Methods for producing immunoglobulin preparations

Abstract

The present invention provides methods for producing IgG preparations. Apparatus for performing the methods of the invention and IgG preparations obtainable or obtained by the methods of the invention are also provided herein.

Description

[0001] METHODS FOR PRODUCING IMMUNOGLOBULIN PREPARATIONS

[0002] Field of Invention

[0003] The present invention provides methods for producing IgG preparations.

[0004] Background

[0005] Human polyclonal Immunoglobulin G (IgG) is widely used to treat a variety of immunodeficiency, inflammatory, autoimmune, and neurological conditions. High doses of IgG are often required for these therapies, therefore an efficient method for purification of this valuable biological is critical. Current commercial processes for production of polyclonal IgG from human plasma are complex and time consuming, and typically achieve recoveries of only 45-65%.

[0006] Traditional methods of IgG purification are based largely upon the Cohn cold-ethanol fractionation process devised in the 1940s, which is associated with high energy costs and, in some instances, might impact the physicochemical and functional properties of the obtained product due to incubation with high concentrations of alcohol.

[0007] Modern methods of chromatography, especially affinity chromatography, afford the potential for improving the yield of IgG products obtained from human plasma. Although most commercially available IgG affinity resins based upon Protein G and camelid antibody-based affinity resins bind all four IgG subclasses, they exhibit relatively low IgG-binding capacity. Further, the vendor ThermoFisher Scientific indicates on the product sheet for CaptureSelect FcXP affinity resins that they have a limited stability to cleaning with NaOH.

[0008] In the context of the above-mentioned drawbacks in prior art methods, there is a need for improved IgG preparation methods, which help satisfy the high demand of clinical IgG.

[0009] Summary of Invention

[0010] In one aspect, the present invention provides a method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0011] (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; and

[0012] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4.In a further aspect, the invention provides a method of producing an IgG preparation from an IgG-containing blood-derived starting material, the method comprising:

[0013] (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; and

[0014] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation.

[0015] In a further aspect, the invention provides a method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0016] (a) contacting the IgG-containing starting material with a first chromatographic solid support comprising a first IgG-binding affinity ligand, wherein the first IgG-binding affinity ligand is Protein A, to obtain a first IgG bound fraction and a first flow-through; (b) contacting the first flow-through with a second chromatographic solid support comprising a second IgG-binding affinity ligand, wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), to obtain a second IgG bound fraction and a second flow-through; and

[0017] (c) eluting the first IgG bound fraction and the second IgG bound fraction to obtain an IgG preparation, wherein the IgG preparation comprises the first IgG bound fraction and the second IgG bound fraction.

[0018] The invention includes apparatus for performing the methods described herein.

[0019] The invention includes an IgG preparation obtainable or obtained by the methods described herein.

[0020] Methods of the invention can be associated with a number of unexpected technical advantages. Methods of the invention may increase the lifespan of the affinity resin, which is beneficial as it reduces the overall cost of the method and decreases the total amount of waste produced by the process. Methods of the invention can provide a cost-effective technology that aids in the challenges on industrial reusability of affinity resins in prior art methods. The methods of the invention can result in reduced energy costs and therefore more sustainable processes, compared to prior art methods, for example due to the lack of use of cold ethanol fractionation. Methods of the invention may be more energy efficient per quantity of IgG prepared. Methods of the invention can result in compact and simple processes compared to prior art methods that contain multiple and complex process steps, producing IgG preparations of high purity in a shorter time, thus allowing higher productivity in manufacturing. Methods of the invention may be more operationally compact than prior art processes. Methods of the invention may require less time to prepare for a given quantity of IgG prepared. Methods of the invention may require less apparatus to prepare for a given quantity of IgG prepared.Advantageously, fibrinogen may be removed in the delipidation step and the removal of fibrinogen in the methods of the invention facilitates the reuse of the resin. Methods of the invention can also be used to produce IgG preparations in high yield and high purity in a short time. The claimed methods can provide good viral clearance, so the IgG preparations prepared by the methods may be safely administered to humans and / or other animals without further purification. The methods of the invention may be used to produce IgG preparations with a low proportion of the cholesterol relative to the starting material. The methods of the invention can be used to produce IgG preparations with a low proportion of the triglycerides relative to the starting material. The methods of the invention can be used to produce IgG preparations with a low proportion of the fatty acids relative to the starting material. Furthermore, embodiments of the methods of the invention using a dual affinity chromatography process of protein A and FcXP may be effective at providing IgG preparations in high yields. Embodiments of the methods of the invention using a dual affinity chromatography process of protein A and FcXP may be effective at providing IgG preparations in high purities. Embodiments of the methods of the invention using a dual affinity chromatography process of protein A and FcXP may be effective at providing IgG preparations at reduced total resin cost. Embodiments of the methods of the invention using a dual affinity chromatography process of protein A and FcXP may be effective at providing IgG preparations with improved stability.

