein the washing uses from about 0.25 column volumes to about 5 column volumes of the wash buffer, for example, about 0.25 to about 4 column volumes, about 0.25 to about 3 column volumes, about 0.25 to about 2 column volumes, about 0.25 to about 1 column volumes, about 0.50 to about 5 columnvolumes, about 0.75 to about 5 column volumes, about 1 to about 5 column volumes, about 2 to about 5 column volumes, or about 3 to about 5 column volumes. In some embodiments the washing uses about 0.25 to about 3 column volumes, about 0.25 to about 2 column volumes, or about 0.25 to about 1 column volumes.

[0028] In various embodiments of the methods described above, storage-stable IgA depleted IgG pharmaceutical formulations (e.g., not more than about 2 pg / mL of IgA) are formed using the method of the present invention.

[0029] In various embodiments, a storage-stable IgA depleted IgG formulation is formatted for injection (IGI), e.g., intravenous immunoglobulin (IVIG) administration. In various embodiments, the formulation is formatted for subcutaneous immunoglobulin (SCIG) administration. In exemplary embodiments, the formulation is useful to treat immunoglobulin- treatable diseases and conditions, particularly in IgA-sensitive patients in need thereof.IL Definitions

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0031] The terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0032] All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic(s) or limitation(s) and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

[0033] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0034] The methods and devices of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional components or limitations described herein or otherwise useful.

[0035] Unless otherwise indicated, all numbers expressing physical dimensions, quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0036] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0037] In the interest of clarity, not all of the routine features of the implementations described herein are shown and described. It will be appreciated that, in the development of any such actual implementation, numerous implementation-specific decisions are made in order to achieve the developer’s specific goals, such as compliance with application- and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking of engineering for those of ordinary skill in the art having the benefit of this disclosure.

[0038] Many modifications and variations of the exemplary embodiments set forth in this disclosure can be made without departing from the spirit and scope of the exemplary embodiments, as will be apparent to those skilled in the art. The specific exemplary embodiments described herein are offered by way of example only, and the disclosure is to belimited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0039] The “Cohn Process”, and “Cohn Fractionation” are used interchangeably herein and as generally understood, refer to a method of separating human plasma through a series of steps, including ethanol precipitation at differing concentrations, changes in pH, changes in temperature, changes in ionic strength, which lead to fractions enriched in certain plasma proteins. See, for example U.S. Pat. No. 2,390,074. As used herein, the terms “Cohn Process” and “Cohn Fractionation” also refers to the many variations and improvements on this pioneering process, e.g., Kistler-Nitschmann Process (Kistler etal. (1952), Vox Sang, 7, 414- 424). Other processes of use in the methods of the invention include the method of isolating IgG set forth in U.S. Pat. No. 8,940,877.

[0040] ‘ ‘Plasma” is the fluid that remains after blood has been centrifuged (for example) to remove cellular materials such as red blood cells, white blood cells and platelets. Plasma is generally yellow-colored and clear to opaque. Blood that is donated and processed to separate the plasma from the other certain blood components, and not frozen is referred to as “never- frozen” plasma. Plasma that is frozen within 8 hours to temperatures, described herein, is referred to herein as “fresh frozen plasma” (“FFP”). It contains the dissolved constituents of the blood such as proteins (6-8%; e.g., serum albumins, globulins, fibrinogen, etc.), glucose, dotting factors (clotting proteins), electrolytes (Na.+, Ca2+, Mg2+, HCOU, CU, etc.), hormones, etc.. Whole blood (WB) plasma is plasma isolated from whole blood with no added agents except anticoagulant(s). Citrate phosphate dextrose (CPD) plasma, as the name indicates, contains citrate, sodium phosphate and a sugar, usually dextrose, which are added as anticoagulants. Plasma includes, but is not limited to, liquid plasma, recovered plasma, thawed plasma, spray dried plasma, and physiologically active reconstituted plasma. In various embodiments, “plasma”, as described herein is the starting material for the IgA depleted IgG formulations of the invention.

[0041] As used herein, the terms "improve", "increase", "inhibit", "decrease", "reduce", or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparableconditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable systems known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0042] As used herein, the term "solvent" encompasses any liquid substance capable of dissolving or dispersing one or more other substances. A solvent may be inorganic in nature, such as water, or it may be an organic liquid, such as ethanol, acetone, methyl acetate, ethyl acetate, hexane, petrol ether, etc. As used in the term "solvent detergent treatment," solvent denotes an organic solvent (e.g., tri-N-butyl phosphate), which is part of the solvent detergent mixture used to inactivate lipid-enveloped viruses in solution.

[0043] As used herein, the term "detergent" is used in this application interchangeably with the term "surfactant" or "surface acting agent." Surfactants are typically organic compounds that are amphiphilic, i.e., containing both hydrophobic groups ("tails") and hydrophilic groups ("heads"), which render surfactants soluble in both organic solvents and water. A surfactant can be classified by the presence of formally charged groups in its head. A non-ionic surfactant has no charge groups in its head, whereas an ionic surfactant carries a net charge in its head. A zwitterionic surfactant contains a head with two oppositely charged groups. Some examples of common surfactants include: Anionic (based on sulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA or PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laureth sulfate (also known as sodium lauryl ether sulfate, or SLES), alkyl benzene sulfonate; cationic (based on quaternary ammonium cations): cetyl trimethylammonium bromide (CTAB) (i.e. hexadecyl trimethyl ammonium bromide), and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), poly ethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); Long chain fatty acids and their salts: including caprylate, caprylic acid, heptanoate, hexanoic acid, heptanoic acid, nonaic acid, decanoic acid, and the like; Zwitterionic (amphoteric): dodecyl betaine; cocamidopropyl betaine; coco ampho glycinate; nonionic: alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and polypropylene oxide) (commercially known as Poloxamers or Poloxamines), alkyl polyglucosides, including octyl glucoside, decyl maltoside, fatty alcohols(e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, and dodecyl dimethylamine oxide.

[0044] As used herein, the term “stable formulation”, such as “stable pharmaceutical formulation”, “storage-stable pharmaceutical formulation”, or “storage-stable IgA depleted IgG pharmaceutical formulation”, as used in connection with the formulations described herein denotes, without limitation, a formulation, which preserves its physical stability / identity / integrity and / or chemical stability / identity / integrity and / or biological activity / identity / integrity during manufacturing, storage, and administration. Various analytical techniques for evaluating protein stability are available in the art and reviewed in Reubsaet, et al. (1998) J Pharm Biomed Anal 17(6-7): 955-78 and Wang, W. (1999) Int J Pharm 185(2): 129-88. Stability can be evaluated by, for example, without limitation, storage at selected climate conditions for a selected time period, by applying mechanical stress such as shaking at a selected shaking frequency for a selected time period, by irradiation with a selected light intensity for a selected period of time, or by repetitive freezing and thawing at selected temperatures. The stability may be determined by, for example, at least one of the methods selected from the group consisting of visual inspection, SDS-PAGE, IEF, size exclusion liquid chromatography (SEC-HPLC), reversed phase liquid chromatography (RP-HPLC), ion-exchange HPLC, capillary electrophoresis, light scattering, particle counting, turbidity, RFFIT, and kappa / lambda ELISA, without limitation. Exemplary characteristics of use with visual inspection include turbidity and aggregate formation.

[0045] In various embodiments, a formulation is considered stable when the immunoglobulins in the formulation (1) retaintheir physical stability, (2) retaintheir chemical stability and / or (3) retain their biological activity.

[0046] Immunoglobulins may be said to “retain their physical stability” in a formulation if, for example, they show limited aggregation (up to about 2% in SEC) or fragmentation (up to about 5% in SEC) even after storage, and no precipitation and / or protein denaturation upon visual examination of color and / or clarity, or as measured by UV light scattering or bysize exclusion chromatography (SEC) or electrophoresis, such as with reference to turbidity or aggregate formation.

[0047] Immunoglobulins may be said to “retain their chemical stability” in a formulation, if, for example, the chemical stability at a given time is such that there is not more than about 5% modification of the immunoglobulins by bond formation or cleavage resulting in a new chemical entity. In a further embodiment, chemical stability can be assessed by detecting and quantifying chemically altered forms of the immunoglobulins. Chemical alteration may involve, for example, size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and / or matrix-assisted laser desorption ionization / time-of-fhght mass spectrometry (MALDI / TOF MS). Other types of chemical alteration include, for example, charge alteration (e.g. occurring as a result of deamidation), which can be evaluated by, e.g. , ion-exchange chromatography.

[0048] Oxidation is another commonly seen chemical modification. In an exemplary embodiment, protein oxidation products account for not more than about 5% of the protein in the formulation.

[0049] In various embodiments, immunoglobulins may be said to “retain their biological activity” relative to native unmodified immunoglobulins in a pharmaceutical formulation, if, for example, the biological activity of the immunoglobulins, at a given time is between about 50% and about 200%, or between about 60% and about 170%, or between about 70% and about 150%, or between about 80% and about 125%, or between about 90% and about 110%, of the biological activity exhibited at the time the formulation was prepared as determined, e.g. , in an antigen binding assay or virus neutralization assay. In a further embodiment, immunoglobulins may be said to “retain their biological activity” relative to native unmodified immunoglobulins in a pharmaceutical formulation, if, for example, the biological activity of the immunoglobulins, at a given time is at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of that of native unmodified immunoglobulins, all measured with respect to monomer and dimer, respectively by HPLC SEC. In anexemplary embodiment, immunoglobulins may be said to “retain their biological activity” relative to native unmodified immunoglobulins in a pharmaceutical formulation, if, for example, the biological activity of the immunoglobulins, at a given time is between about 90% and about 100% monomer and dimer content as measured by HPLC SEC.

[0050] In various embodiments, a formulation of the present invention may be said to be stable, e.g., “storage stable”, when IgG does not substantially aggregate, denature, or fragment such that at least about 90% of the IgG is present as monomers or oligo- / dimers, with a molecular weight of IgG between at or about greater than about 70 kDa and less than 450 kDa. Thus, less than about 10%, for example, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% of the IgG protein is present as an aggregate (i.e., has a molecular size greater than or equal to 450 kDa in size) in the formulation. Similarly, no more than about 5% to about 7%, for example, about 7%, 6%, 5%, 4%, 3%, 2%, 1%, or about 0.5% or less of the IgG in the formulation is fragmented (i.e., has a molecular size less than 70 kDa). In an exemplary embodiment less than about 5% of the IgG in the formulation is fragmented at the end of the shelf-life.

[0051] In various embodiments, a formulation of the present invention may be said to be stable when the protein(s), immunoglobulins, and / or IgG essentially retain their physical and chemical stability and integrity upon storage for at least six months at temperatures up to about 32 °C. For purposes herein, “stability at room temperature” means stability at the upper range of typical room temperatures for warmer locales (i.e., about 28 °C to about 32 °C, e.g., for Italy or Texas). The formulations are stable, e.g., “storage stable”, over the range of refrigerated and room temperatures, i.e., 0 °C-32 °C, or up to about 32 °C for at least six months. Assays for assessing the stability of each are well known to one of skill in the art and described herein.

[0052] As used herein, “storage” means that a formulation is not immediately administered to a subject once prepared, but is kept for a period of time under particular conditions (e.g., particular temperature; liquid or lyophilized form) prior to use. For example, a liquid formulation can be kept for days, weeks, months, or years, generally at least six months, prior to administration to a subject under varied temperatures such as refrigerated (about 0 °C to about 10 °C) or room temperature (e.g., temperature up to about 32 °C).

[0053] As used herein, the term “ultrafiltration” (abbreviated “UF”) encompasses a variety of membrane filtration methods in which hydrostatic pressure forces a liquid against a semi- permeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane. This separation process is often used for purifying and concentrating macromolecular (103-l 06Da) solutions, especially protein solutions. A number of ultrafiltration membranes are available depending on the size of the molecules they retain. Ultrafiltration is typically characterized by a membrane pore size between 1 and 1000 kDa and operating pressures between 0.01 and 10 bar, and is particularly useful for separating colloids like proteins from small molecules like sugars and salts.

[0054] As used herein, the term "diafiltration" is performed with the same membranes as ultrafiltration and is a tangential flow filtration. During diafiltration, buffer is introduced into the recycle tank while filtrate is removed from the unit operation. In processes where the product is in the retentate (e.g., IgG), diafiltration washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species.

[0055] The term "chromatography" refers to any kind of technique which separates a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Usually, the protein of interest is separated from other molecules (e.g., contaminants) as a result of differences in rates at which the individual molecules of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes. The term "matrix" or" chromatography matrix" are used interchangeably herein and refer to any kind of sorbent, resin or solid phase which in a separation process separates a protein of interest (e.g., an Fc region containing protein such as an immunoglobulin) from other molecules present in a mixture. Non-limiting examples include particulate, monolithic or fibrous resins as well as membranes that can be put in columns or cartridges. Examples of materials for forming the matrix include polysaccharides (such as agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of the above. Examples for typical matrix types suitable for the method of the present disclosure are cation exchange resins, affinity resins, anion exchange resins or mixed mode resins. A "ligand" is a functional group that isattached to the chromatography matrix and that determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interactions groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the aforementioned). Some preferred ligands that can be used herein include, but are not limited to, strong cation exchange groups, such as sulphopropyl, sulfonic acid; strong anion exchange groups, such as trimethylammonium chloride; weak cation exchange groups, such as carboxylic acid; weak anion exchange groups, such as N5N di ethylamino or DEAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl; and affinity groups, such as Protein A, Protein G, and Protein L. An exemplary anion exchange resin is an ANX resin (Cytivia).

[0056] The term "chromatography column" or "column" in connection with chromatography as used herein, refers to a container, frequently in the form of a cylinder or a hollow pillar which is filled with the chromatography matrix or resin. The chromatography matrix or resin is the material which provides the physical and / or chemical properties that are employed for purification.

[0057] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the degree of purity of a protein of interest from a composition or sample comprising the protein of interest and one or more impurities. Typically, the degree of purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition. “Purifying” and its equivalents refer to one or more step performed to isolate a therapeutic protein from one or more other impurities (e.g., bulk impurities) or components present in a fluid containing a therapeutic protein (e.g., plasma, Cohn fraction, liquid culture medium proteins or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.) present in or secreted from a mammalian cell). For example, purifying can be performed during or after an initial capturing step. Purification can be performed using a resin, membrane, or any other solid support that binds either a therapeutic protein or contaminants (e.g., through the use of affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). A therapeutic protein can be purified from a fluid containing the therapeutic protein using at least one chromatography column and / or chromatographic membrane (e.g., anyof the chromatography columns or chromatographic membranes described herein). In exemplary embodiments, these terms refer to separating IgG from essentially all IgA present in a mixture of IgG and IgA according to a process of the invention.

[0058] For example, immunoglobulin, e.g., IgG, can be purified by the removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin other than IgG, e.g., IgA. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulin other than IgG results in an increase in the percent of desired IgG in the purified mixture. Purity can be measured by standard assays known in the art or described herein, examples of which include SDS-PAGE followed by Coomassie blue staining as well as chromatographic methods (e.g., size exclusion chromatography (SEC) on a HPLC system). Purity of the IgG sample can be calculated from an SDS PAGE gel after scanning using a Kodak Image Station 1000 or equivalent system, or by analysis of SEC chromatogram by software on a Shimadzu HPLC system. A sample is considered pure if it is at least 90%, 95%, or 99% free of components other than the desired product (e.g., immunoglobulin). An exemplary component removed from IgG by a process of the invention is IgA.

[0059] Preferably, the method of the invention yields a preparation of IgG that is at least about 80%, 85%, 90%, 95%, or 99% or more pure. Pure refers to a mixture of IgG isotypes in which contaminating non-IgG proteins are reduced. Alternatively, “pure” refers to a preparation containing one or more IgG isotypes in which one or more other isotypes have been substantially completely removed.