[0021] In one embodiment, the method of the invention comprises the use of an IgG-binding affinity ligand in the chromatography column as part of a single affinity chromatography process, wherein the IgG-binding affinity ligand is VHH. Optionally, the method of the invention comprises the use of dextran sulfate as the polyanionic compound for the delipidation step and an IgG-binding affinity ligand in the chromatography column as part of a single affinity chromatography process, wherein the IgG-binding affinity ligand is VHH. In such an embodiment, the VHH may be the VHH of FcXP resin, optionally wherein the chromatographic resin is CaptureSelect™ FcXP Affinity with a matrix, for example wherein the matrix is an Agarose matrix or a POROS-based matrix. In other words, method of the invention may comprise the use of dextran sulfate as the polyanionic compound for the delipidation step and a chromatography column comprising FcXP resin as part of a single affinity chromatography process, optionally wherein the chromatographic resin is CaptureSelect™ FcXP Affinity with a matrix, for example wherein the matrix is an Agarose matrix or a POROS-based matrix. These embodiments can provide the surprising technical advantage that the delipidation step can be performed at relatively mild pH (for example at pH of from about 6.5 to about 8.5), in contrast to prior art performed at acidic pH. The need for adjusting the pH following the delipidation step may also be reduced or eliminated, and thus the IgG preparation may show higherstability, as the IgGs are maintained as close to physiological pH throughout most of the process. Additionally, these single affinity processes may be associated with the benefit of higher lgG3 recovery as a proportion of the total IgG content, see for example Tables 5 and 6. Disclosed herein is a higher yield method of producing an IgG preparation from an IgG-containing starting material. Disclosed herein is a higher yield method of producing an IgG preparation from an IgG-containing blood-derived starting material. It will be understood that all methods disclosed herein may be higher yield methods, for example higher yield methods than prior art methods.

[0022] Except for where the context requires otherwise, the considerations set out in this disclosure should be applicable to all methods described herein.

[0023] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0024] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. Where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0025] Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0026] Various aspects of the invention are described in further detail below.

[0027] Brief Description of Drawings

[0028] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0029] Figure 1 displays an overview of an embodiment of the methods of the invention.

[0030] Figure 2 displays a chromatogram of a single-affinity Capture Select FcXP column IgG capture step. Chromatogram displays absorbance at 280 nm and pH.

[0031] Figure 3 displays a chromatogram of a dual-affinity column IgG capture step. Chromatogram displays absorbance at 280 nm, and pH.

[0032] Figure 4 displays SDS-PAGE (NuPAGE4-12% Bis-Tris gels with MES running buffer) analysis of proteins in intermediates from the 4-step affinity chromatography-based IgG purificationprocess. Samples were analyzed under both non-reducing and reducing conditions. Lane 1: Mark12 molecular weight standard; Lane 2: Delipidated cryoprecipitate supernatant; Lane 3: Clarified & filtered supernatant from Dextran Sulfate / CaCh treatment of the delipidated cryoprecipitate supernatant, also known as dual-affinity load material; Lane 4: Dual affinity flow-through / wash pool; Lane 5: Dual affinity elution pool; Lane 6: Fractogel TMAE Hicap pH 5.35 IEX polishing step flow-through pool; Lane 7: Final IgG product (~5%) from dual-affinity 4-step process; Lane 8: commercial IVIG control; and Lane 9: Mark12 molecular weight standard.

[0033] Figure 5 displays a chromatogram of a Fractogel TMAE Hicap IgG polishing step. Chromatogram displays absorbance at 280 nm and conductivity.

[0034] Figure 6 displays a chromatogram of a POROS XQ IgG polishing step. Chromatogram displays absorbance at 280 nm, conductivity, and pH.

[0035] Figure 7 displays IgG recovery from a CaptureSelect FcXP column (Column 1) during repetitive loadings with delipidated cryoprecipitate supernatant (30 g IgG / L resin) and sanitizations with 0.1 M NaOH, as well as IgG recovery from this same column repetitively overloaded with purified IgG (80 g IgG / L resin) at indicated intervals. A second, identical column (Column 2) was overloaded repetitively with purified IgG (80 g IgG / L resin) and exposed to 0.1 M NaOH for the indicated time.

[0036] Figure 8 shows the effect of different concentrations of dextran sulfate on IgG recovery in the methods of the invention. Notably, it is shown that low dextran sulfate concentrations improve IgG yield.

[0037] Figure 9 shows the impact of dextran sulfate concentration on IgG recovery, FNG removal, cholesterol removal over time.

[0038] Detailed Description

[0039] A method of preparing an IgG preparation with a delipidation step

[0040] The present invention is based on the inventors’ development of a novel method for purifying immunoglobulin G (IgG) from an IgG-containing starting material, such as an IgG-containing biological sample. The method is particularly useful in the context of blood-derived samples, which generally have a high lipid content, and which contain a complex mixture of proteins. The novel method involves the step of delipidating a sample prior to subjecting the sample to affinity chromatography. Delipidating a sample with a polyanionic compound may be particularly useful in the context of the methods described herein. Surprisingly, the step ofdelipidating (in particular, with polyanionic compounds) may extend the life span of the chromatographic resins used in affinity chromatography, thereby reducing the overall costs of the method. For example, delipidation may remove fibrinogen from the starting material, which prevents fouling of the resins used in affinity chromatography, facilitating re-use of the resins. Experimental data is provided in the application demonstrating that methods of the invention with a delipidation step result in (i) removal of lipids and e.g. fibrinogen from the starting material (see example 1); and (ii) prolonged resin lifespans (see example 9).