[0060] The term "flow- through" or "flow-through mode" as used herein refers to the general purification approach wherein contaminants are removed from a mixture during chromatography because they are retained by a chromatographic medium, e.g., bound to a resin in a column. A protein of interest is purified because it does not bind (or it binds less strongly than contaminants) to a chromatographic medium, usually a resin in a column, and instead flows through to be collected. After the protein of interest is removed from contact with the chromatographic medium, the impurities bound to the column are generally "stripped", or removed from the column, so that the column can then be regenerated for another chromatographic run. This approach differs from "bind-and-elute" or "bind-and-elute mode" wherein the target protein of interest is retained on a column and impurities flow through thecolumn. This process then involves specific elution of the protein of interest using different column conditions that interfere with the binding of the protein of interest to the chromatographic medium, usually a resin in a column.

[0061] As used herein the term "contaminant" is used in its broadest sense to cover any undesired component or compound within a mixture. Contaminant proteins include, without limitation, those naturally produced by a donor, or a host cell, as well as proteins related to or derived from the protein of interest (e.g., proteolytic fragments) and other process related contaminants. In certain embodiments, the contaminant precipitate is separated from the cell culture using another means, such as centrifugation, sterile filtration, depth filtration and tangential flow filtration. An exemplary contaminant is a non-IgA immunoglobulin, e.g., IgA.

[0062] "Eluate," as used herein, refers to a fluid that is emitted from a chromatography column or chromatographic membrane that contains a detectable amount of a protein.

[0063] In some embodiments, the chromatographic system disclosed herein is on a skid. "Skid," as used herein, refers to a three-dimensional solid structure that can act as a platform or support for a system described herein. A skid can, if it comprises one or more structures that enable movement (e.g., wheels, rollers, or the like), confer mobility on the system or a portion thereof. An exemplary skid includes two or more chromatography columns linked in parallel, or in series. A preferred skid includes two or more columns linked in parallel.

[0064] In some embodiments, the methods and systems have a recovery from the feedstock of a desired protein, e.g., IgG or a selected IgG isoform, of at least about 30%, 35%, 40%, 45%, 50%, 55%, or at least about 60%, and up to about 65%, 70%, 75%, 80%, 85%, 90%, or up to about 95% of the amount of the protein contained in the feedstock. In some embodiments, the purification recovery is at least about 50%. An exemplary feedstock is an anion exchange precursor solution, which is applied to an anion exchange chromatography resin.

[0065] As used in this application, the term "spraying" refers to a means of delivering a liquid substance into a system, e.g., during an alcohol precipitation step, such as a modified Cohn fractionation I or II+III precipitation step, in the form of fine droplets or mist of the liquid substance. Spraying may be achieved by any pressurized device, such as a container (e.g., a spray bottle), that has a spray head or a nozzle and is operated manually or automatically togenerate a fine mist from a liquid. Typically, spraying is performed while the system receiving the liquid substance is continuously stirred or otherwise mixed to ensure rapid and equal distribution of the liquid within the system.

[0066] Generally, “Immunoglobulin for Injection” (IGI), refers to a therapeutic formulation of IgG formatted for administration via IV, subcutaneous, or intramuscular routes. As used herein, the term "Intravenous IgG" or "IVIG" treatment refers generally to a therapeutic formulation of IgG formatted for intravenous injection and its use in a method of intravenously administering a composition of IgG immunoglobulins to a patient for treating a number of conditions such as immune deficiencies, inflammatory diseases, and autoimmune diseases. The IgG immunoglobulins are typically pooled and prepared from plasma. Whole antibodies or fragments can be used. IgG immunoglobulins can be formulated in higher concentrations (e.g., greater than 10%) for subcutaneous administration, or formulated for intramuscular administration. This is particularly common for specialty IgG preparations which are prepared with higher than average titers for specific antigens (e.g., Rho D factor, pertussis toxin, tetanus toxin, botulism toxin, rabies, etc.). For ease of discussion, such subcutaneously or intramuscularly formulated IgG compositions are also included in the term "IVIG" in this application. The term “Immunoglobulin for Injection” (IGI) is sometimes used synonymously with IVIG.

[0067] By "therapeutically effective amount or dose" or "sufficient / effective amount or dose," it is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0068] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health may continue to deteriorate.

[0069] As used herein, "pharmaceutically acceptable carrier" includes any material, which when combined with the conjugate retains the activity of the conjugate activity and is non-reactivewith the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions. Typically, such carriers contain excipients.

[0070] Excipients can be used in the invention for a wide variety of purposes, such as adjusting physical, chemical, or biological properties of formulations, such as adjustment of viscosity, and or processes of the invention to further improve effectiveness and or to further stabilize such formulations and processes against degradation and spoilage due to, for instance, stresses that occur during manufacturing, shipping, storage, pre-use preparation, administration, and thereafter. The term "excipient" generally includes fillers, binders, disintegrants, coatings, sorbents, anti-adherents, glidants, preservatives, antioxidants, solvents, co-solvents, buffering agents, chelating agents, viscosity imparting agents, surface active agents, diluents, humectants, carriers, diluents, preservatives, emulsifiers, stabilizers and tonicity modifiers.

[0071] Acceptable excipients are preferably pharmaceutically acceptable, i.e. nontoxic to recipients at the dosages and concentrations employed.

[0072] Exemplary excipients include, without limitation: amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine, including charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine preservatives, including antimicrobials such as antibacterial and antifungal agents antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium hydrogen-sulfite; buffers, buffer systems and buffering agents which are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8 or 9; examples of buffers are borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids, succinate, phosphate, histidine and acetate; for example Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5; non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; aqueous carriers including water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media; biodegradable polymers such as polyesters; bulking agents such as mannitol or glycine; chelating agents such as ethylenediamine tetraacetic acid (EDTA); isotonic and absorption delaying agents; complexing agents such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin) fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol; (low molecular weight) proteins, polypeptides or proteinaceous carriers such as human or bovine serum albumin, gelatin or immunoglobulins, preferably of human origin; coloring and flavouring agents; sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monothioglycerol, and sodium thio sulfate diluting agents; emulsifying agents; hydrophilic polymers such as polyvinylpyrrolidone) salt-forming counter-ions such as sodium; preservatives such as antimicrobials, anti-oxidants, chelating agents, inert gases and the like; examples are: benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); metal complexes such as Zn-protein complexes; solvents and co-solvents (such as glycerin, propylene glycol or polyethylene glycol); sugars and sugar alcohols, including polyols, trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol stachyose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; and polyhydric sugar alcohols; suspending agents; surfactants or wetting agents such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal; surfactants may be detergents, preferably with a molecular weight of >1.2 KD and / or a polyether, preferably with a molecular weight of >3 KD; non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples for preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000; stability enhancing agents such as sucrose or sorbitol; tonicity enhancing agents such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol; parenteral delivery vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils; intravenous delivery vehicles including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0073] It is evident to those skilled in the art that the different excipients of the pharmaceutical composition ( .g, those listed above) can have different effects, for example, and amino acid can act as a buffer, a stabilizer and / or an antioxidant; mannitol can act as a bulking agent and / or a 1tonicity enhancing agent; sodium chloride can act as delivery vehicle and / or tonicity enhancing agent; etc.

[0074] Polyols are useful stabilizing agents in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes, and are also useful for adjusting the tonicity of formulations. Polyols include sugars, e.g., mannitol, sucrose, and sorbitol and polyhydric alcohols such as, for instance, glycerol and propylene glycol, and, for purposes of discussion herein, polyethylene glycol (PEG) and related substances. Mannitol is commonly used to ensure structural stability of the cake in lyophilized formulations. It ensures structural stability to the cake. It is generally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucrose are commonly used agents for adjusting tonicity and as stabilizers to protect against freeze-thaw stresses during transport or the preparation of bulks during the manufacturing process. PEG is useful to stabilize proteins and as a cryoprotectant.

[0075] Surfactants routinely are used to prevent, minimize, or reduce surface adsorption. Protein molecules may be susceptible to adsorption on surfaces and to denaturation and consequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects generally scale inversely with protein concentration. These deleterious interactions generally scale inversely with protein concentration and typically are exacerbated by physical agitation, such as that generated during the shipping and handling of a product. Commonly used surfactants include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188. Surfactants also are commonly used to control protein conformational stability. The use of surfactants in this regard is protein-specific since, any given surfactant typically will stabilize some proteins and destabilize others.

[0076] Antioxidants can — to some extent — prevent deleterious oxidation of proteins in pharmaceutical formulations by maintaining proper levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can be used as well to prevent oxidative degradation of proteins. Among useful antioxidants in this regard are reducing agents, oxygen / free-radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations are preferably water-soluble and maintain their activity throughout the shelf life of a product. EDTA is a useful example.

[0077] Metal ions can act as protein co-factors and enable the formation of protein coordination complexes. Metal ions also can inhibit some processes that degrade proteins.

[0078] Salts may be used in accordance with the invention to, for example, adjust the ionic strength and / or the isotonicity of the pharmaceutical formulation and / or to further improve the solubility and / or physical stability of the antibody construct or other ingredient. As is well known, ions can stabilize the native state of proteins by binding to charged residues on the protein's surface and by shielding charged and polar groups in the protein and reducing the strength of their electrostatic interactions, attractive, and repulsive interactions. Furthermore, ionic interaction with charged and polar groups in a protein also can reduce intermolecular electrostatic interactions and, thereby, prevent or reduce protein aggregation and insolubility. Ionic species differ in their effects on proteins. A number of categorical rankings of ions and their effects on proteins have been developed that can be used in formulating pharmaceutical compositions in accordance with the invention. One example is the Hofmeister series, which ranks ionic and polar non-ionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are referred to as "kosmotropic." Destabilizing solutes are referred to as "chaotropic." Kosmotropes commonly are used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting-out"). Chaotropes commonly are used to denture and / or to solubilize proteins ("salting-in"). The relative effectiveness of ions to "salt-in" and "salt-out" defines their position in the Hofmeister series.

[0079] Free amino acids can be used in the pharmaceutical composition as stabilizers, and antioxidants, as well as other standard uses. Lysine, proline, serine, and alanine can be used for stabilizing proteins in a formulation. Glycine is useful in lyophilization to ensure correct cake structure and properties. Arginine may be useful to inhibit protein aggregation, in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0080] Exemplary useful excipients for formulating the pharmaceutical composition include sucrose, trehalose, mannitol, sorbitol, arginine, lysine, polysorbate 20, polysorbate 80, poloxamer 188, pluronic and combinations thereof. Said excipients may be present in the pharmaceutical composition in different concentrations, as long as the composition exhibits the desirable properties as exemplified herein, and in particular promotes stabilization of the contained bispecific single chain antibody constructs. For instance, sucrose may be present in thepharmaceutical composition in a concentration from about 2% (w / v) to about 12% (w / v), i.e. in a concentration of about 12% (w / v), 11% (w / v), 10% (w / v), 9% (w / v), 8% (w / v), 7% (w / v), 6% (w / v), 5% (w / v), 4% (w / v), 3% (w / v) or 2% (w / v). Preferred sucrose concentrations range from about 4% (w / v) to about 10% (w / v) and more preferably from about 6% (w / v) to about 10% (w / v). Polysorbate 80 may be present in the pharmaceutical composition in a concentration from about 0.001% (w / v) to about 0.5% (w / v), i.e. in a concentration of about 0.5% (w / v), 0.2% (w / v), 0.1% (w / v), 0.08% (w / v), 0.05% (w / v), 0.02% (w / v), 0.01% (w / v), 0.008% (w / v), 0.005% (w / v), 0.002% (w / v) or 0.001% (w / v). Preferred Polysorbate 80 concentrations range from about 0.002% (w / v) to about 0.5% (w / v), and preferably from about 0.005% (w / v) to about 0.02% (w / v).

[0081] The pharmaceutical composition provided herein may comprise one or more preservatives. Useful preservatives for formulating pharmaceutical compositions generally include antimicrobials (e.g. anti-bacterial or anti-fungal agents), anti-oxidants, chelating agents, inert gases and the like; examples are: benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide). Antimicrobial preservatives are substances which are used to extend the shelf-life of medicines by reducing microbial proliferation. Preservatives that particularly useful for formulating the pharmaceutical composition of the invention include benzyl alcohol, chlorobutanol, phenol, meta-cresol, methylparaben, phenoxyethanol, propylparaben thiomerosal. The structure and typical concentration for the use of these preservatives are described in Table 1 of Meyer et al. J Pharm Sci. 96(12), 3155. Compositions comprising such carriers are formulated by well-known conventional methods.

[0082] IgG-based therapeutics are generally administered alone at doses within a range of about 100 mg to about 2 g / kg / patient / dose of protein agent per infusion. The invention further provides for “facilitated” formulations, “facilitated” infusion of these formulations, and systems containing, and used for infusion of “facilitated” formulations. As used herein, “facilitated” refers to the co-administration or contemporaneous administration of a formulation of hyaluronidase (e.g., rHuPH20) and the IgA depleted IgG pharmaceutical formulation.

[0083] As used herein, “Hyaluronidase” refers to an enzyme catalyzing hydrolysis of glycosaminoglycans including hyaluronans. Included in this definition are naturally occurringhyaluronidases, and recombinant hyaluronidases, both human and from other sources. For clarity reference to Hyaluronidase refers to all forms. Hyalruonidases are included in the formulations, methods and combinations provides provided herein.

[0084] As used herein, “soluble hyaluronidase” refers to a polypeptide characterized by its solubility under physiologic conditions. Soluble hyalurondases include any, that, upon expression and secretion from a cell, exist in soluble form. Such soluble hyaluronidases include, but are not limited to, non-human soluble hyaluronidases, including those referred to as sHASEPGs), bacterial soluble hyaluronidases, bovine PH20, ovine PH20, and variants thereof. Included among the soluble hyaluronidases are human PH20 polypeptides that have been been modified, generally by C-terminal truncation, so that they are secreted when expressed and are soluble. For example, hyaluronidases, such as human PH20, that contain a glycophophatidylinositol (GPI) anchor can be made soluble by truncation of and removal of all or a portion of the GPI anchor. In one example, the human hyaluronidase PH20, which is normally membrane anchored via a GPI anchor, is made soluble by truncation of and removal of all or a portion of the GPI anchor at the C-terminus. Exemplary of a soluble hyaluronidase is soluble human PH20. Soluble forms of recombinant human PH20 have been produced and can be used in the compositions, combinations and methods described herein. The description of and production of such soluble forms of PH20 is described, for example, in U.S. Patent Nos. 7,767,429, 8,202,517, 8,431,380, 8,431,124, 8,450,470 8,765,685, 8,772,246, 7,871,607, 7,846,431, 7,829,081, 8,105,586, 8,187,855, 8,257,699, 8,580,252, 9,677,061, and 9,677,062 which are incorporated by reference herein.

[0085] An exemplary hyaluronidase of use in the kits and methods disclosed herein is “HuPH20”, referring to human hyaluronidase, e.g., human recombinant hyaluronidase (rHuPH20).III. Compositions

[0086] As routinely practiced in modern medicine, sterilized preparations of concentrated immunoglobulins (especially IgGs) are used for treating medical conditions that fall into three main classes: immune deficiencies, inflammatory and autoimmune diseases, and acute infections. One commonly used IgG product (IGI), intravenous immunoglobulin or IVIG, is formulated for intravenous administration, for example, at a concentration of at or about 10%IgG. Concentrated immunoglobulins may also be formulated for subcutaneous or intramuscular administration, for example, at a concentration at or about 20% IgG. For ease of discussion, such subcutaneously or intramuscularly formulated IgG compositions are also included in the term "IVIG" or “IGI” in this application.