[0041] The step of affinity chromatography may involve contacting the delipidated sample with one or more chromatographic solid support comprising at least one IgG-binding affinity ligand. By using more than one chromatographic solid support, the costs of purifying IgG from a sample may be even further reduced. Specifically, a first, more cost-effective solid support may be used to purify a large portion of IgG (for example lgG1, lgG2 and lgG-4), followed by a second column, that may purify lgG3. In such an example, the IgG-binding affinity ligand in the first column may be Protein A, and the IgG-binding affinity ligand in the second column, may be Protein G or a ligand based on recombinant single-domain antibody fragments (e.g. VHH) such as the VHH in FcXP resin or a synthetic mimic of Protein A which binds lgG3. By removing a large portion of the IgG using the first column, a significantly lower amount of affinity ligand is required in the second column, therefore reducing the overall cost of the method.

[0042] Where, for example, a first, more cost-effective solid support comprising at least one IgG-binding affinity ligand is used to purify a large portion of IgG (for example lgG1, lgG2 and lgG-4), followed by a second column, that may purify lgG3, it may be possible to use a reduced volume of resin in the second column. This may apply, for example, when the resin in the first column is Protein A and the resin in the second column is a Protein G or a VHH-based resin such as FcXP resin or a synthetic mimic of Protein A which binds lgG3. In these embodiments, the volume ratio of the resin in the first column to the second column may be 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15. The optimal volume ratio of the first chromatographic solid support to the second chromatographic solid support will depend on inter alia the binding capacities of the first and second resin and the relative abundance of each IgG subclass within the starting material. Where the first IgG-binding affinity ligand is Protein A and where the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15.. Where the first IgG-binding affinity ligand is Protein A and where the secondIgG-binding affinity ligand is the resin FcXP, the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15.

[0043] Additionally, the method described herein was shown by the inventors to provide a high yield of highly pure IgG in a surprisingly short time of 2 days or less (see e.g. example 7). As can be seen in the examples, the delipidation step can be performed in 1 to 4 hours. The method described herein results in a large increase in yield of IgG from plasma (from ~ 65% up to ~ 90%), thus maximizing recovery of IgG per unit of costly plasma. Specifically, solely from the dual-affinity column chromatography, the yield was 99.6%, while the overall IgG yield from the method comprising delipidation, dual-affinity column chromatography, flow-through anion-exchange chromatography, and a concentration step was up to ~ 90%. Similar results were observed for delipidation, single-affinity chromatography and flow-through anion-exchange chromatography. Importantly, the IgG preparation that has undergone delipidation, single- or dual-affinity column chromatography, flow-through anion-exchange chromatography, and a concentration step is sufficiently pure for clinical use.

[0044] Use of affinity chromatography has also allowed design of a much faster process, which will allow more IgG to be produced per unit of time.

[0045] Moreover, the two-step method of the present disclosure (comprising the step of delipidating the starting material and the step of affinity chromatography) has been found by the inventors to provide two distinct steps of viral clearance, thereby effectively reducing viral contamination in the starting material. Specifically, the step of delipidation may reduce viral contamination by precipitation (especially when the delipidating agent is a polyanionic compound, more especially when the delipidating agent is dextran sulfate), and the step of affinity chromatography (especially when the IgG-binding affinity ligand comprises Protein A, for example MabSelect PrismA™ Protein A, or the resin FcXP, for example FcXP used in a singleaffinity method) may reduce viral contamination by binding only to a target protein (such as an IgG) and allowing the virus to flow through the column. As it will be appreciated, in the context of blood or blood-derived starting materials, a reduction of viral particles will typically be highly desirable. Accordingly, the methods described herein are advantageous for a number of reasons.

[0046] In one aspect, the present invention provides a method of producing an IgG preparation from an IgG-containing (e.g. blood-derived) starting material. The term “preparing” as used herein means “obtaining”, “producing” or “purifying”, and these terms may be used interchangeably.An “IgG enriched preparation” as used herein refers to a composition that is not naturally occurring (but may be obtained from a naturally occurring IgG-containing starting material, such as a blood-derived starting material). For example, a composition prepared by the step of delipidating the starting material may be referred to herein as an IgG enriched preparation. The phrase “IgG enriched preparation” encompasses a composition prepared by the step of delipidating the starting material which has not undergone the step of affinity chromatography. In the context of Figure 1 , an IgG enriched preparation is therefore obtained after step 1 of the depicted method. The term “IgG preparation” used herein refers to a composition derived from a process of the invention as claimed (e.g. a method comprising the step of delipidating the starting material and the step of affinity chromatography), and it will be understood that an IgG preparation may undergo other processing steps. In the context of Figure 1, an IgG preparation therefore encompasses the composition obtained after step 2. Typically, an IgG preparation is a composition wherein the concentration of the IgG compared to other proteins in the composition is significantly higher as compared to the concentration of IgG compared to other proteins in the starting material (where the starting material may be for example a blood-derived sample). As an example, IgG in human serum accounts for about 10 to 20% of plasma proteins. Therefore, in the context of the present disclosure, an IgG preparation obtained by the method described herein may be a composition wherein the IgG accounts for more than 70% of plasma proteins in the IgG preparation. More suitably, the IgG may account for at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of plasma protein in the IgG preparation. Most suitably, the IgG preparation may be substantially devoid of other plasma proteins. By substantially devoid it is meant that other plasma proteins (i.e. proteins that are not IgG) account for less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of plasma proteins in the IgG preparation. In this context, reference to “IgG” refers to all IgG subclasses (i.e. the total amount of all IgG subclasses) that are present. An IgG preparation may also be referred to as an IgG concentrate, an IgG enriched preparation, or an IgG eluate.