[0087] In various embodiments, the present invention provides pharmaceutical compositions and formulations of IgG compositions prepared according to the improved manufacturing methodologies provided herein. In certain embodiments, these compositions and formulations provide improved properties as compared to other IGI, e.g., IVIG, compositions currently on the market. For example, in certain embodiments, the compositions and formulations provided herein are stable for an extended period of time. In some embodiments, the improved property is an IgA content reduced relative to the starting IgG solution, e.g., an anion exchange precursor solution

[0088] In exemplary embodiments, the present invention provides a storage-stable IgA depleted IgG pharmaceutical formulation derived from human plasma. In some embodiments, IgA is not more than about 2 pg / mL, not more than about 1.9 pg / mL, not more than about 1.8 pg / mL, not more than about 1.7 pg / mL, not more than about 1.6 pg / mL, not more than about 1.5 pg / mL, not more than about 1.4 pg / mL, not more than about 1.3 pg / mL, not more than about 1.2 pg / mL, not more than about 1.1 pg / mL, not more than about 1 pg / mL, not more than about 0.9 pg / mL, not more than about 0.8 pg / mL, not more than about 0.7 pg / mL, not more than about 0.6 pg / mL, not more than about 0.5 pg / mL, not more than about 0.4 pg / mL, not more than about 0.3 pg / mL, not more than about 0.2 pg / mL, not more than about 0.1 pg / mL, not more than about 0.01 pg / mL, or less. In exemplary embodiments, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 2 pg / mL. In another exemplary embodiments, IgA content of a pharmaceutical formulation of IgG of the invention is not more than about 1.8 pg / mL.

[0089] In exemplary embodiments, IgA content in the IgA is not more than about 2 pg / mL. In some embodiments, IgA is from about 0 pg / mL to about 2 pg / mL, for example, about 0 pg / mL to about 1.9 pg / mL, about 0 pg / mL to about 1.8 pg / mL, about 0 pg / mL to about 1.7 pg / mL, about 0 pg / mL to about 1.6 pg / mL, about 0 pg / mL to about 1.5 pg / mL, about 0 pg / mL to about 1.4 pg / mL, about 0 pg / mL to about 1.3 pg / mL, about 0 pg / mL to about 1.2 pg / mL, about 0 pg / mL to about 1.1 pg / mL, about 0 pg / mL to about 1.0 pg / mL, about 0 pg / mL to about 0.9gg / mL, about 0 gg / mL to about 0.8 gg / mL, about 0 gg / mL to about 0.7 gg / mL, about 0 gg / mL to about 0.6 gg / mL, about 0 gg / mL to about 0.5 gg / mL, about 0.5 gg / mL to about 2 gg / mL, about 0.5 gg / mL to about 1.9 gg / mL, about 0.5 gg / mL to about 1.8 gg / mL, about 0.5 gg / mL to about 1.7 gg / mL, about 0.5 gg / mL to about 1.6 gg / mL, about 0.5 gg / mL to about 1.5 gg / mL, about 0.5 gg / mL to about 1.4 gg / mL, about 0.5 gg / mL to about 1.3 gg / mL, about 0.5 gg / mL to about 1.2 gg / mL, about 0.5 gg / mL to about 1.1 gg / mL, or about 0.5 gg / mL to about 1.0 gg / mL. In some embodiments, IgA is not measurable by conventional analytical methods.

[0090] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 gg / mL of IgA) comprises the purified IgG in an aqueous pharmaceutically acceptable carrier.

[0091] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 gg / mL of IgA) has a property selected from a pH of from about 4.0 to about 5.5, from about 0.01 M to about 1.0 M glycine, and a combination thereof. In some embodiments, the formulation has a property selected from a pH of from about 4.4 to about 5.1, from about 0.1 M to about 0.4 M glycine, and a combination thereof. In exemplary embodiments, the storage-stable IgA depleted IgG pharmaceutical formulation has a property selected from a pH of from about 4.6 to about 5.1, from about 0.2 M to about 0.3 M glycine, and a combination thereof. The pH value always refers to a ready to use formulation.

[0092] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 gg / mL of IgA) has a pH of from about 4.0 to about 5.5, for example, about 4.1 to about 5.5, about 4.2 to about 5.5, about 4.3 to about 5.5, about 4.4 to about 5.5, about 4.4 to about 5.4, about 4.4 to about 5.3, about 4.4 to about 5.2, about 4.4 to about 5.1, about 4.4 to about 5.0, about 4.4 to about 4.9, about 4.0 to about 5.4, about 4.0 to about 5.3, about 4.0 to about 5.2, about 4.0 to about 5.1, about 4.0 to about 5.3, about 4.0 to about 5.2, about 4.0 to about 5.1, about 4.0 to about 5.0, or about 4.0 to about 4.9. In some embodiments, the pH is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. In some embodiments, the pH is not more than about 5.1. In some embodiments, the pH is not less than about 4.4. In some embodiments, the pH is from about 4.4 to about 5.1. Inexemplary embodiments, the pH is from about 4.4 to about 4.9. In some embodiments, the pH is from about 4.6 to about 5.1

[0093] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) contains glycine from about 0.01 M to about 1.0 M, for example, about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments glycine is about 0.01 M, about 0.05 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, or about 1.0 M. In some embodiments, glycine is not more than about 0.4 M. In some embodiments, glycine is not less than about 0.1 M. In exemplary embodiments, glycine is from about 0.2 M to about 0.3 M. In exemplary embodiments, glycine is from about 0.1 M to about 0.4 M.

[0094] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) has a pH of from about 4.0 to about 5.5 and contains glycine from about 0.01 M to about 1.0 M. In some embodiments, the pH is about 4.0 to about 5.5 and contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.1 to about 5.5 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.2 to about 5.5 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M,about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.3 to about 5.5 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 5.5 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 5.4 and contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 5.3 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 5.2 and contains it glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 5.1 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.4 to about 5.0 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.4 to about 4.9 and contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.0 to about 5.4 and contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.0 to about 5.3 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is from about 4.0 to about 5.2 and contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.0 to about 5.1 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.0 to about 5.3 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pHof the formulation is about 4.0 to about 5.2 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.0 to about 5.1 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.0 to about 5.0 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In some embodiments, the pH of the formulation is about 4.0 to about 4.9 and it contains glycine from about 0.1 M to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.2 M to about 0.3 M, or about 0.2 M to about 0.4 M. In exemplary embodiments, the formulation has a property selected from a pH of from about 4.6 to about 5.1, from about 0.2 M to about 0.3 M glycine, and a combination thereof.

[0095] In various embodiments of the present invention, the storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) contains at least about 5% (w / v) and up to about 25% (w / v) IgG and / or total protein, for example, about 5% (w / v) to about 20% (w / v), about 5% (w / v) to about 15% (w / v), about 5% (w / v) to about 10% (w / v), about 5% (w / v) to about 15% (w / v), about 7% (w / v) to about 13% (w / v), about 9% (w / v) to about 11% (w / v). In some embodiments, IgG and / or total protein is about 9% (w / v), about 10% (w / v), about 11% (w / v), about 12% (w / v), about 13% (w / v), about 14% (w / v), about 15% (w / v), about 16% (w / v), about 17% (w / v), about 18% (w / v), about 19% (w / v), or about 20% (w / v). In exemplary embodiments, IgG and / or total protein is about 10% (w / v). In exemplaryembodiments, IgG and / or total protein is from about 9% (w / v) to about 11% (w / v). In exemplary embodiments, IgG and / or total protein is about 20% (w / v). In exemplary embodiments, IgG and / or total protein is from about 19% (w / v) to about 21% (w / v).

[0096] In various embodiments, the methods provided herein allow for the preparation of IgG pharmaceutical compositions having very high levels of purity. For example, in one embodiment, at least about 95% of the total protein in a composition provided herein will be IgG. In other embodiments, at least about 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5%, or more of the total protein of the composition will be IgG. In a preferred embodiment, at least 97% of the total protein of the composition will be IgG. In another preferred embodiment, at least 98% of the total protein of the composition will be IgG. In another preferred embodiment, at least 99% of the total protein of the composition will be IgG.

[0097] In various embodiments, the methods provided herein allow for the preparation of IgG pharmaceutical compositions having very high ratios of IgG to other Igs. For example, in one embodiment, at least about 95% of the total Ig in a composition provided herein will be IgG. In other embodiments, at least about 96% of the Ig is IgG, or at least about 97%, 98%, 99%, 99.5%, or more of the total Ig in the composition will be IgG. In a preferred embodiment, at least 97% of the total Ig in the composition will be IgG. In another preferred embodiment, at least 98% of the total Ig in the composition will be IgG. In another preferred embodiment, at least about 99% of the total Ig in the composition will be IgG.

[0098] In some embodiments, the pharmaceutical compositions provided herein may optionally further comprise an agent for adjusting the osmolarity of the composition. Non-limiting examples of osmolarity agents include mannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose, fructose, lactose, polyethylene glycols, phosphates, sodium chloride, potassium chloride, calcium chloride, calcium gluconoglucoheptonate, dimethyl sulfone, and the like.

[0099] Exemplary formulations provided herein will have osmolarities that are comparable to physiologic osmolarity, about 285 to about 295 mOsmol / kg (Lacy et al., Drug Information Handbook— Lexi-Comp 1999:1254. In certain embodiments, the osmolarity of the formulation will be between about 200 mOsmol / kg and about 350 mOsmol / kg, preferably between about 240 and about 300 mOsmol / kg. In particular embodiments, the osmolarity of the formulation will beabout 200 mOsmol / kg, or 210 mOsmol / kg, 220 mOsmol / kg, 230 mOsmol / kg, 240 mOsmol / kg, 245 mOsmol / kg, 250 mOsmol / kg, 255 mOsmol / kg, 260 mOsmol / kg, 265 mOsmol / kg, 270 mOsmol / kg, 275 mOsmol / kg, 280 mOsmol / kg, 285 mOsmol / kg, 290 mOsmol / kg, 295 mOsmol / kg, 300 mOsmol / kg, 310 mOsmol / kg, 320 mOsmol / kg, 330 mOsmol / kg, 340 mOsmol / kg, 340 mOsmol / kg, or 350 mOsmol / kg.

[0100] The pharmaceutical compositions provided herein will typically comprise one or more buffering agents or pH stabilizing agents suitable for intravenous, subcutaneous, and / or intramuscular administration. Non-limiting examples of buffering agents suitable for formulating an IgG composition provided herein include glycine, citrate, phosphate, acetate, glutamate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, malate, succinate, formate, propionate, carbonate, or any combination thereof adjusted to an appropriate pH. Generally, the buffering agent will be sufficient to maintain a suitable pH in the formulation for an extended period of time. In an exemplary embodiment, the buffering agent is glycine.

[0101] In various embodiments of the present invention, a storage-stable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) is stable at from about 28 °C to about 32 °C for about 6 months to about two years. In some embodiments, the formulation is stable for at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, or more. In various embodiments, the formulation is stable at from about 0 °C to about 10 °C for at least about 6 months to about 3 years. In some embodiments, the formulation is stable for at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years or more at from about 2°C to about 8°C.

[0102] In various embodiments, a storage-stable IgA depleted IgG formulation (IGI) is formatted for intravenous immunoglobulin (IVIG) administration. In various embodiments, the formulation (IGI) is formatted for subcutaneous immunoglobulin (SCIG) administration. In exemplary embodiments, the formulation is useful to treat immunoglobulin-treatable diseases and conditions, particularly in IgA-sensitive patients in need thereof.

[0103] In various embodiments, the invention provides IgA depleted IgG pharmaceutical formulations analogous to marketed IgG formulations, or formulations in the clinic in every way with the exception of the lower IgA concentration of the inventive formulations.

[0104] Exemplary of an IgG preparation is Immune Globulin Intravenous (Human), 10% (IVIG, 10%, marketed as GAMMAGARD® LIQUID, Takeda Pharmaceuticals U.S.A., Inc.), which is a liquid unmodified IgG preparation, with a distribution of IgG subclasses similar to that of normal plasma. The preparations contain 100 mg / mL protein, with at least about 98% being IgG. The formulation has an osmolality similar to physiologic osmolality, and contains no added sugars, sodium, or preservatives. It is formulated with glycine for stabilization at a pH of 4.6 to 5.1. The manufacturing process employs a modified Cohn-Oncley cold alcohol fractionation procedure and further purifications by a continuous process through the use of weak cation exchange chromatography and weak anion exchange chromatography. The manufacturing process also includes 3 independent viral inactivation or removal steps: solvent / detergent (S / D) treatment, nanofiltration, and incubation at a low pH and elevated temperature. in. Kits

[0105] As a means to improve and facilitate the overall patient experience of subjects infusing a concentrated IgG formulation, in various embodiments, the invention provides a kit including a first container comprising a pharmaceutical formulation of hyaluronidase, e.g., human hyaluronidase, e.g., recombinant human hyaluronidase in a pharmaceutically acceptable carrier, a second container comprising a storage-stable IgA depleted IgG pharmaceutical formulation in a pharmaceutically acceptable carrier (e.g., an aqueous pharmaceutically acceptable carrier), and instructions providing guidance for sequentially subcutaneously infusing into a first infusion site, (i), a first aliquot of a pre-determined dosage of the pharmaceutical formulation of recombinant human hyaluronidase and, (ii), following (i), a first aliquot of a pre- determined dosage of the storage-stable IgA depleted IgG pharmaceutical formulation described herein.

[0106] In various embodiments, pre- determined dosage of the pharmaceutical formulation of recombinant human hyaluronidase contains about 160 U / mL hyaluronidase, e.g, recombinant human hyaluronidase. In an exemplary embodiment, the recombinant human hyaluronidase is rHuPH20.

[0107] In some embodiments, the kit includes instructions on how to infuse from about 100 mb to about 300 mb of the IgA depleted IgG formulation of the invention at a rate of at least about 120 mL / hr, at least about 150 mL / hr, at least about 200 mL / hr, at least about 250 mL / hr, or at least about 300 mL / hr. In exemplary embodiments, the instructions provide guidance for subcutaneously infusing at least about 120 mb of the storage-stable IgA depleted IgG pharmaceutical formulation to the first infusion site at a rate of at least about 120 mL / hr, at least about 150 mL / hr, at least about 200 mL / hr, at least about 250 mL / hr, or at least about 300 mL / hr.V. Methods of Manufacture

[0108] In exemplary embodiments, the present invention provides methods of forming a storagestable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) of the invention.

[0109] Generally, immunoglobulin preparations according to the present invention can be prepared from any suitable starting materials, for example, recovered plasma or source plasma. In an exemplary embodiment, the plasma is a spray dried plasma, which is reconstituted. In a typical example, blood or plasma is collected from healthy donors. Usually, the blood is collected from the same species of animal as the subject to which the immunoglobulin preparation will be administered (typically referred to as “homologous” immunoglobulins). The immunoglobulins are isolated from the blood by suitable procedures, such as, for example, precipitation (alcohol fractionation or polyethylene glycol fractionation), chromatographic methods (ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, etc. ultracentrifugation, and electrophoretic preparation, and the like. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459-75 (1946); Oncley etal., J. Am. Chem. Soc. 71:541- 50 (1949); Barundern et al. , Vox Sang. 7:157-74 (1962); Koblet eta / ., Vox Sang. 13:93-102 (1967); U.S. Patent Nos. 5,122,373 and 5,177,194; the disclosures of which are hereby incorporated by reference in their entireties for all purposes).

[0110] In many cases, immunoglobulins are prepared from gamma globulin-containing products produced by alcohol fractionation and / or ion exchange and affinity chromatography methods well known to those skilled in the art. For example, purified Cohn Fraction II is commonly used as a starting point for the isolation of immunoglobulins. The starting CohnFraction II paste is typically about 95 percent IgG and is comprised of the four IgG subtypes. The different subtypes are present in Fraction II in approximately the same ratio as they are found in the pooled human plasma from which they are obtained. The Fraction II is further purified before formulation into an administrable product. For example, the Fraction II paste can be dissolved in a cold purified aqueous alcohol solution and impurities removed via precipitation and filtration. Following the final filtration, the immunoglobulin suspension can be dialyzed or diafiltered (e.g., using ultrafiltration membranes having a nominal molecular weight limit of less than or equal to 100,000 Daltons) to remove the alcohol. The solution can be concentrated or diluted to obtain the desired protein concentration and can be further purified by techniques well known to those skilled in the art.

[0111] Methods for purifying and concentrating IgG using ion exchange chromatography are well known in the art. For example, U.S. Patent No. 5,886,154 describes a method in which a Fraction II+III precipitate is extracted at low pH (between about 3.8 and 4.5), followed by precipitation of IgG using caprylic acid, and finally implementation of two anion exchange chromatography steps. U.S. Patent No. 6,069,236 describes a chromatographic IgG purification scheme that does not rely on alcohol precipitation at all. PCT Publication No. WO 2005 / 073252 describes an IgG purification method involving the extraction of a Fraction II+III precipitate, caprylic acid treatment, PEG treatment, and a single anion exchange chromatography step. U.S. Patent No. 7,186,410 describes an IgG purification method involving the extraction of either a Fraction 1+11+111 or a Fraction II precipitate followed by a single anion exchange step performed at an alkaline pH. U.S. Patent No. 7,553,938 describes a method involving the extraction of either a Fraction I+II+III or a Fraction II+III precipitate, caprylate treatment, and either one or two anion exchange chromatography steps. U.S. Patent No. 6,093,324 describes a purification method comprising the use of a macroporous anion exchange medium operated at a pH between about 6.0 and about 6.6. U.S. Patent No. 6,835,379 describes a purification method that relies on cation exchange chromatography in the absence of alcohol fractionation. The disclosures of the above publications are hereby incorporated by reference in their entireties for all purposes.

[0112] Furthermore, additional preparative steps can be used to enrich a particular isotype or subtype of immunoglobulin. For example, protein A, protein G or protein H Sepharose chromatography can be used to enrich a mixture of immunoglobulins for IgG, or for specific IgG31subtypes. See generally Harlow and Lane, Using Antibodies, Cold Spring Harbor Laboratory Press (1999); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); and U.S. Patent No. 5,180,810, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0113] In one aspect, the present invention provides methods of preparing concentrated IgG compositions that utilize a cryo-poor starting material. Exemplary methods provided herein utilize both modified Cohn-Oncley alcohol fractionation steps and ion exchange chromatography to provide superior IgG yields, while maintaining the same, if not improved, quality as found in currently available commercial IGI, e.g., IVIG, preparations. An exemplary improved quality is a lower content of non-IgG immunoglobulins, e.g., IgA.

[0114] In various embodiments, the present invention provides methods of forming a storagestable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA), e.g., by using anion exchange on anion exchange precursor solution, with an IgG yield compared to IgG present in a starting plasma sample of at least about 50%. In some embodiments, the yield is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater.

[0115] In various embodiments, the present invention provides methods of forming a storagestable IgA depleted IgG pharmaceutical formulation (e.g., not more than about 2 pg / mL of IgA) by (a) performing an anion exchange step on an anion exchange precursor solution comprising the IgG, the step comprising: (i) submitting the anion exchange precursor solution to anion exchange chromatography in a single pass through a first portion of an anion exchange medium contained in a first column and collecting a first anion exchange flowthrough from the first column.

[0116] In various embodiments of the methods described above, the step further comprises (ii), following (i), washing the anion exchange medium with a wash buffer, wherein the washing uses from about 0.25 column volumes to about 5 column volumes of the wash buffer, for example, about 0.25 to about 4 column volumes, about 0.25 to about 3 column volumes, about 0.25 to about 2 column volumes, about 0.25 to about 1 column volumes, about 0.50 to about 5 columnvolumes, about 0.75 to about 5 column volumes, about 1 to about 5 column volumes, about 2 to about 5 column volumes, or about 3 to about 5 column volumes. In some embodiments, the washing uses about 0.25, about 0.50, about 0.75, about 1, about 2, about 3, about 4, or about 5 column volumes of the wash buffer. In exemplary embodiments, the washing uses from about 0.25 column volumes to about 3 column volumes of the wash buffer.

[0117] In various embodiments of the methods described above, the single pass through the anion exchange medium occurs in both a first column, and in a second column containing a second portion of the anion exchange medium. In some embodiments, the first and second columns are run in serial mode. In some embodiments, the first and second columns are run in parallel mode. In some embodiments, in parallel mode a second anion exchange flowthrough is collected from the second column.

[0118] In various embodiments of the methods described above, the IgG is loaded onto the anion exchange medium at a maximum loading of about 150 mg protein / mL of medium. In some embodiments, the maximum loading is about 150 mg protein / mL, about 140 mg / mL, about 130 mg / mL, about 120 mg / mL, about 110 mg / mL, about 100 mg / mL, about 90 mg / mL, about 80 mg / mL, about 70 mg / mL, about 60 mg / mL, about 50 mg protein / mL, or less. In some embodiments, the maximum loading is from about 25 mg protein / mL to about 150 mg / mL of protein, from about 50 mg protein / mL to about 150 mg / mL of protein, from about 25 mg protein / mL to about 125 mg / mL of protein, from about 25 mg protein / mL to about 100 mg / mL of protein, or from about 25 mg protein / mL to about 75 mg / mL of protein. In exemplary embodiments, the maximum loading is less than about 100 mg protein / mL of medium. In exemplary embodiments, the maximum loading is equal to and / or is less than about 70 mg protein / mL of medium. In exemplary embodiments, the maximum loading is equal to and / or is less than about 50 mg protein / mL of medium. The person skilled in the art will acknowledge that the explicitly maximum loading is also dependent on the individual anion exchange medium used in the method and will adjust the maximum loading accordingly.

[0119] In various embodiments of the methods described above, the first and second anion exchange flowthrough are pooled. In some embodiments, the first and / or second and / or additional anion exchange flowthrough are pooled. In some embodiments, several anionexchange flowthrough are pooled. In some embodiments of the method described above, the first and second anion exchange flowthrough are not pooled.

[0120] As will be appreciated by those of skill in the art, any reference to “a first” and “a second” are exemplary and the steps of the disclosed procedure can be repeated any desirable number of times, using any useful number and / or configuration of components in a combination and order. Thus, for example, anion exchange can be performed using “a third”, and / or “a fourth” portion of anion exchange medium with columns arranged in serial, parallel or a combination thereof.

[0121] In various embodiments of the methods described above, the pH of the anion exchange precursor solution is from about 6.2 to about 7.0, for example from about 6.2 to about 6.9, about6.3 to about 7.0, from about 6.4 to about 7.0, from about 6.4 to about 7.0, from about 6.5 to about 7.0, from about 6.7 to about 7.0, from about 6.8 to about 7.0, about 6.3 to about 6.9, from about6.4 to about 6.9, from about 6.4 to about 6.9, from about 6.5 to about 6.9, or from about 6.7 to about 6.9. In some embodiments, the pH is about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In exemplary embodiments, the pH is from about6.4 to about 7.0. In exemplary embodiments, the pH is from about 6.7 to about 6.9. In exemplary embodiments, the pH is about 6.8.

[0122] In various embodiments of the methods described above, the conductivity of the anion exchange precursor solution is from about 0.5 mS / cm to about 2.5 mS / cm. In some embodiments, the conductivity is about 0.5 mS / cm, about 0.6 mS / cm, about 0.7 mS / cm, about 0.8 mS / cm, about 0.9 mS / cm, about 1.0 mS / cm, about 1.1 mS / cm, about 1.2 mS / cm, about 1.3 mS / cm, about 1.4 mS / cm, about 1.5 mS / cm, about 1.6 mS / cm, about 1.7 mS / cm, about 1.8 mS / cm, about 1.9 mS / cm, about 2.0 mS / cm, about 2.1 mS / cm, about 2.2 mS / cm, about 2.3 mS / cm, about 2.4 mS / cm, or about 2.5 mS / cm. In exemplary embodiments, the conductivity is from about 0.7 mS / cm to about 1.5 mS / cm. In exemplary embodiments, the conductivity is from about 0.8 mS / cm to about 1.0 mS / cm. In exemplary embodiments, the conductivity is about 0.9 mS / cm.

[0123] Any of the features of the anion exchange precursor described herein may be combined in any practical manner. For example, in various embodiments of the methods described above, thepH of the anion exchange precursor solution is from about 6.2 to about 7.0, the buffer salt concentration from about 60 mM to about 140 mM, and the conductivity of the anion exchange precursor solution is from about 0.5 mS / cm to about 2.5 mS / cm. In exemplary embodiments, the pH is from about 6.4 to about 7.0, the buffer salt concentration from about 80 mM to about 120 mM, and the conductivity is from about 0.7 mS / cm to about 1.5 mS / cm. In exemplary embodiments, the pH is from about 6.7 to about 6.9, the buffer salt concentration from about 90 mM to about 110 mM, and the conductivity is from about 0.8 mS / cm to about 1.0 mS / cm. In exemplary embodiments, the pH is about 6.8, the buffer salt concentration about 100 mM, and the conductivity is about 0.9 mS / cm.

[0124] Exemplary anion exchange materials of use in the invention include those with strong anion exchange groups, such as trimethylammonium chloride and weak anion exchange groups, such as N5N diethylamino or DEAE.

[0125] Though various levels of protein loading on the column are of use, in an exemplary embodiment, the anion exchange precursor solution is loaded onto the column at a protein loading of from about 20 to about 40 mg protein / mL resin, e.g. from about 25 to about 35 mg protein / mL resin.

[0126] In an exemplary embodiment, about 25 to about 35 mg protein / mL resin is loaded on a column at a pH from about 6.4 to about 6.8. The column is washed with a first equilibration buffer having a concentration of from about 80 mM to about 120 mM of a phosphate buffer salt, e.g., monosodium phosphate, and a pH of from about 6 to about 7, e.g. about 6.4 to about 6.8. The wash solution from the column is collected and contains at least a portion of the IgG loaded onto the column in the anion exchange precursor, and contains an IgA content reduced relative to the IgA content of the anion exchange precursor.

[0127] In some embodiments, to maintain an appropriate protein loading and a convenient column size, the anion exchange precursor is loaded onto more than one column, e.g., 2, 3, 4, or more columns, e.g., linked in parallel.

[0128] Following loading onto the column, the anion exchange precursor is processed with a first equilibration buffer. The characteristics of the anion exchange precursor, and the first and second equilibration buffers are as set forth herein.

[0129] In various embodiments of the methods described above, the first equilibration buffer comprises monobasic sodium phosphate from about 60 mM to about 120 mM, for example, from about 65 mM to about 115 mM, from about 70 mM to about 115 mM, from about 75 mM to about 110 mM, from about 80 mM to about 100 mM, from about 85 mM to about 95 mM.

[0130] In various embodiments of the methods described above, the first equilibration buffer has a pH of from about 6.0 to about 8.0, for example from about 6.2 to about 7.8, from about 6.4 to about 7.6, from about 6.6 to about 7.4, from about 6.8 to about 7.2. In some embodiments, the first equilibration buffer has a pH of about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 7.0, about 7.2, about 7.4. In exemplary embodiments, the first equilibration buffer has a pH of from about 6.6 to about 7.2. In exemplary embodiments, the first equilibration buffer has a pH of about 6.8 to about 7.0.

[0131] In various embodiments of the methods described above, the first equilibration buffer has a conductivity of from about 0.1 mS / cm to about 2.0 mS / cm, for example, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.4 mS / cm, from about 0.4 mS / cm to about 1.3 mS / cm, from about 0.5 mS / cm to about 1.2 mS / cm, from about 0.6 mS / cm to about 1.0 mS / cm, from about 0.7 mS / cm to about 0.9 mS / cm. In some embodiments, the first equilibration buffer has a conductivity of about 0.6 mS / cm, about 1.2 mS / cm. In exemplary embodiments, the anion exchange elution buffer has a conductivity of about 0.9 ± 0.1 mS / cm.

[0132] Any of the features of the first equilibration buffer described hereinabove may be combined in any practical manner. For example, in various embodiments of the methods described herein, the first equilibration buffer has a pH of from about 5.5 to about 7.5 and a conductivity of about 0.5 mS / cm to about 1.5 mS / cm. In some embodiments, the anion exchange elution buffer has a pH of from about 6.0 to about 7.0 and a conductivity of about 0.6 mS / cm to about 1.2 mS / cm. In some embodiments, the first equilibration buffer has a pH of from about 6.2 to about 7.2 and a conductivity of about 0.6 mS / cm to about 1.0 mS / cm. In exemplary embodiments, the first equilibration buffer comprises monobasic sodium phosphate (e.g., from about 50 mM to about 150 mM) with a pH of about 6.8 ± 0.2 and / or a conductivity of about 0.9 ± 0.2 mS / cm.

[0133] In an exemplary embodiment, the anion exchange precursor is loaded on one or more ANX columns as one or more aliquots at a pH of from about 6 to about 7, e.g. about 6.4 to about 6.8. An exemplary anion chromatography precursor has a pH of about 6.8. An exemplary ANX column is equilibrated with a first equilibration buffer. An exemplary first equilibration buffer has a concentration of from about 80 mM to about 120 mM of the buffer salt. An exemplary buffer salt is phosphate, e.g., monosodium phosphate. An exemplary buffer has a pH of from about 6 to about 7, e.g. about 6.4 to about 6.8. An exemplary first equilibration buffer has a pH of about 6.8.

[0134] In an exemplary embodiment, the IgG is collected in the column flow-through in the first equilibration buffer over from about 1 to about 5 column volumes, e.g., about 3 column volumes.

[0135] In an exemplary embodiment, the column(s) is / are further contacted with a second equilibration buffer. An exemplary second equilibration buffer includes two or more buffer salts. An exemplary buffer salt pair is phosphate, e.g., monosodium phosphate and acetate, e.g., sodium acetate. An exemplary second equilibration buffer has a pH of from about 6 to about 7, e.g. about 6.4 to about 6.8. An exemplary second equilibration buffer has a pH of 6.8. An exemplary second equilibration buffer has a conductivity of from about 0.7 to about 1.2 mS / cm, e.g., from about 0.8 to about 1.0 mS / cm. An exemplary second equilibration buffer has a conductivity of about 0.9 mS / cm.

[0136] In an exemplary embodiment, the IgG comes off the column during washing with the first equilibration buffer, and is collected. In an exemplary embodiment, the IgG comes off the column during washing with the second equilibration buffer, and is collected. In an exemplary embodiment, the IgG comes off the column during washing with both the first and second equilibration buffer, and is collected. In an exemplary embodiment, the wash with the second equilibration buffer is discarded.

[0137] In various embodiments of the methods described above, the IgG passes from the anion chromatography medium in the flow-through with the first equilibration buffer and is collected in the anion exchange flow-through.

[0138] In various embodiments of the methods described above, the protein concentration of the anion exchange flowthrough is from about 1 g / L to about 20 g / L, for example, from about 1 g / Lto about 19 g / L, from about 1 g / L to about 18 g / L, from about 1 g / L to about 17 g / L, from about 1 g / L to about 16 g / L, from about 1 g / L to about 15 g / L, from about 1 g / L to about 14 g / L, from about 1 g / L to about 13 g / L, from about 1 g / L to about 12 g / L, from about 1 g / L to about 10 g / L, from about 2 g / L to about 20 g / L, from about 3 g / L to about 20 g / L, from about 4 g / L to about 20 g / L, from about 1 g / L to about 15 g / L, from about 2 g / L to about 15 g / L, or from about 3 g / L to about 15 g / L. In some embodiments, the protein concentration is about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L, about 14 g / L, about 15 g / L, about 16 g / L, about 17 g / L, about 18 g / L, about 19 g / L, or about 20 g / L. In exemplary embodiments, the protein concentration is from about 3 g / L to about 15 g / L. An exemplary protein concentration is an IgG concentration.

[0139] In various embodiments of the methods described herein, the anion exchange precursor solution is an eluate from a cation exchange step of an IgG containing suspension preceding (a). In exemplary embodiments, the eluate from a cation exchange step preceding (a) is an eluate from one or more cation exchange columns. In some embodiments, one or more cation exchange columns is about 1, about 2, about 3, about 4, about 5, about 6, or more cation exchange columns.