[0047] An IgG enriched preparation and / or an IgG preparation may comprise an elution buffer. An elution buffer is a buffer that enables the protein of interest (such as IgG), which is immobilized on a chromatographic solid support, to be passed through the chromatographic column. Many elution buffers are known to those skilled in the art and may depend upon the nature of the immobilized protein as well as the affinity ligand. Merely by way of example, the elution buffer may be 50 mM acetic acid (pH 3.0).Elution buffers of the invention may be of low pH. In some embodiments, the elution buffer is of pH 4.0 or less, pH 3.5 or less, pH 3.0 or less, pH 2.5 or less, or pH 2.0 or less. In some embodiments, the elution buffer is of pH 1.5-4.0, pH 1.5-3.5, pH 1.5-3.0, pH 1.5-2.5, or pH 1.5-2.0. In embodiments where a first chromatographic solid support and a second chromatographic solid support are used, an elution buffer may be added to the first chromatographic solid support resulting in a first eluate wherein the first eluate comprises the first IgG bound fraction, and the first eluate passes directly onto the second chromatographic solid support resulting in an IgG preparation comprising the first IgG bound fraction and the second IgG bound fraction. In such a way, the first chromatographic solid support and the second chromatographic solid support may be eluted concurrently. Used herein the terms “concurrently” and “in tandem” are synonymous. In some embodiments the composition of the elution buffer is such (e.g. it is of a sufficiently low pH) that the first IgG bound fraction substantially does not bind to the second chromatographic solid support during the concurrent elution process. As will be appreciated by the skilled person, an IgG preparation comprising additional components (such as e.g. elution buffer) may undergo subsequent processing step(s), for example in order to be suitable for administration to a human or animal. Merely by way of example such a subsequent processing step may include separating the IgG from the elution buffer and / or purifying the IgG.

[0048] Suitably, IgG may be separated from the elution buffer. Methods for removing the elution buffer are well known in the art, and may include dialysis, diafiltration or precipitation, followed by removal of supernatant. The term “IgG preparation” as used herein encompasses IgG preparations that comprise additional components (such as e.g. elution buffer) and also IgG preparations that are obtained by subsequent processing step(s), for example in order to be suitable for administration to a human or animal.

[0049] Suitably, the IgG preparation may be purified by subjecting the IgG preparation to flow-through anion-exchange chromatography. Such a purification step may remove substantially all non-IgG proteins in the preparation, such as IgA and / or IgM. Accordingly, prior to this purification step, IgG may account for at least about 95% of the proteins in the IgG enriched preparation, whereas after this purification step, IgG may account for at least 98% of the proteins in the IgG preparation. Suitably, methods of the disclosure may comprise: (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4; and (c) subjecting the IgG preparation to flow-through anion-exchange chromatography to obtain a purified IgG preparation. Suitably, methods of thedisclosure may comprise: (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation; and (c) subjecting the IgG preparation to flow-through anion-exchange chromatography to obtain a purified IgG preparation. Suitably, methods of the disclosure may comprise: (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4; (c) subjecting the IgG preparation to flow-through anion-exchange chromatography to obtain a purified IgG preparation; and (d) formulating and concentrating the purified IgG preparation. Suitably, methods of the disclosure may comprise: (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation; (c) subjecting the IgG preparation to flow-through anion-exchange chromatography to obtain a purified IgG preparation; and (d) formulating and concentrating the purified IgG preparation.

[0050] The IgG preparation may be purified by flow-through mode anion-exchange chromatography using a number of different anion-exchange resins, which may have different optimal operating conditions. The skilled person will be aware of a number of suitable anion-exchange resins for this purpose.

[0051] Suitably, the IgG preparation may have a pH of from about 5 to about less than 6, for example from about 5.25 to about 5.5, for example about 5.35, prior to being loaded into an anion-exchange column (for example comprising Fractogel® EMD TMAE Hicap (M) resin (EMD Millipore)). The pH of the IgG preparation may be adjusted to the desired pH (for example to about 5.35) by acetate buffer. Adjusting the pH of the IgG preparation to from about 5 to about less than 6, for example from about 5.25 to about 5.5, for example about 5.35, is associated with reduced turbidity of the preparation.

[0052] Suitably, the IgG preparation may have a pH of from about 5.5 to about 6.5, for example from about 6 to about 6.5, for example 6.2, prior to being loaded into an anion-exchange column (for example comprising POROS XQ resin (Thermofisher)). The pH of the IgG preparation may be adjusted to the desired pH (for example about 6.2) by acetate buffer.

[0053] Suitably, the IgG preparation may be purified by flow-through mode anion-exchange chromatography using other resins, such as Q Sepharose (Cytiva), ANX Sepharose (Cytiva), DEAE Sepharose Fast Flow (Cytiva), Poros 50 HQ (Thermofisher) or Nuvia HP-Q (Bio-Rad).Suitably, the IgG preparation may be purified by flow-through mode anion-exchange chromatography using single-use or reusable membrane chromatography, as well as by different types of protein adsorbers.

[0054] Suitably, the purified IgG preparation may be subsequently formulated and concentrated. Merely by way of example, the IgG preparation may be formulated through buffer exchange by diafiltration and concentrated by ultrafiltration. Suitably, the diafiltration buffer may be a buffer such as 0.2 M glycine-HCI (pH 4.2) or a related buffer. Suitably, concentration may be carried out before diafiltration or before and after diafiltration. Suitably, ultrafiltration may be performed through 30-100 kDa filters (for example Millipore Labscale TFF system equipped with three Pellicon XL 50 kDa filters (EMD Millipore)).