[0140] In various embodiments of the methods described herein, the IgG containing suspension is from Precipitate G. In exemplary embodiments, the anion exchange precursor solution is an eluate from a cation exchange step of Precipitate G preceding (a). In various embodiments of the methods described above, Precipitate G is prepared according to U.S. Patent No. 8,304,524, U.S. Patent No. 8,993,734, or U.S. Patent No. 8,772,462.

[0141] In various embodiments of the methods described above, a precursor solution for the cation exchange step is a mixture of Precipitate G, a solvent, and a detergent. In some embodiments, Precipitate G is dissolved and / or suspended in the mixture. In some embodiments, Precipitate G is clarified. In some embodiments, the solvent and the detergent are mixture of octoxynol 9, polysorbate 80, and tri-(n-butyl) phosphate, and / or equivalent.

[0142] In various embodiments of the methods described herein, octoxynol 9 and / or equivalent is present at from about 0.1% (v / v) to about 2.0% (v / v). In some embodiments, octoxynol 9and / or equivalent is present at about 0.1% (v / v), about 0.2% (v / v), about 0.3%, about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), about 1.0% (v / v), about 1.1% (v / v), about 1.2% (v / v), about 1.3% (v / v), about 1.4% (v / v), about 1.5% (v / v), about 1.6% (v / v), about 1.7% (v / v), about 1.8% (v / v), about 1.9% (v / v), or about 2.0% (v / v). In exemplary embodiments, octoxynol 9 and / or equivalent is present at about 1.0 ± 0.5% (v / v),

[0143] In various embodiments of the methods described herein, polysorbate 80 and / or equivalent is present at from about 0.1% (v / v) to about 1% (v / v). In some embodiments, polysorbate 80 and / or equivalent is present at about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), or about 1.0% (v / v). In exemplary embodiments, polysorbate 80 and / or equivalent is present at about 0.3 ± 0.15% (v / v).

[0144] In various embodiments of the methods described herein, tri-(n-butyl) phosphate and / or equivalent is present at from about 0.1% (v / v) to about 1% (v / v). In some embodiments of the method described above, tri-(n-butyl) phosphate and / or equivalent is present at about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), about 0.5% (v / v), about 0.6% (v / v), about 0.7% (v / v), about 0.8% (v / v), about 0.9% (v / v), or about 1.0% (v / v). In exemplary embodiments, tri-(n-butyl) phosphate and / or equivalent is present at about 0.3 ± 0.15% (v / v).

[0145] In exemplary embodiments of the methods described above, octoxynol 9 is present at about 1.0 ±0.5% (v / v), polysorbate 80 is present at about 0.3 ± 0.15% (v / v), and tri-(n-butyl) phosphate is present at about 0.3 ± 0.15% (v / v), and / or equivalent.

[0146] In various embodiments, in order to solubilize the IgG content of the Precipitate G, a cold extraction buffer is used to re-suspend the Precipitate G. Briefly, the Precipitate G is dissolved 1 to 3.5 in Water for Injection (WFI) (w / v or equal to w / w when assuming p(WFI) being 1) at from about above 0 °C to about 8 °C (e.g., from about 2 °C to about 8 °C) to achieve an AU280-320 value of between about 40 to 95. The final pH of the solution, which is stirred for at least 2 hours, is then adjusted to at or about 5.2 ± 0.2. In some embodiments, this pH adjustment is performed with 1 M acetic acid. To increase the solubility of IgG, the conductivity of the suspension is increased to from about 2.5 to about 6.0 mS / cm. In one embodiment, theconductivity is increased by the addition of sodium chloride. The suspended Precipitate G solution is then filtered with a suitable depth filter having a nominal pore size of from about 0.1 pm to about 0.4 pm in order to remove undissolved particles. In one embodiment, the nominal pore size of the depth filter is about 0.2 pm (e.g., Cuno VR06 filter or equivalent) to obtain a clarified filtrate. In another embodiment, the suspended Precipitate G solution is centrifuged to recover a clarified supernatant. Post-wash of the filter is performed using a sodium chloride solution with a conductivity of between about 2.5 and about 6.0 mS / cm. Typically, suitable solutions for the extraction of Precipitate G include, WFI and low conductivity buffers. In one embodiment, a low conductivity buffer has a conductivity of less than about 10 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 6 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 4 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 2 mS / cm.

[0147] In various embodiments, in order to inactivate various viral contaminants which may be present in plasma-derived products, the clarified Precipitate G filtrate is next subjected to a solvent detergent (S / D) treatment. Methods for the detergent treatment of plasma derived fractions are well known in the art (for review see, Pelletier JP et al. , Best Pract Res Clin Haematol. 2006; 19(1 ):205-42). Generally, any standard S / D treatment may be used in conjunction with the methods provided herein. For example, an exemplary protocol for an S / D treatment is provided below.

[0148] Briefly, Triton X-100, Tween-20, and tri(n-butyl)phosphate (TNBP) are added to the clarified Precipitate G filtrate at final concentrations of about 1.0% (w / v), 0.3% (w / v), and 0.3% (w / v), respectively. The mixture is then stirred at a temperature from about 18 °C to about 25 °C for at least about an hour.

[0149] In one embodiment, a process improvement is realized by adding the S / D reagents (e.g., Triton X-100, Tween-20, and TNBP) by spraying rather than by fluent addition. In other embodiments, the detergent reagents may be added as solids to the clarified Precipitate G filtrate, which is being mixed to ensure rapid distribution of the S / D components. In certain embodiments, it is preferable to add solid reagents by sprinkling the solids over a delocalizedsurface area of the filtrate such that local overconcentration does not occur, such as in fluent addition.

[0150] In various embodiments of the methods described above, prior to (a), (b) a cation exchange chromatography eluate is generated by submitting a cation exchange chromatography precursor solution to a cation exchange medium in one or more columns with a cation exchange buffer and collecting the cation exchange chromatography eluate. In some embodiments, one or more columns is 1 column, or 2 columns, or 3 columns, or 4 columns, or 5 columns, or more. In exemplary embodiments, one or more columns is one column. In exemplary embodiments, one or more columns is 2 columns. In exemplary embodiments, one or more columns is 4 columns. In another exemplary embodiments, one or more columns is 6 columns. The person skilled in the art will acknowledge that the number of columns will also dependent on the individual column size and / or column size used in the method and will adjust the number of columns accordingly in view of the above teaching.

[0151] Exemplary cation exchange media for this step include strong cation exchange groups, such as sulphopropyl, sulfonic acid and weak cation exchange groups, such as carboxylic acid, carboxymethyl (CM), and the like.

[0152] In various embodiments of the methods described above, the cation exchange medium has a protein loading of from about 10 mg protein / mL medium to about 200 mg protein / mL of medium. In some embodiments, the protein loading is about 10 mg protein / m , about 15 mg protein / mL, about 20 mg protein / mL, about 25 mg protein / mL, about 30 mg protein / mL, about 35 mg protein / mL, about 40 mg protein / mL, about 45 mg protein / mL, about 50 mg protein / mL, about 55 mg protein / mL, about 60 mg protein / mL, about 65 mg protein / mL, about 70 mg protein / mL, about 75 mg protein / mL, about 80 mg protein / mL, about 85 mg protein / mL, about 90 mg protein / mL, about 95 mg protein / mL, about 100 mg protein / mL, about 105 mg protein / mL, about 110 mg protein / mL L, about 115 mg protein / mL, about 120 mg protein / mL, about 125 mg protein / mL, about 130 mg protein / mL, about 135 mg protein / mL, about 140 mg protein / mL, about 145 mg protein / mL, about 150 mg protein / mL, about 155 mg protein / mL, about 160 mg protein / mL, about 165 mg protein / mL, about 170 mg protein / mL, about 180 mg protein / mL, about 190 mg protein / mL, about 195 mg protein / mL, or about 200 mg protein / mL.In exemplary embodiments, the protein loading is from about 50 mg protein / mL medium to about 125 mg protein / mL of medium.

[0153] In various embodiments of the methods described above, the IgG is bound to the cation exchange medium within the one or more column. In various embodiments, the bound IgG is eluted from the cation chromatography medium to which it is bound with a cation exchange elution buffer and collecting the cation exchange eluate.

[0154] In various embodiments of the methods described above, the IgG bound to the cation exchange medium is washed with from about 5 to about 40 column volumes of a wash buffer prior to eluting the bound IgG from the cation exchange medium. In some embodiments, the IgG bound to the cation exchange medium is washed with about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more column volumes of wash buffer. In exemplary embodiments, the IgG bound to the cation exchange medium is washed with from about 10 to about 30 column volumes of a wash buffer.

[0155] In various embodiments of the methods described above, the bound IgG is eluted from the cation chromatography medium to which it is bound with a cation exchange elution buffer and collecting the cation exchange eluate.

[0156] In various embodiments of the methods described above, the cation exchange elution buffer comprises monobasic sodium phosphate from about 20 mM to about 60 mM, for example, from about 20 mM to about 55 mM, from about 20 mM to about 50 mM, from about 20 mM to about 45 mM, from about 20 mM to about 40 mM, from about 20 mM to about 35 mM, from about 25 mM to about 60 mM, from about 30 mM to about 60 mM, or from about 35 to about 60 mM. In some embodiments, the cation exchange elution buffer comprises monobasic sodium phosphate from about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, or about 60 mM. In exemplary embodiments, the cation exchange elution buffer comprises monobasic sodium phosphate from about 35 mM to about 55 mM.

[0157] In various embodiments of the methods described above, the cation exchange elution buffer comprises tris from about 5 mM to about 15 mM, for example, from about 5 mM to about14 mM, from about 5 mM to about 13 mM, from about 5 mM to about 12 mM, from about 5 mM to about 11 mM, from about 5 mM to about 10 mM, from about 6 mM to about 15 mM, from about 7 mM to about 15 mM, from about 8 mM to about 15 mM, from about 9 mM to about 15 mM, or from about 10 mM to about 15 mM. In some embodiments, the cation exchange elution buffer comprises tris is from about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM. In exemplary embodiments, the cation exchange elution buffer comprises about 10 mM tris.

[0158] In various embodiments of the methods described above, the cation exchange elution buffer has a pH of from about 7.5 to about 8.7, for example from about 7.5 to about 8.6, from about 7.5 to about 8.5, from about 7.6 to about 8.7, from about 7.7 to about 8.7, from about 7.8 to about 8.7, from about 7.9 to about 8.7, from about 8.0 to about 8.7, from about 8.1 to about 8.7, from about 8.2 to about 8.7, or from about 8.3 to about 8.7. In some embodiments, the cation exchange elution buffer has a pH of no more than about 8.7. In some embodiments, the cation exchange elution buffer has a pH of about 8.7, about 8.6, about 8.5, about 8.4, about 8.3, about 8.2, about 8.1, about 8.0, about 7.9, about 7.8, about 7.7, about 7.6, about 7.5, or less. In exemplary embodiments, the cation exchange elution buffer has a pH of from about 7.9 to about 8.7. In exemplary embodiments, the cation exchange elution buffer has a pH of about 8.3 to about 8.7. In exemplary embodiments, the cation exchange elution buffer comprises a pH of about 8.5 ± 0.2.

[0159] In various embodiments of the methods described above, the cation exchange elution buffer has a conductivity of from about 2.0 mS / cm to about 10.0 mS / cm, for example, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a conductivity of about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm,about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, or about 10.0 mS / cm. In exemplary embodiments, the cation exchange elution buffer has a conductivity of about 5.0 ± 0.2 mS / cm.

[0160] In various embodiments of the methods described herein, the cation exchange elution buffer has a pH of from about 7.5 to about 8.7 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.5 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.5 to about 8.5 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.6 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.7 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.8 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.9 to about 8.6 and a conductivity ofabout 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 8.0 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 8.1 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 8.2 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 8.3 to about 8.6 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In some embodiments, the cation exchange elution buffer has a pH of from about 7.9 to about 8.7 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5 mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. the cation exchange elution buffer has a pH of from about 8.3 to about 8.7 and a conductivity of about 2.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 10.0 mS / cm, from about 3.0 mS / cm to about 10.0 mS / cm, from about 3.5 mS / cm to about 10.0 mS / cm, from about 4.0 mS / cm to about 10.0 mS / cm, from about 4.5 mS / cm to about 10.0 mS / cm, from about 5.0 mS / cm to about 10.0 mS / cm, from about 2.5 mS / cm to about 9.5 mS / cm, from about 2.5 mS / cm to about 9.0 mS / cm, from about 2.5 mS / cm to about 8.5 mS / cm, from about 2.5 mS / cm to about 8.0 mS / cm, from about 2.5 mS / cm to about 7.5 mS / cm, from about 2.5 mS / cm to about 7.0 mS / cm, from about 2.5 mS / cm to about 6.5 mS / cm, from about 2.5 mS / cm to about 6.0 mS / cm, from about 2.5mS / cm to about 5.5 mS / cm, or from about 2.5 mS / cm to about 5.0 mS / cm. In exemplary embodiments, the cation exchange elution buffer comprises monobasic sodium phosphate (e.g., from about 35 mM to about 55 mM) and / or tris (e.g., about 10 mM) with a pH of about 8.5 ± 0.2 and / or a conductivity of about 5.0 ± 0.2 mS / cm.

[0161] In various embodiments of the methods described herein, prior to being loaded onto the anion exchange column, the cation exchange eluate is adjusted to a pH of from about 6.0 to about 7.0, for example, from about 6.0 to about 6.9, from about 6.0 to about 6.8, from about 6.1 to about 7.0, from about 6.2 to about 7.0, from about 6.3 to about 7.0, from about 6.4 to about 7.0, from about 6.5 to about 7.0, from about 6.6 to about 7.0, from about 6.7 to about 7.0, or from about 6.8 to about 7.0. In some embodiments, the cation exchange eluate is adjusted to a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In exemplary embodiments, the cation exchange eluate is adjusted to a pH of from about 6.5 to about 7.0. In exemplary embodiments, the cation exchange eluate is adjusted to a pH of from about 6.7 to about 6.9. In exemplary embodiments, the cation exchange eluate is adjusted to a pH of about 6.8 ± 0.1. The person skilled in the art will acknowledge that based on the above outlined teaching the pH of the cation exchange eluate will have to be adjusted to the properties of anion exchange medium as used in the described method.

[0162] In various embodiments of the methods described herein, prior to being loaded onto the anion exchange column, the cation exchange eluate is adjusted to a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, or from about 0.9 mS / cm to about 1.5 mS / cm. In some embodiments, the cation exchange eluate is adjusted to a conductivity of about 0.1 mS / cm, about 0.2 mS / cm, about 0.3 mS / cm, about 0.4 mS / cm, about 0.5 mS / cm, about 0.6 mS / cm, about 0.7 mS / cm, about 0.8 mS / cm, about 0.9 mS / cm, about 1.0 mS / cm, about 1.1 mS / cm, about 1.2 mS / cm, about 1.3 mS / cm, about 1.4 mS / cm, or about 1.5 mS / cm. In exemplary embodiments,the cation exchange eluate is adjusted to a conductivity of from about 0.7 mS / cm to about 1.1 mS / cm. In exemplary embodiments, the cation exchange eluate is adjusted to a conductivity of from about 0.8 mS / cm to about 1.0 mS / cm. In exemplary embodiments, the cation exchange eluate is adjusted to a conductivity of about 0.9 ± 0.1 mS / cm.

[0163] As will be appreciated, the cation exchange eluate can be adjusted to any combination of pH and conductivity according to the parameters set forth above. For example, in various embodiments of the methods described above, prior to being loaded onto the anion exchange column, the cation exchange eluate is adjusted to a pH of from about 6.0 to about 7.0 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.0 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.0 to about 6.8 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cmto about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.1 to about 7.0 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.2 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.3 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, fromabout 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.0 to about 6.4 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.5 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.6 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.7 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.8 to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.5 to about 7.0 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In some embodiments, the cation exchange eluate is adjusted to a pH of from about 6.7to about 6.9 and a conductivity of from about 0.1 mS / cm to about 1.5 mS / cm, for example, from about 0.1 mS / cm to about 1.4 mS / cm, from about 0.1 mS / cm to about 1.3 mS / cm, from about 0.1 mS / cm to about 1.2 mS / cm, from about 0.1 mS / cm to about 1.1 mS / cm, from about 0.1 mS / cm to about 1.0 mS / cm, from about 0.1 mS / cm to about 0.9 mS / cm, from about 0.2 mS / cm to about 1.5 mS / cm, from about 0.3 mS / cm to about 1.5 mS / cm, from about 0.4 mS / cm to about 1.5 mS / cm, from about 0.5 mS / cm to about 1.5 mS / cm, from about 0.6 mS / cm to about 1.5 mS / cm, from about 0.7 mS / cm to about 1.5 mS / cm, from about 0.8 mS / cm to about 1.5 mS / cm, from about 0.9 mS / cm to about 1.5 mS / cm, 0.8 mS / cm to about 1.0 mS / cm, or about 0.7 mS / cm to about 1.1 mS / cm to form the anion exchange precursor solution. In exemplary embodiments, the cation exchange eluate is adjusted to a pH of about 6.8 ± 0.1 and / or a conductivity of about 0.9 ± 0.1 mS / cm to form the anion exchange precursor solution. In exemplary embodiments, the cation exchange eluate is adjusted to a pH of about 6.8 ± 0.1 and / or a conductivity of about 0.8 ± 0.1 mS / cm to form the anion exchange precursor solution.

[0164] In various embodiments of the methods described herein, the method further comprises submitting the anion exchange flowthrough to nanofiltration, forming a first nanofiltered IgG formulation precursor solution.

[0165] In some embodiments, the anion exchange column effluent may be nanofiltered using a suitable nanofiltration device. In some embodiments, nanofiltration is used to reduce the viral load of the IgG formulations provided herein. In some embodiments, the nanofiltration device will have a mean pore size of between about 15 nm and about 200 nm. Examples of nanofilters suitable for this use include, without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In a specific embodiment, the nanofilter may have a mean pore size of between about 15 nm and about 72 nm, or between about 19 nm and about 35 nm, or of about 15 nm, 19 nm, 35 nm, or 72 nm. In a preferred embodiment, the nanofilter will have a mean pore size of about 35 nm, such as an Asahi PLANOVA 35N filter or equivalent thereof.

[0166] In various embodiments, ultrafiltration / diafiltration may performed to further concentrate the nanofiltrate. In some embodiments, an open channel membrane is used with a specifically designed post-wash and formulation near the end the production process render the resulting IgG compositions about twice as high in protein concentration (200 mg / mL) comparedto state-of-the-art IVIGs (e.g., GAMMAGARD® LIQUID) without affecting yield and storage stability. With most of the commercially available ultrafiltration membranes a concentration of 200 mg / mL IgG cannot be reached without major protein losses. These membranes will be blocked early and therefore adequate post-wash is difficult to achieve. Therefore, open channel membrane configurations have to be used. Even with open channel membranes, a specifically designed post-wash procedure has to be used to obtain the required concentration without significant protein loss (less than 2% loss). Even more surprising is the fact that the higher protein concentration of 200 mg / mL does not affect the virus inactivation capacity of the low pH storage step.

[0167] In various embodiments of the methods described herein, a protein load for nanofiltration is not more than about 8 kg protein / m2of nanofilter membrane area. In some embodiments, the protein load for nanofiltration is not more than about 8 kg protein / m2, not more than about 7 kg protein / m2, not more than about 6 kg protein / m2, not more than about 5 kg protein / m2, not more than about 4 kg protein / m2, not more than about 3 kg protein / m2, not more than about 2 kg protein / m2, not more than about 1 kg protein / m2, or less. In some embodiments, the protein load for nanofiltration is not more than about 8 kg protein / m2.

[0168] In various embodiments of the methods described herein, a differential pressure during the nanofiltration is not more than about 1.0 bar. In some embodiments, the differential pressure during the nanofiltration is not more than about 1.0 bar, not more than about 0.9 bar, not more than about 0.8 bar, not more than about 0.7 bar, not more than about 0.6 bar, not more than about 0.5 bar, or less.

[0169] In various embodiments of the methods described herein, the method further comprises submitting the first nanofiltered IgG formulation precursor solution to ultrafiltration. In various embodiments, submitting the first nanofiltered IgG formulation precursor solution to ultrafiltration concentrates total protein in the first nanofiltered IgG formulation precursor. In various embodiments, submitting the first nanofiltered IgG formulation precursor solution to ultrafiltration concentrates total protein in the first nanofiltered IgG formulation precursor, forming an ultrafiltered IgG formulation precursor solution.

[0170] In various embodiments of the method described herein, the total protein concentration in the ultrafiltered IgG formulation precursor is from about 1% (w / v) to about 10% (w / v), for example, from about 1% (w / v) to about 9% (w / v), 1% (w / v) to about 8% (w / v), 1% (w / v) to about 7% (w / v), 1% (w / v) to about 6% (w / v), 1% (w / v) to about 5% (w / v), 2% (w / v) to about 10% (w / v), 3% (w / v) to about 10% (w / v), 4% (w / v) to about 10% (w / v), or 5% (w / v) to about 10% (w / v). In some embodiments, the total protein concentration in the ultrafiltered IgG formulation precursor is about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), or about 10% (w / v). In exemplary embodiments, the total protein concentration in the ultrafiltered IgG formulation precursor is from about 2% (w / v) to about 8% (w / v). In exemplary embodiments, the total protein concentration in the ultrafiltered IgG formulation precursor is from about 3% (w / v) to about 7% (w / v). In exemplary embodiments, the total protein concentration in the ultrafiltered IgG formulation precursor is about 5 ± 2% (w / v).

[0171] In various embodiments of the methods described herein, the ultrafiltration is performed with a membrane with a nominal molecular weight cutoff of not more than about 50K Daltons. In various embodiments of the method described above, the ultrafiltration is performed with a membrane with a nominal molecular weight cutoff of not more than about 5 OK Daltons, not more than about 40K Daltons, not more than about 3 OK Daltons, not more than about 20K Daltons, not more than about 10K Daltons, or less.

[0172] In various embodiments of the methods described herein, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to from about 10.0% (w / v) to about 22.0% (w / v), about 10.0% (w / v) to about 20.0% (w / v), from about 10.0% (w / v) to about 17.0% (w / v), for example, from about 10.5% (w / v) to about 17.0% (w / v), 11.0% (w / v) to about 17.0% (w / v), 11.5% (w / v) to about 17.0% (w / v), 12.0% (w / v) to about 17.0% (w / v), 12.5% (w / v) to about 17.0% (w / v), 13.0% (w / v) to about 17.0% (w / v), 13.5% (w / v) to about 17.0% (w / v), 10.0% (w / v) to about 16.5% (w / v), 10.0% (w / v) to about 16.0% (w / v), 10.0% (w / v) to about 15.5% (w / v), 10.0% (w / v) to about 15.0% (w / v), 10.0% (w / v) to about 14.5% (w / v), 10.0% (w / v) to about 14.0% (w / v), or 10.0% (w / v) to about 13.5% (w / v). In some embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 10.0% (w / v), about 10.5% (w / v), about 11.0% (w / v), about 11.5% (w / v), about 12.0% (w / v),about 12.5% (w / v), about 13.0% (w / v), about 13.5% (w / v), about 14.0% (w / v), about 14.5% (w / v), about 15.0% (w / v), about 15.5% (w / v), about 16.0% (w / v), about 16.5% (w / v), about 17.0% (w / v), about 20.0% (w / v) or about 22.0% (w / v). In exemplary embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to from about 13.0% (w / v) to about 14.0% (w / v). In exemplary embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 13.5% (w / v). In exemplary embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 10.0% (w / v). In another exemplary embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 20.0% (w / v).

[0173] In various embodiments of the methods described herein, the method further comprises formulating the ultrafiltered IgG formulation precursor as an IgG formulation by adjusting the IgG and / or total protein concentration and / or adjusting the pH of the ultrafiltered IgG formulation precursor, thereby forming the IgG formulation. In some embodiments, adjustment of the IgG and / or total protein concentration and / or pH is directly measured in the IgG formulation (i.e., concentrated product). In some embodiments, adjustment of the IgG and / or total protein concentration and / or pH is not directly measured in the IgG formulation.

[0174] In various embodiments of the methods described herein, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to at least about 5.0% (w / v) to about 20.0% (w / v), for example, from about 5.0% (w / v) to about 19.0% (w / v), from about 5.0% (w / v) to about 18.0% (w / v), from about 5.0% (w / v) to about 17.0% (w / v), from about 5.0% (w / v) to about 16.0% (w / v), from about 5.0% (w / v) to about 15.0% (w / v), from about 5.0% (w / v) to about 14.0% (w / v), from about 5.0% (w / v) to about 13.0% (w / v), from about 5.0% (w / v) to about 12.0% (w / v), from about 5.0% (w / v) to about 11.0% (w / v), from about 5.0% (w / v) to about 10.0% (w / v), from about 6.0% (w / v) to about 20.0% (w / v), from about 7.0% (w / v) to about 20.0% (w / v), from about 8.0% (w / v) to about 20.0% (w / v), from about 9.0% (w / v) to about 20.0% (w / v), or from about 10.0% (w / v) to about 20.0% (w / v). In some embodiments, IgG and / or total protein is about 5.0% (w / v), about 6.0% (w / v), about 7.0% (w / v), about 8.0% (w / v), about 9.0% (w / v), about 10.0% (w / v), about 11.0% (w / v), about 12.0% (w / v), about 13.0% (w / v), about 14.0% (w / v), about 15.0% (w / v), about 16.0% (w / v), about17.0% (w / v), about 18.0% (w / v), about 19.0% (w / v), or about 20.0% (w / v). In exemplary embodiments, IgG and / or total protein is adjusted to about 10.0% (w / v) or about 20.0% (w / v). In exemplary embodiments, IgG and / or total protein is adjusted to from about 9.0% (w / v) to about 11.0% (w / v). In exemplary embodiments, IgG and / or total protein is adjusted to about 10.0 ± 0.1% (w / v).

[0175] In various embodiments of the methods described herein, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of from about 4.0 to about 5.5, for example, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5. In some embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. In some embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of not more than about 5.1. In some embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of not less than about 4.4. In some embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of from about 4.4 to about 5.1. In exemplary embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of from about 4.4 to about 4.9. In some embodiments, the pH of the ultrafiltered IgG formulation precursor is adjusted to a pH of from about 4.6 to about 5.1.

[0176] In various embodiments of the methods described herein, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about 4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 19.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 toabout 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about 4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 18.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 17.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 16.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 15.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 14.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, fromabout 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 13.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 12.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 11.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 5.0% (w / v) to about 10.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 6.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 7.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 8.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 9.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 10.0% (w / v) to about 20.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In some embodiments, IgG and / or total protein concentration of the ultrafiltered IgG formulation precursor is adjusted to from about 9.0% (w / v) to about 11.0% (w / v) and pH is adjusted to from about 4.0 to about 5.5, from about 4.0 to about 5.4, from about 4.0 to about 5.3, from about 4.0 to about 5.2, from about 4.0 to about 5.1, from about 4.0 to about 5.0, from about 4.0 to about 4.9, from about 4.1 to about 5.5, from about 4.2 to about 5.5, from about 4.3 to about 5.5, or from about 4.4 to about 5.5, from about 4.4 to about 5.1, or from about4.4 to about 4.9. In exemplary embodiments, IgG and / or total protein in the ultrafiltered IgGformulation precursor solution is concentrated to about 10.0 ± 0.1% (w / v) and / or pH is adjusted to about 4.4 to about 4.9. In exemplary embodiments, IgG and / or total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 10.0 ± 0.1% (w / v) and / or pH is adjusted to from about 4.6 to about 5.1. In some embodiments, adjustment of the IgG and / or total protein concentration and / or pH is directly measured in the concentrated product. In some embodiments, adjustment of the IgG and / or total protein concentration and / or pH is not directly measured in the concentrated product.

[0177] In various embodiments of the methods described herein, the method further comprises transferring the IgG formulation into a container. In various embodiments, after transferring the IgG formulation into a container, the container containing the IgG formulation is incubated for a selected period of time at a selected temperature.

[0178] In various embodiments of the methods described herein, the container containing the IgG formulation is incubated at from about 25 °C to about 35 °C, for example, from about 26 °C to about 35 °C, from about 27 °C to about 35 °C, from about 28 °C to about 35 °C, from about 29 °C to about 35 °C, from about 30 °C to about 35 °C, from about 25 °C to about 34 °C, from about 25 °C to about 33 °C, from about 25 °C to about 32 °C, from about 25 °C to about 31 °C. In some embodiments, the container containing the IgG formulation is incubated at about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, or about 35 °C. In exemplary embodiments, the container containing the IgG formulation is incubated at from about 30 °C to about 32 °C.

[0179] In various embodiments of the methods described herein, the container containing the IgG formulation is incubated for from about 15 days to about 30 days, for example, from about 16 days to about 30 days, from about 17 days to about 30 days, from about 18 days to about 30 days, from about 19 days to about 30 days, from about 20 days to about 30 days, from about 21 days to about 30 days, from about 15 days to about 29 days, from about 15 days to about 28 days, from about 15 days to about 27 days, from about 15 days to about 28 days, from about 15 days to about 27 days, from about 15 days to about 26 days, from about 15 days to about 25 days, from about 15 days to about 24 days, from about 15 days to about 23 days, from about 15 days to about 22 days, or from about 15 days to about 21 days. In some embodiments, the container containing the IgG formulation is incubated for about 15 days, about 16 days, about 17days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or more. In exemplary embodiments, the container containing the IgG formulation is incubated for about 21 to about 24 days. In some embodiments, the container containing the IgG formulation is incubated for from about 2 weeks to about 4 weeks. In exemplary embodiments, the container containing the IgG formulation is incubated for about 3 weeks.

[0180] In various embodiments of the methods described herein, the method provides the IgG formulation with an IgG yield compared to IgG present in a starting plasma sample of at least about 50%. In some embodiments, the yield is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater.

[0181] In various embodiments of the methods described herein, storage-stable IgA depleted IgG pharmaceutical formulations (e.g., not more than about 2 pg / mL of IgA) are formed using the method of the present invention.VI. Methods of Use

[0182] As routinely practiced in the modern medicine, sterilized preparations of concentrated immunoglobulins (especially IgGs) are used for treating medical conditions that fall into these three main classes: immune deficiencies, inflammatory and autoimmune diseases, and acute infections. These IgG preparations may also be useful for treating multiple sclerosis (especially relapsing-remitting multiple sclerosis or RRMS), Alzheimer's disease, and Parkinson's disease. The purified IgG preparation of this invention is suitable for these purposes, as well as other clinically accepted uses of IgG preparations.

[0183] The FDA has approved the use of IGI, e.g., IVIG, to treat various indications, including allogeneic bone marrow transplant, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), and kidney transplant with a high antibody recipient or with an ABO incompatible donor. In certain embodiments, the IGI, e.g., IVIG, compositions provided herein are useful for the treatment or management of these diseases and conditions.

[0184] Furthermore, off-label uses for IGI, e.g., IVIG are commonly provided to patients for the treatment or management of various indications, for example, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves' ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, Lupus erythematosus, multifocal motor neuropathy, multiple sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus B19 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous Abortion / Miscarriage, stiff person syndrome, opsoclonus Myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, and hypogammaglobulinemia. In certain embodiments, the IGI, e.g., IVIG, compositions provided herein are useful for the treatment or management of these diseases and conditions.