[0055] The IgG preparation may comprise a pharmaceutically acceptable excipient, carrier, or diluent.

[0056] The term “IgG-containing starting material” as used herein refers to a sample (for example a biological sample) that contains IgG. For avoidance of doubt, the term "IgG" as used herein means immunoglobulin G type. As it will be clear to a person of skill in the art, IgG is a class of antibodies that are substantially encoded by recognized immunoglobulin gamma genes. Suitably, the IgG may be selected from the group consisting of lgG1, lgG2, lgG3, and lgG4. The structure of IgG is described elsewhere herein.

[0057] IgG is typically present in the blood of mammals, for example, primates such as humans and chimpanzees, experimental animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats. Accordingly, the IgG-containing starting material may be obtained from any organism that contains IgG, such as those listed herein. Alternatively, the IgG-containing starting material may be obtained from a cell line such as CHO cell line, hybridoma, baby hamster kidney cells (such as including BHK-21), HEK (such as including HEK293T), and others.

[0058] In the context of the present disclosure, the term IgG-containing starting material may be a blood sample, blood-derived sample (such as plasma or serum), or ascites. The term “blood plasma” or “plasma” refers to the liquid portion of blood and lymphatic fluid. Plasma generally accounts for about half of the blood volume (e.g. about 50% to 60% by volume). There are substantially no cells in the plasma. Plasma may contain coagulation factors, in particular fibrinogen, and water. Plasma components include electrolytes, lipid metabolites, biomarkers such as markers for infection or cancer cells, enzymes, substrates, proteins, and additional molecular components. As used herein, the term “serum” is used in accordance with its plain and ordinary meaning and refers to the fluid and solute component of blood which does not play a role in clotting. It may be defined as blood plasma without fibrinogen. Suitably, theplasma may be selected from the group consisting of plasma cryoprecipitate supernatant, plasma cryoprecipitate filtrate, anti-coagulated plasma, citrated plasma or a precipitated fraction containing IgG (also known as paste), further optionally wherein the plasma is a plasma fraction (for example fraction l+ll+lll or ll+lll from Cohn method). Used herein the terms “precipitated plasma fraction containing IgG” and “paste” are used synonymously.

[0059] More suitably, the plasma may be plasma cryoprecipitate filtrate. Suitably the plasma cryoprecipitate filtrate may be obtained by filtering plasma cryoprecipitate through a porous filter. The filter may have a pore size of from about 0.1 pm to about 0.3 pm. More suitably the pore size may be about 0.2 pm, for example 0.22 pm. The term “pore size” refers to the average pore diameter of the pores in the filter.

[0060] Suitably, the IgG may be selected from the group consisting of human, non-human, humanized and chimeric IgG. The term “human IgG” as used herein includes IgG having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the IgG contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human IgG may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human IgG”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Suitably, human IgG may be isolated from a human sample.

[0061] By the same token, a non-human IgG is one that may be isolated from non-human plasma. Suitably, the non-human IgG may be an anti-venom IgG. Such an anti-venom IgG may be a horse IgG (isolated from a horse).

[0062] The term “humanized IgG” as used herein includes IgG in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.

[0063] The term “chimeric IgG” as used herein includes IgG in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an IgG in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human IgG.Suitably, the IgG may be monospecific (i.e. having the ability to bind only one epitope) or multispecific (i.e. having the ability to bind more than one, for example two or more, epitopes). Suitably, the IgG may be monoclonal or polyclonal. The term "monoclonal" as used herein has the meaning typically ascribed to it in the art, namely an antibody arising from a single clone of an antibody-producing cell, recognizing a single epitope on the antigen bound. The term “polyclonal” as used herein refers to a population comprising multiple clones recognizing different epitopes of the same antigen or of different antigens. The examples section of the present disclosure shows that the present invention is highly suitable for obtaining highly pure polyclonal IgG preparations.

[0064] The method of the invention may comprise the step of delipidating the IgG-containing starting material (e.g. the blood-derived starting material). Advantageously, delipidation (in particular, with polyanionic compounds) of the starting material reduces the amount of fibrinogen present in the IgG preparation that is contacted with one or more chromatographic solid supports (see e.g. example 1, table 1). This increases the life span of the chromatographic resins used in the affinity chromatography.

[0065] The step of delipidating may comprise combining (for example by mixing) the starting material with a polyanionic compound (for example dextran sulfate or polyphosphate) and optionally, a divalent cation. This step may result in the formation of a precipitate. Suitably, the precipitate may be removed prior to contacting the delipidated sample with one or more chromatographic solid supports.

[0066] Suitably, when the polyanionic compound is dextran sulfate the divalent cation may be combined with the starting material prior to the dextran sulfate being combined with the starting material. Suitably, when the polyanionic compound is dextran sulfate the divalent cation may be combined with the starting material after the dextran sulfate being combined with the starting material.

[0067] Among the polyanionic compounds of the disclosure, dextran sulfate is especially preferred. This is because, inter alia, dextran sulfate is especially effective in removal of phospholipids. Suitably, when the polyanionic compound is polyphosphate the divalent cation may be combined with the starting material after the polyphosphate being combined with the starting material.