[0185] Finally, experimental use of IGI, e.g., IVIG, for the treatment or management of diseases including primary immune deficiency, RRMS, Alzheimer's disease, and Parkinson's disease has been proposed (U.S. Patent Application Publication No. U.S. 2009 / 0148463, which is herein incorporated by reference in its entirety for all purposes). In certain embodiments, the IGI, e.g., IVIG compositions provided herein are useful for the treatment or management of primary immune deficiency, RRMS, Alzheimer's disease, or Parkinson's disease. In certain embodiments comprising daily administration, an effective amount to be administered to the subject can be determined by a physician with consideration of individual differences in age, weight, disease severity, route of administration (e.g., intravenous v. subcutaneous) and response to the therapy. In certain embodiments, an immunoglobulin preparation of this invention can be administered to a subject at about 5 mg / kilogram to about 2000 mg / kilogram each day. In additional embodiments, the immunoglobulin preparation can be administered in amounts of at least about 10 mg / kilogram, at last 15 mg / kilogram, at least 20 mg / kilogram, at least 25 mg / kilogram, at least 30 mg / kilogram, or at least 50 mg / kilogram. In additional embodiments, the immunoglobulin preparation can be administered to a subject at doses up to about 100 mg / kilogram, to about 150 mg / kilogram, to about 200 mg / kilogram, to about 250 mg / kilogram, to about 300 mg / kilogram, to about 400 mg / kilogram each day. In other embodiments, the doses of the immunoglobulin preparation can be greater or less. Further, the immunoglobulin preparations can be administered in one or more doses per day. Clinicians familiar with thediseases treated by IgG preparations can determine the appropriate dose for a patient according to criteria known in the art.

[0186] An effective amount of an IGI, e.g., IVIG preparation is administered to the subject by intravenous means. The term "effective amount" refers to an amount of an IGI preparation that results in an improvement or remediation of disease or condition in the subject. An effective amount to be administered to the subject can be determined by a physician with consideration of individual differences in age, weight, the disease or condition being treated, disease severity and response to the therapy. In certain embodiments, an IGI preparation can be administered to a subject at dose of about 5 mg / kilogram to about 2000 mg / kilogram per administration. In certain embodiments, the dose may be at least about 5 mg / kg, or at least about 10 mg / kg, or at least about 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1300 mg / kg, 1400 mg / kg, 1500 mg / kg, 1600 mg / kg, 1700 mg / kg, 1800 mg / kg, 1900 mg / kg, or at least about 2000 mg / kg.

[0187] The dosage and frequency of IGI treatment will depend upon, among other factors, the disease or condition being treated and the severity of the disease or condition in the patient. Generally, for primary immune dysfunction a dose of between about 100 mg / kg and about 400 mg / kg body weight will be administered about every 3 to 4 weeks. For neurological and autoimmune diseases, up to 2 g / kg body weight is implemented for three to six months over a five day course once a month. This is generally supplemented with maintenance therapy comprising the administration of between about 100 mg / kg and about 400 mg / kg body weight about once every 3 to 4 weeks. Generally, a patient will receive a dose or treatment about once every 14 to 35 days, or about every 21 to 28 days. The frequency of treatment will depend upon, among other factors, the disease or condition being treated and the severity of the disease or condition in the patient.

[0188] In a preferred embodiment, a method of treating an immunodeficiency, autoimmune disease, or acute infection in a human in need thereof is provided, the method comprising administering a pharmaceutical IGI composition of the present invention. In a relatedembodiment, the present invention provides IGI compositions manufactured according to a method provided herein for the treatment of an immunodeficiency, autoimmune disease, or acute infection in a human in need thereof.

[0189] In certain embodiments, the immunodeficiency, autoimmune disease, or acute infection is selected from allogeneic bone marrow transplant, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), kidney transplant with a high antibody recipient or with an ABO incompatible donor, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves' ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, Lupus erythematosus, multifocal motor neuropathy, multiple sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus Bl 9 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous Abortion / Miscarriage, stiff person syndrome, opsoclonus Myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia, primary immune deficiency, RRMS, Alzheimer's disease, and Parkinson's disease.

[0190] As noted above IgG therapeutics are often administered through intravenous infusions, which are costly, inconvenient for patients and require a high level of patient compliance. It is also desirable for IgG to be administered via subcutaneous or intramuscular injection. While the SubQ and IM routes can offer clear advantages in ease of administration and cost when compared to intravenous infusions, they can also present challenges that may arise, for example, from limited infusion volume tolerance.VIL Exemplary Embodiments

[0191] In various embodiments, the invention provides a storage-stable IgG pharmaceutical formulation containing not more than about 2 pg / mL of IgA in an aqueous pharmaceutically acceptable carrier, the formulation having a property selected from:(i) a pH of from about 4.4 to about 4.9;(ii) from about 0.1 M to about 0.4 M glycine, and a combination thereof.

[0192] In various embodiments, the invention provides a storage-stable IgG pharmaceutical formation according to paragraph

[0189] above having about 10% (w / v) IgG.

[0193] In various embodiments, the invention provides a method of forming the storage-stable IgG pharmaceutical formulation of any preceding claim, the method comprising:(a) performing an anion exchange step on an anion exchange precursor solution comprising the IgG, the step comprising:(i) submitting the anion exchange precursor solution to anion exchange chromatography in a single pass through a first portion of an anion exchange medium contained in a first column and collecting a first anion exchange flowthrough from the first column; and(ii) optionally, following (i), washing the anion exchange medium with a wash buffer, wherein the washing uses from about 0.25 column volumes to about 1 column volume of the wash buffer.

[0194] In various embodiments, the invention provides a method according to paragraph

[0191] above wherein the single pass through the anion exchange medium occurs in the first column, and in a second column containing a second portion of the anion exchange medium, the first and second columns run: (i) in serial mode; or (ii) in parallel mode and in (ii), collecting a second anion exchange flowthrough from the second column.

[0195] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0192] above wherein the IgG is loaded onto the anion exchange material at a maximum loading of about 70 mg / mL of resin.

[0196] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0193] above wherein the IgG is loaded onto the anion exchange material at a maximum loading of about 50 mg / mL of resin.

[0197] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0194] above wherein the IgG is loaded onto the anion exchange material at a maximum loading of about 30 mg / mL of resin.

[0198] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0195] above, wherein the first, and second anion exchange flowthrough are optionally pooled.

[0199] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0196] above, wherein the pH of the anion exchange precursor solution is about from about 6.2 to about 7.0, e.g., from about 6.4 to about 7.0, e.g., from about 6.7 to about 6.9 and the conductivity is from about 0.5 to about 2.5, e.g., from about 0.7 to about 1.5, e.g., from about 0.8 to about 1.0 mS / cm.

[0200] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0197] above, wherein the protein concentration of the anion exchange flowthrough is from about 3 g / L to about 15 g / L.

[0201] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0198] above, wherein the wash buffer flowthrough is pooled with the anion exchange flowthrough.

[0202] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0199] above, wherein the anion exchange precursor solution is an eluate from a cation exchange step of an IgG containing suspension (e.g., from precipitate G) preceding (a).

[0203] In various embodiments, the invention provides a method according to paragraph

[0200] above, wherein the eluate from a cation exchange step preceding (a) is an eluate from one or more cation exchange columns.

[0204] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0201] above, wherein a precursor solution for the cation exchange step is a mixture of dissolved Precipitate G, a solvent and a detergent.

[0205] In various embodiments, the invention provides a method according to paragraph

[0202] above, wherein the solvent and the detergent are mixture of octoxynol 9, polysorbate 80 and tri- (n-butyl) phosphate or equivalent.

[0206] In various embodiments, the invention provides a method according to paragraph

[0203] above, wherein the octoxynol 9 is present at about 1.0 ±0.5% (v / v); the polysorbate 80 is presentat about 0.3 ± 0.15% (v / v); and the tri-(n-butyl) phosphate is present at about 0.3 ± 0.15% (v / v) or equivalent.

[0207] In various embodiments, the invention provides a method according to any of paragraphs

[0191] -

[0204] above, the method further comprising: prior to (a), (b) a cation exchange chromatography eluate is generated by submitting a cation exchange chromatography precursor solution to a cation exchange medium in one or more column with a cation exchange buffer and collecting the cation exchange chromatography eluate.

[0208] In various embodiments, the invention provides a method according to paragraph

[0205] above, wherein the cation exchange medium has a protein loading of from about 50 mg / mL to about 125 mg / mL of medium.

[0209] In various embodiments, the invention provides a method according to an of paragraphs

[0205] -

[0206] above, wherein the IgG is bound to the cation exchange medium within the one or more column.

[0210] In various embodiments, the invention provides a method according to any of paragraphs

[0205] -

[0207] above, wherein IgG bound to the cation exchange medium is washed with from about 10 to about 30 column volumes of a wash buffer prior to eluting the bound IgG from the cation exchange material.

[0211] In various embodiments, the invention provides a method according to any of paragraphs

[0205] -

[0208] above, the method comprising, eluting the bound IgG from the cation chromatography medium to which it is bound with a cation exchange elution buffer and collecting the cation exchange eluate.

[0212] In various embodiments, the invention provides a method according to any of paragraphs

[0204] -

[0209] above, wherein the cation exchange elution buffer comprises: monobasic sodium phosphate (from about 35 mM to about 55 mM); and tris (about 10 mM), with a pH of 8.5 ± 0.2, and a conductivity of 5.0 ± 0.2 mS / cm.

[0213] In various embodiments, the invention provides a method according to any of paragraphs

[0204] -

[0210] above, wherein the cation exchange eluate is adjusted to a pH of 6.8 ± 0.1, and a conductivity of 0.9 ± 0.1 mS / cm, forming the anion exchange precursor solution.

[0214] In various embodiments, the invention provides a method according to any paragraphs

[0191] -

[0211] above, the method further comprising, submitting the anion exchange flowthrough to nanofiltration, forming a first nanofiltered IgG formulation precursor solution.

[0215] In various embodiments, the invention provides a method according to paragraph

[0212] above, wherein a protein load for nanofiltration is not more than about 8 kg protein / m2of nanofilter membrane area.

[0216] In various embodiments, the invention provides a method according to paragraph

[0213] above, wherein a differential pressure during the nanofiltration is not more than about 1.0 bar.

[0217] In various embodiments, the invention provides a method according to any one of paragraphs

[0212] -

[0214] above, the method further comprising, submitting the first nanofiltered IgG formulation precursor solution to ultrafiltration, concentrating total protein in the first nanofiltered IgG formulation precursor, forming an ultrafiltered IgG formulation precursor solution.

[0218] In various embodiments, the invention provides a method according to any of paragraphs

[0212] -

[0215] above, wherein the total protein concentration in the ultrafiltered IgG formulation precursor is about 5 ± 2% (w / v).

[0219] In various embodiments, the invention provides a method according to any of paragraphs

[0212] -

[0216] above, wherein the ultrafiltration is performed with a membrane with a nominal molecular weight cutoff of not more than about 50K Daltons.

[0220] In various embodiments, the invention provides a method according to any of paragraphs

[0212] -

[0217] above, wherein the total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 13.5% (w / v).

[0221] In various embodiments, the invention provides a method according to any of paragraphs

[0212] -

[0218] above, further comprising, formulating the ultrafiltered IgG formulation precursor as an IgG formulation by adjusting IgG concentration of the ultrafiltered IgG formulationprecursor to 10.0 ± 0.1% (w / v), and adjusting pH to about 4.4 to about 4.9 directly measured in the concentrated product.

[0222] In various embodiments, the invention provides a method according to any of paragraphs

[0212] -

[0219] above, the method further comprising, transferring the IgG formulation into a container and incubating the IgG formulation in the container at from about 30°C to about 32°C for from about 21 to about 24 days.

[0223] In various embodiments, the invention provides a method according to any one of paragraphs

[0191] -

[0220] above, wherein the method provides the IgG formulation with an IgG yield of at least about 50% IgG compared to IgG present in a starting plasma sample.

[0224] In various embodiments, the invention provides a storage-stable IgG pharmaceutical formulation containing not more than about 2 pg / mL of IgA, in an aqueous pharmaceutically acceptable carrier, the formulation having a property selected from:(i) a pH of from about 4.4 to about 4.9;(ii) from about 0. IM to about 0.4M glycine, and a combination thereof, wherein the storage-stable IgG pharmaceutical formulation is made by a method according to any one of paragraphs

[0179] -

[0209] above.

[0225] In various embodiments, the invention provides a storage-stable IgG pharmaceutical formulation according to paragraph

[0222] above having about 10% (w / v) IgG.

[0226] As will be appreciated, any combination of the above parameters achieving the practical result of the instant invention is encompassed by this disclosure.VII. Examples

[0227] The following examples are offered to illustrate exemplary embodiments of the invention and do not define or limit its scope.EXAMPLE 1Preparation Precipitate G a. Summary

[0228] Previously frozen pooled plasma from blood donors was separated into a cryo-poor plasma sample for isolation of various crude coagulation factors and inhibitors prior to subsequent cold fractionation procedure was described by Teschner etal. (2007) Vox Sang. 92: 42-55. The alcohol fractionation procedure gave a principal intermediate IgG fraction, referred to as Precipitate G, which was further processed to the final product using chromatographic purification.

[0229] For pre-clinical IGI 10% production, Cohn starting materials derived from various absorption step options (plasma without adsorption steps, plasma after FEIBA, AT-III adsorption and (plasma after F-IX, F-VII, AT-III adsorption) were chosen to cover a broad variety of different adsorption steps prior to alcohol fractionation. Various adsorption processes are described in Teschner et al. (2007) Vox Sang. 92:42-55; Polsler et al. (2008) Vox Sang. 94: 184- 192; U.S. Pat. Nos. 6,395,880 and 5,409,990; and Prothrombin complex: Brummelhuis in Methods of Plasma Protein Fractionation (J. M. Curling Editor, Academic Press, 1980). c. Fractionation i. Obtain Supernatant of Fractionation I

[0230] While the plasma was being stirred, pre-cooled ethanol was added, to a target concentration of 8% v / v ethanol, and the temperature was further lowered to -2 °C to 0 °C to allow precipitation. Supernatant (or Fractionation I) was collected after centrifugation. ii. Precipitate of Fractionations II and III

[0231] Fractionation I was adjusted to pH 7 and 20% (v / v) to 25% (v / v) ethanol concentration, while the temperature was further lowered. Subsequently, centrifugation was performed to separate liquid (Fractionation 11+111 supernatant) and solid.Hi. Extraction from Fractionations II and III Precipitate

[0232] A cold extraction buffer (5 mM monobasic sodium phosphate, 5 mM acetate, pH 4.5±0.2, conductivity of 0.7 to 0.9 mS / cm) was used to re-suspend Fractionations II+III at a ratio of 1 : 15 precipitate: extraction buffer. The extraction process was performed at 2 °C to 8° C. iv. Fumed Silica Treatment and Filtration

[0233] Fumed silica (e.g., Aerosil 380 or equivalent) was added to the suspension to a concentration of about 40 g / kg of suspension (or equivalent to 1.8 g / L of cryo-poor plasma) and was mixed at 2 °C to 8 °C for 50 to 70 minutes. Liquids and solids were separated by filtration at 2 °C to 8 °C using a filter aid (Hyflo Super-Cel, World Minerals Inc., 0.5 kg / kg of suspension), followed by post-washing of the filter press with extraction buffer. v. Fractionation of Precipitate G

[0234] The filtrate was mixed with a tenside at a substantial low quantity with stirring for at least 30 minutes at 2 °C to 8 °C. Sodium citrate dihydrate was then mixed into the solution at 8 g / L for another 30 minutes of stirring at 2 °C to 8 °C. The pH was then adjusted to 7.0±0.1 with either IM sodium hydroxide or IM acetic acid. Cold alcohol was then added to the solution to a concentration of about 25% v / v, and a precipitation step similar to Cohn II was performed (Cohn et al. (1946) J. Am. Chem. Soc. 68:459-467). vz. Suspension of Precipitate G and Solvent / Detergent Treatment

[0235] The precipitate was dissolved and filtered with a depth filter of a nominal pore size of 0.2 pm (e.g., Cuno VR06 filter or equivalent) to obtain a clear filtrate which was used for the solvent / detergent (S / D) treatment.

[0236] The first of the steps in viral inactivation is S / D treatment of the re-suspended Precipitate G. The S / D treatment mixture contained 1.0% (v / v) Triton X-100, 0.3% (v / v) Tween-80, and 0.3% (v / v) tri-n- butyl phosphate, and the mixture was held at 18 °C to 25 °C for at least 60 minutes.Example 2: Process Performance Qualification of an IgA depleted IgG composition Manufacturing Process

[0237] In order to provide an additional treatment option for IgA-sensitive patients, a new version of an immune globulin infusion (such as the Takeda GAMMAGARD® LIQUID 10%) product containing lower levels of IgA has been developed.

[0238] Several small-scale studies were performed to define the process parameters leading to a reduction of the IgA concentration in final container without any major changes to other impurities. The ANX chromatography step was enhanced to achieve an ultra-low IgA antibody content IgG product with the same product safety (equivalent viral inactivation steps) and product quality (all quality attributes remain equivalent). After the optimization of the ANX chromatography conditions, the specifications of the purified IgG remain within the licensed parameters of GAMMAGARD® LIQUID [Immune Globulin Infusion (Human)] 10%. Thus, the proposed manufacturing process optimization generates a product equivalent to GAMMAGARD® LIQUID [Immune Globulin Infusion (Human)] 10% with a lower IgA content.

[0239] This validation was supported by the execution of an engineering run at manufacturing scale based on the small-scale development studies. This engineering run has been concluded positively and conforms to the acceptance criteria of less than or equal to 2 pg / mL for the IgA concentration in the final container after incubation.IgA Depleted IgG Composition Manufacturing Process

[0240] The following describes an exemplary IgA depleted IgG composition manufacturing process.Suspension of Precipitate G

[0241] Precipitate G was dissolved in Water for Injection. The target for OD280-320 value was between 40 AU and 95 AU (OD280-320 = absorbance unit at 280 nm - absorbance unit at 320 nm). The solution was stirred for at least 2 hours at a pH of 5.2 ± 0.2. The pH was adjustment was performed with 1 M acetic acid. The temperature during the dissolution was set at 0-8 °C. The conductivity of the solution was adjusted to 2.5-6.0 mS / cm, if necessary, with the addition ofsodium chloride. The solution was filtered through Cuno 90SP05A filter or equivalent (i.e., a depth filter that has a nominal pore size of 0.2 pm). Postwash was performed using a sodium chloride solution with a conductivity of 2.5-6.0 mS / cm.Solvent Detergent (S / D) Treatment

[0242] The filtrate was collected in a jacketed stainless steel reaction tank containing a predetermined amount of solvent detergent in a dilute sodium chloride solution with conductivity at 2.5-6.0 mS / cm. The target concentrations (acceptable ranges for the S / D components were ±50% of the target concentrations) of solvent / detergent components in the final mixture were: 1.0 ± 0.5% (v / v) octoxynol 9, 0.3 ± 0.15% (v / v) polysorbate 80, and 0.3 ± 0.15% (v / v) tri-(n- butyl) phosphate (TNBP). The calculated target OD280-320 value is 14 AU to 70 AU (for protein solution with an extinction coefficient of 14.1, the OD280-320 of 14 to 70 represents a protein concentration range of 1% to 5% (w / v)). The temperature during S / D treatment was maintained at 18.0 to 25.0 °C. The time required was a minimum of 60 minutes. The maximum exposure time to S / D was 24 hours.Cation Exchange Chromatography

[0243] Removal of the S / D mixture was performance by weak cation exchange chromatography with CM columns. Two columns are connected in parallel and equilibrated but the S / D treated protein solution is loaded on one column to reach a protein load of about 100 mg protein / mL resin (CM column equilibration II buffer 10 mM acetate buffer, pH 5.0 ± 0.2, conductivity at column outlet 1.00 to 1.50 mS / cm). The column used was washed with a minimum of 30 column volumes of wash buffer (10 mM acetate buffer, pH 5.5 ± 0.2). The adsorbed proteins were eluted with CM elution buffer (CM elution buffer: 35 mM monobasic sodium phosphate, 10 mM tris, pH 8.5 ± 0.2, conductivity 5.0 ± 0.2 mS / cm). The eluate was collected in a jacketed stainless steel reaction tank for further processing.Anion Exchange Chromatography

[0244] The eluate of the CM column was adjusted to a pH of 6.8 ± 0.1 by addition of acetic acid. The conductivity of the solution was reduced to 0.9 ± 0.1 mS / cm by dilution with Water for Injection.

[0245] The diluted solution was loaded as two aliquots onto two equilibrated ANX columns connected in parallel (ANX column equilibration I buffer 100 mM monosodium phosphate pH 6.8 ± 0.1; ANX column equilibration II buffer monosodium phosphate and acetate pH 6.8 ± 0.1 and conductivity 0.9 ± 0.1 mS / cm).

[0246] Once the first aliquot was completed, the columns were washed with buffer (ANX column equilibration II buffer monosodium phosphate and acetate pH 6.8 ± 0.1 and conductivity 0.9 ± 0.1 mS / cm) (3 CV) and the ANX washing was discarded. At the end of the first ANX washing, both ANX columns were cleaned and equilibrated. Just before loading the second aliquot of CM eluate solution, a pH / conductivity check was performed. Then, the second aliquot was processed. The total protein load applied was 30 + / - 5 mg protein / mb resin. Finally, the two ANX columns were washed with buffer (pH 6.8 ± 0.1, conductivity 0.9 ± 0.1 mS / cm) (3 CV). The second ANX washing was discarded as well. Both columns were cleaned. During the first and the second processing of the aliquots, the column flow-through was collected in the same jacketed stainless steel reaction tank for further processing.Nanofiltration

[0247] The ANX column effluent was filtered through a clarifying filter, Cuno VR06 or equivalent (depth filter with nominal pore size of 0.2 pm) prior to nanofiltration. Nanofiltration was performed using Asahi Planova 35N filters or equivalent (filtration mode for Asahi Planova 35N filter is tangential). The maximum protein load for nanofiltration was 8 kg protein per square meter of nanofilter membrane area. Each nanofilter was tested for pressure hold before and after use and tested for membrane integrity after use by gold particle test or equivalent as recommended by the manufacturer. The nanofiltration was started when the second ANX equilibration starts. The differential pressure (e.g., inlet filter pressure minus filtrate pressure) during nanofiltration was 1.0 bar maximum.Ultrafiltration

[0248] The glycine concentration of the nanofiltrate was adjusted to a target of 0.25 M. The solution was concentrated to a protein concentration of 5 ± 2% (w / v) (protein concentration was determined by measurement of OD280-320; extinction coefficient is 14.1) though ultrafiltration (UF). The pH was adjusted to 5.2 ± 0.2. The UF membrane used had a nominal molecular weightcut off (NMWCO) of 50,000 Daltons or less (e.g., Millipore Pellicon Biomax with a NMWCO of 50K Daltons or less; membrane material is poly ether sulphone).

[0249] The concentrate was diafiltered against a 0.25 M glycine solution, pH 4.2 ± 0.2. The minimum exchange volume was 10 times of the original concentrate volumes. Throughout the ultrafiltration / diafiltration operation, the solution was maintained at 4-20 °C.

[0250] After diafiltration, the solution was concentrated to a protein concentration (may be determined by UV reading through the use of an extinction coefficient value of 13.99) of 13.5% (w / v). The solution temperature was adjusted to 2-8 °C.Formulation

[0251] The protein concentration was further adjusted to 10.0 ± 0.1% (w / v) with a diafiltration buffer. The pH was adjusted to 4.4 to 4.9, if necessary (pH of the sample is determined with no prior dilution with saline).Sterile FiltrationThe bulk solution was aseptically filtered through a membrane filter cartridge of 0.2 pm absolute pore size (Durapore provided by Millipore Corporation, or equivalent). The process samples before the sterile filtration have a bioburden action limit of 10 CFU / mL. The filter must be tested and found to be satisfactory for integrity before and after use. Bulk solution could be re-sterile filtered (reprocessing) if one or more of the following are encountered: (1) if the sterile filter cartridge was clogged, (2) if the integrity test on the sterile filter failed, (3) the air filter on the sterile tank failed the integrity test, or (4) the integrity of the sterile tank was compromised.Aseptic FillingThe sterile bulk solution was aseptically dispensed into final containers, stoppered and sealed under aseptic conditions and capped. Representative samples of the lot were removed and tested.Low pH Incubation

[0252] The filled vials were incubated at 30 °C to 32 °C for 21 to 24 days. Representative samples of the lot were removed and tested. The pH of the product after incubation was 4.4 to 4.9 (pH of the sample was determined with no prior dilution with saline).IgA depleted IgG composition was subject to standard release criteria for intravenous human normal Immunoglobulin products. The IgA content was 1 to 2 pg / ml.

[0253] The final container of IgA depleted IgG composition after incubation was further characterized and met the set test parameters to fulfil requirements for intravenous or subcutaneous normal human immunoglobulin preparations.

Claims

WHAT IS CLAIMED IS:

1. A method of forming a storage-stable IgG pharmaceutical formulation, wherein the pharmaceutical formulation is containing not more than about 2 pg / mL of IgA in an aqueous pharmaceutically acceptable carrier, the formulation having a property selected from:(i) a pH of from about 4.4 to about 4.9;(ii) from about 0. IM to about 0.4M glycine, and a combination thereof, the method comprising:(a) performing an anion exchange step on an anion exchange precursor solution comprising the IgG, the step comprising:(i) submitting the anion exchange precursor solution to anion exchange chromatography in a single pass through a first portion of an anion exchange medium contained in a first column and collecting a first anion exchange flowthrough from the first column; and(ii) optionally, following (i), washing the anion exchange medium with a wash buffer, wherein the washing uses from about 0.25 column volumes to about 1 column volume of the wash buffer.

2. The method according to claim 1 , wherein the single pass through the anion exchange medium occurs in the first column, and in a second column containing a second portion of the anion exchange medium, the first and second columns run in parallel mode and collecting a second anion exchange flowthrough from the second column.

3. The method according to claim 1 , wherein the single pass through the anion exchange medium occurs in the first column, and in a second column containing a second portion of the anion exchange medium, the first and second columns run in serial mode and collecting a second anion exchange flowthrough from the second column.

4. The method according to any preceding claim wherein the IgG is loaded onto the anion exchange material at a maximum loading of about 70 mg / mL of resin.

5. The method according to any of claims 1-4, wherein the first, and second anion exchange flowthrough are optionally pooled.

6. The method according to any of claims 1 -5, wherein the pH of the anion exchange precursor solution is about from about 6.2 to about 7.0, e.g., from about 6.4 to about 7.0, e.g., from about 6.7 to about 6.9 and the conductivity is from about 0.5 to about 2.5, e.g., from about 0.7 to about 1.5, e.g., from about 0.8 to about 1.0 mS / cm.

7. The method according to any of claims 1 -6, wherein the protein concentration of the anion exchange flowthrough is from about 3 g / L to about 15 g / L.

8. The method according to any of claims 1-7, wherein the wash buffer flowthrough is pooled with the anion exchange flowthrough.

9. The method according to any of claims 1-8, wherein the anion exchange precursor solution is an eluate from a cation exchange step of an IgG containing suspension (e.g., from precipitate G) preceding (a).

10. The method according to claim 9, wherein the eluate from a cation exchange step preceding (a) is an eluate from one or more cation exchange columns.

11. The method according to claim 9, wherein a precursor solution for the cation exchange step is a mixture of dissolved Precipitate G, a solvent and a detergent.

12. The method according to claim 11 , wherein the solvent and the detergent are mixture of octoxynol 9, polysorbate 80 and tri-(n-butyl) phosphate or equivalent.

13. The method according to claim 12, wherein the octoxynol 9 is present at about 1.0 ±0.5% (v / v); the polysorbate 80 is present at about 0.3 ± 0.15% (v / v); and the tri-(n-butyl) phosphate is present at about 0.3 ± 0.15% (v / v) or equivalent.

14. The method of any of claims 1-13, the method further comprising: prior to (a), (b) a cation exchange chromatography eluate is generated by submitting a cation exchange chromatography precursor solution to a cation exchange medium in one or more column with a cation exchange buffer and collecting the cation exchange chromatography eluate.

15. The method according to claim 14, wherein the cation exchange medium has a protein loading of from about 50 mg / mL to about 125 mg / mL of medium.

16. The method according to claim 14, wherein the IgG is bound to the cation exchange medium within the one or more column.

17. The method according to claim 16, wherein IgG bound to the cation exchange medium is washed with from about 10 to about 30 column volumes of a wash buffer prior to eluting the bound IgG from the cation exchange material.

18. The method according to claim 14, the method comprising, eluting the bound IgG from the cation chromatography medium to which it is bound with a cation exchange elution buffer and collecting the cation exchange eluate.

19. The method according to claim 18, wherein the cation exchange elution buffer comprises: monobasic sodium phosphate (from about 35 mM to about 55 mM); and tris (about 10 mM), with a pH of 8.5 ± 0.2, and a conductivity of 5.0 ± 0.2 mS / cm.

20. The method according to claim 18, wherein the cation exchange eluate is adjusted to a pH of 6.8 ± 0.1, and a conductivity of 0.9 ± 0.1 mS / cm, forming the anion exchange precursor solution.

21. The method according to claim 18, the method further comprising, submitting the anion exchange flowthrough to nanofiltration, forming a first nanofiltered IgG formulation precursor solution.

22. The method according to claim 21, wherein a protein load for nanofiltration is not more than about 8 kg protein / m2of nanofilter membrane area.

23. The method according to claim 21, wherein a differential pressure during the nanofiltration is not more than about 1.0 bar.

24. The method according to claims 1-23, the method further comprising, submitting the first nanofiltered IgG formulation precursor solution to ultrafiltration, concentrating total protein in the first nanofiltered IgG formulation precursor, forming an ultrafiltered IgG formulation precursor solution.

25. The method according to claim 24, wherein the total protein concentration in the ultrafiltered IgG formulation precursor is about 5 ± 2% (w / v).

26. The method according to claim 24, wherein the ultrafiltration is performed with a membrane with a nominal molecular weight cutoff of not more than about 5 OK Daltons.

27. The method according to claim 24, wherein the total protein in the ultrafiltered IgG formulation precursor solution is concentrated to about 13.5% (w / v).

28. The method according to claim 24, further comprising, formulating the ultrafiltered IgG formulation precursor as an IgG formulation by adjusting IgG concentration of the ultrafiltered IgG formulation precursor to 10.0 ± 0.1% (w / v), and adjusting pH to about 4.4 to about 4.9 directly measured in the concentrated product.

29. The method according to claim 28, the method further comprising, transferring the IgG formulation into a container and incubating the IgG formulation in the container at from about 30°C to about 32°C for from about 21 to about 24 days.

30. The method according to any of claims 1-29, wherein the method provides the IgG formulation with an IgG yield of at least about 50% IgG compared to IgG present in a starting plasma sample.

31. The method according to any of claims 1 -30, wherein the storage-stable IgG pharmaceutical formulation is having about 10% (w / v) IgG.

32. A storage stable IgG pharmaceutical formulation prepared by the method of any preceding claim.

33. A storage-stable IgG pharmaceutical formulation containing not more than about 2 pg / mL of IgA, in an aqueous pharmaceutically acceptable carrier, the formulation having a property selected from:(i) a pH of from about 4.4 to about 4.9;(ii) from about 0. IM to about 0.4M glycine, and a combination thereof, wherein the storage-stable IgG pharmaceutical formulation is made by a method of any of claims 1-30.

34. The storage-stable IgG pharmaceutical formulation according to claim 33, having about10% (w / v) IgG.

35. A storage-stable IgG pharmaceutical formulation containing not more than about 2 pg / mL of IgA in an aqueous pharmaceutically acceptable carrier, the formulation having a property selected from:(i) a pH of from about 4.4 to about 4.9;(ii) from about 0. IM to about 0.4M glycine, and a combination thereof.

36. The storage-stable IgG pharmaceutical formulation according to claim 35, having about 10% (w / v) IgG.