[0068] The term “delipidating” as used herein means reducing the lipid content in the starting material to obtain a sample with reduced lipid content (i.e. a delipidated sample).Lipids are hydrophobic and / or amphipathic molecules, which are characterised as being substances soluble in organic solvents and insoluble or poorly soluble in aqueous solutions. The term “lipid” refers to one or more fatty acids (including free fatty acids and fatty acid esters), phospholipids, triacylglycerols (triglycerides), diacylglycerides, monoacylglycerides, lysophospholipids, soaps, phosphatides, waxes, sterols, sphingolipids, glycerolipids, glycerophospholipids, and other lipids known to those skilled in the art. Lipids include polar lipids and neutral lipids.

[0069] Suitably, in the context of the present disclosure, the lipids may be lipids that are commonly found in blood, i.e. blood lipids. As used herein, the term "blood lipids" may be present in the blood in free form or as part of a protein complex, such as a lipoprotein complex. Nonlimiting examples of serum lipids may include fatty acids, triglycerides, cholesterol and phospholipid, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C), and intermediate density lipoprotein cholesterol (IDL-C).

[0070] Suitably, the step of delipidating may remove at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50% or more of the lipids in the starting material. For example, the step of delipidating may remove at least 60%, at least 70%, at least 80%, at least 90% or more of the lipids in the starting material. More suitably, the step of delipidating may remove substantially all lipids from the sample, i.e. it may remove at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the lipids in the starting material. It will be appreciated that in the context of the present disclosure, a delipidated sample need not be completely devoid of lipids. A delipidated sample may have a significantly reduced amount of lipids as compared to the starting material.

[0071] Experimental data showing that methods of the invention are effective in removing cholesterol, triglycerides, phospholipids and fatty acids are shown e.g. in example 1. Suitably the step of delipidating may remove about 75% of cholesterol from the starting material. Suitably, the step of delipidating may remove at least about 75% of cholesterol from the starting material.

[0072] Suitably, the step of delipidating may remove about 70% of triglycerides from the starting material. Suitably, the step of delipidating may remove at least about 70% of triglycerides from the starting material. Suitably the step of delipidating may remove about 80% of triglycerides from the starting material. Suitably, the step of delipidating may remove at least about 80% of triglycerides from the starting material.

[0073] Suitably, the step of delipidating may remove about 40% of phospholipids from the starting material. Suitably, the step of delipidating may remove at least about 40% of fatty acids from the starting material. Suitably, the step of delipidating may remove about 50% of phospholipidsfrom the starting material. Suitably, the step of delipidating may remove at least about 50% of fatty acids from the starting material. Suitably, the step of delipidating may remove about 60% of phospholipids from the starting material. Suitably, the step of delipidating may remove at least about 60% of fatty acids from the starting material.

[0074] Suitably, the step of delipidating may remove about 15% of fatty acids from the starting material. Suitably, the step of delipidating may remove at least about 15% of fatty acids from the starting material.

[0075] Suitably, the step of delipidating may remove about 75% of cholesterol, about 80% of triglycerides, and about 15% of fatty acids from the starting material. Suitably, the step of delipidating will remove at least about 75% of cholesterol, at least about 80% of triglycerides, and at least about 15% of fatty acids from the starting material. Suitably the step of delipidating may remove about 75% of cholesterol, about 70% of triglycerides, and about 15% of fatty acids from the starting material. Suitably, the step of delipidating will remove at least about 75% of cholesterol, at least about 70% of triglycerides, and at least about 15% of fatty acids from the starting material.

[0076] Several means for delipidating a starting material are known in the art. Merely by way of example, the delipidating may be performed with a compound selected from the group consisting of a polyanionic compound; Amberlite XAD-2; Amberlite XAD-4 (as described in US 4,775,482 and C.S. Burden et al. (2012) J. Chromatogr. B 880, 82-89); CUNO Zeta Plus DELIP series filter cartridge; Sorbents with primary and / or secondary amines; graphitized carbon black; strong anion-exchange resins; C18 particles (as described in D.Y. Bang et al. (2014) J. Chromatogr. A 1331, 19-26); CytoSorb® (CytoSorbents Corp, USA); Silica-based compounds (such as Cab-O-Sil (fumed silica)); caprylate; Lipid Removal Agent (Lipid Removal Agent (LRA), MilliporeSigma™ Supelco™); Cleanascite™ (Biotech Support Group LLC); and CataKlear Lipid Clearing Agent (Vetlab Supplies Ltd).

[0077] Suitably, the step of delipidating the starting material may be performed with a polyanionic compound. Where delipidation is performed with a polyanionic compound a divalent cation is present in the delipidation step. The polyanionic compound may comprise a divalent cation or, alternatively, where the polyanionic compound does not comprise a divalent cation the step of delipidating the starting material comprises the polyanionic compound and a divalent cation source (for example, the divalent cation source is a salt of a divalent cation). The polyanionic compound may comprise a divalent cation. Suitably the polyanionic compound does not comprise a divalent cation and the step of delipidating the starting material comprises combining the starting material, the polyanionic compound and a divalent cation source, optionally wherein the divalent cation source is a salt of a divalent cation. Suitably, the step ofdelipidating the IgG-containing starting material (e.g. blood-derived starting material) may be performed with a polyanionic compound and a divalent cation compound.

[0078] In the present invention, the term “polyanionic compound” means a compound having a plurality of anionic functional groups in its molecule. Examples of the anionic functional groups include functional groups negatively charged at neutral pH, such as a carboxyl group, a sulfonate group, a sulfate group, and a phosphate group. Mixtures of different functional groups negatively charged at neutral pH may be present within the molecule of the polyanionic compound. The polyanionic compound may be polyelectrolyte. The polyanionic compound may be a polyacid, i.e. a polyelectrolyte composed of macromolecules containing acid groups on a substantial fraction of the constitutional units.

[0079] The term “divalent cation source” means a compound capable of providing one or more divalent cations when dissolved in the aqueous environment of the delipidation step. In some embodiments the divalent cation is Mg++, Ca++, Mn++, Zn++, Sr++, Ba++, Fe++, V++, Cr++, Co++, Ni++, Cu++, or combinations thereof. Preferably the divalent cation is Mg++, Ca++, Mn++, or combinations thereof. More preferably the divalent cation is Ca++. The term “divalent cation” means a metal ion having a 2+ charge, for example the alkaline earth metals in the 2+ oxidation state.

[0080] The polyanionic compound may be a macromolecule having a plurality of anionic functional groups, for example, may be a polysaccharide having a plurality of anionic functional groups. The polyanionic compound may be a sulfated polysaccharide.

[0081] Representative examples of the polyanionic compound include a dextran polymer, polyphosphate, and hydroxyapatite resin; optionally wherein the polyanionic compound is combined with a divalent cation. Experimental data for the polyanionic compound being either a dextran polymer or polyphosphate are provided in the examples. Alternatively, the polyanionic compound may be selected from the group consisting of carrageenans, kappa carrageenan, iota carrageenan, lambda carrageenan, algin, alginates, carboxymethyl cellulose, and salts thereof. Further, the polyanionic compound may be selected from the group consisting of heparin, heparin sulfate, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, polyaspartic acid, polyglutamic acid, nucleic acid including DNA and RNA, and anionic chromatographic resins.

[0082] As used herein the term “dextran polymer” refers to dextran or a derivative or salt thereof having a plurality of anionic functional groups, including, but not limited to, dextran sulfate and dextran sulfate sodium salt. Dextran is a polysaccharide with a main polymer main chain consisting of a-1,6 glycosidic linkages between glucose monomers, with branches from a-1,3 linkages. For example, the molecular weight of dextran polymer may range from 8 kDa togreater than or about 500 kDa. Suitably, the dextran polymer is dextran sulfate or dextran sulfate, sodium salt. Suitably, the dextran sulfate may be isolated from the bacterium Leuconostoc mesenteroides, strain B 512. Various molecular weight fractions of dextran sulfate may be produced by limited hydrolysis and fractionation. The present inventors believe that a molecular weight in the range of 36 kDa or larger may be most effective for delipidation. In one example, the polyanionic compound is dextran sulfate. As shown herein, delipidation of the starting material using dextran sulfate significantly reduces the amount of fibrinogen that is present in the IgG preparation that is contacted with one or more chromatographic solid supports (see example 1, table 1). Specifically, the inventors found that delipidation with dextran sulfate removed approximately 60% of fibrinogen. This serves to increase the life span of the chromatographic resins used in the affinity chromatography. The inventors found that, unexpectedly, dextran sulfate was especially effective at removing triglycerides and phospholipids during delipidation (see e.g. example 1).

[0083] The term “polyphosphate” as used herein refers to a salt, ester or derivative of polymeric oxyanion formed from tetrahedral PO4 (phosphate) structural units linked together by sharing oxygen atoms via phosphoanhydride bonds. Polyphosphates can adopt linear or a cyclic (also called, ring) structures. Polyphosphates can be linear or branched. In some embodiments, the number of phosphate residues in the polyphosphate chain or ring is 3 to 1000, 4 to 1000, 5 to 1000, 6 to 1000, 10 to 1000, 20 to 1000, 25 to 1000, 30 to 1000, 40 to 1000, 50 to 1000, 100 to 1000, 150 to 1000, 200 to 1000, 300 to 1000, 400 to 1000, or 500 to 1000. In some embodiments the polyphosphate is >10 kDa.

[0084] In one example, the polyanionic compound is polyphosphate. In some embodiments, the polyanionic compound is sodium polyphosphate. In some embodiments, the polyanionic compound is ammonium polyphosphate (for example ammonium polyphosphate from BOC Sciences).

[0085] As shown herein, delipidation of the starting material using polyphosphate may be particularly effective at reducing the amount of fibrinogen that is present in the IgG preparation that is contacted with one or more chromatographic solid supports. Surprisingly, the inventors found that polyphosphate removed at least 90% of fibrinogen, whereas dextran sulfate removed 60% of fibrinogen (see example 1, table 1). As such, although both dextran sulfate and polyphosphate were effective at removing fibrinogen, polyphosphate (e.g. ammonium polyphosphate) shows the unexpected technical advantage of being especially suitable for fibrinogen removal. Delipidation using polyphosphate was also surprisingly found by the inventors to be particularly good at reducing the levels of Apolipoprotein B.The step of delipidation may be carried out at any suitable temperature for example from about 23°C to about 2°C. However, the present inventors also surprisingly identified that polyphosphate removed factor XI and fibrinogen particularly effectively when the step of delipidation was performed at a low temperature (see e.g. table 1). The increased effectiveness of fibrinogen removal at low temperature as opposed to room temperature can be seen in example 1, e.g. in table 2. As it will be clear to a person of skill in the art, the delipidation step may be performed at a low temperature by reducing the temperature of the reagents and / or performing the delipidation step in low temperature conditions (for example in a cold room or in a cold environment). The term “low temperature” refers to a temperature 12 °C or less. For example, the low temperature may be 10°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, 4°C or less, 3°C or less. Suitably the low temperature may be from about 2 to about 8 °C. Accordingly, in the context of the present disclosure, the step of delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample may be performed at a temperature of 12 °C or less, 10°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, 4°C or less, 3°C or less. Suitably, the step of delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample may be performed at a temperature of from about 2 to about 8 °C.

[0086] The term “hydroxyapatite resin” as used herein refers to a compound that is generally made of calcium phosphate and is a naturally occurring mineral. Various hydroxyapatite resins are available commercially, and any available form of the material can be used in the practice of this invention. In one embodiment, the hydroxyapatite is in a crystalline form. Hydroxyapatites for use in the method described herein may be those that are agglomerated to form particles and sintered at high temperatures into a stable porous ceramic mass. The particle size of the hydroxyapatite may vary widely, but a typical particle size ranges from 1 pm to 1 ,000 pm in diameter, and may be from 10 pm to 100 pm. In one embodiment, the particle size is 20 pm. In another embodiment, the particle size is 40 pm. In yet another embodiment, the particle size is 80 pm. The choice of a particular hydroxyapatite type can be determined by the skilled artisan.

[0087] Suitably, the hydroxyapatite resin may be loose (e.g. for use in batch mode), packed in a column, or packed in multiple columns as part of a set up for continuous chromatography. In one embodiment, ceramic hydroxyapatite resin is packed in a column. The choice of column dimensions can be determined by the skilled artisan. In one embodiment of the invention, a column diameter of at least 0.5 cm with a bed height of about 20 cm may be used for small scale purification. In an additional embodiment of the invention, a column diameter of from about 35 cm to about 60 cm may be used. In yet another embodiment of the invention, a column diameter of from 60 cm to 85 cm may be used.As mentioned herein, the polyanionic compound may be combined with a divalent cation source. The divalent cation source may be selected from a salt of magnesium, a salt of calcium, or a salt of manganese. The divalent cation source may be a salt which liberates divalent calcium when dissolved in an aqueous solution, a salt which liberates divalent magnesium when dissolved in an aqueous solution or a salt which liberates divalent manganese when dissolved in an aqueous solution. Merely by way of example the divalent cation source may be selected from the group consisting of MgCh, CaCh, MnCh, MgBr2, CaBr2, MnBr2, MgSCU, MnSC calcium acetate, calcium citrate, calcium gluconate, calcium lactate, magnesium acetate, magnesium citrate, magnesium gluconate, magnesium lactate, manganese acetate, manganese citrate, manganese gluconate, manganese lactate, and hydrates and / or mixtures thereof. Preferably the divalent cation source is MgCh, CaCh, MnCh and hydrates and / or mixtures thereof. More preferably the divalent cation source is CaCh. Suitably, CaCh may be at a concentration of from about 25 to 75 mM, for example about 50 mM. Additionally, or alternatively, solid CaCh may be used. Additionally, or alternatively, CaCh may be added to the plasma as a solution, for example a concentrated solution.

[0088] Suitably, the polyanionic compound may be a solution of the polyanionic compound, preferably an aqueous solution of the polyanionic compound. Suitably, the polyanionic compound may be added as a solid during the delipidation step.

[0089] In a suitable embodiment, when the polyanionic compound is dextran sulfate (for example at a concentration of 0.1%) the divalent cation source may be CaCh at a concentration of 50mM.

[0090] In a suitable embodiment, when the polyanionic compound is ammonium polyphosphate (for example at a concentration of 0.1%) the divalent cation source may be CaCh at a concentration of 50mM. In such an embodiment, the ammonium polyphosphate may have a M.W. > 10 kDa. In some embodiments, the polyanionic compound is polyphosphate, preferably ammonium polyphosphate, and the divalent cation source is CaCh and in the step of delipidation the polyphosphate is added to the solution to be delipidated several minutes, for example 10 to 30 minutes (e.g., 20 minutes) before the calcium chloride is added to the solution to be delipidated. Suitably, polyphosphate may be added as a slurry during the delipidation step.

[0091] Suitably, in the context of the methods described herein, the step of delipidating may be performed for several minutes, for example for at least 10 minutes, at least 20 minutes or at least 30 minutes. In one embodiment, the step of delipidating may be performed for 10 to 30 minutes (e.g., 20 minutes). In one embodiment, the step of delipidating is performed for atleast one hour. In another embodiment, the step of delipidating is performed at least 2 hours or at least 3 hours. In one embodiment, the step of delipidating may be performed for 10 minutes up to 3 hours.

[0092] Suitably, in the context of the methods described herein, the step of delipidating may be performed at a pH of from about 6.5 to about 8.5. More suitably, the pH may be from around 7 to about 8, for example around 7.5 to about 8. Surprisingly, when the step of delipidation is performed at a pH from around 7 to 8, the present inventors found that IgG precipitation did not occur, enabling a high yield of IgG to be obtained in this delipidation step, and ultimately also upon completion of the affinity chromatography step. Experimental data showing the high IgG recovery at these pHs are provided, e.g. in example 2. These data also demonstrate the surprising effectiveness of delipidation at these pHs, as the turbidity of the samples delipidated at these pHs is low. Further advantageously, delipidation at a pH of from around 7 to about 8 may reduce the need for further delipidated sample processing steps, as this may also be the pH at which the delipidated sample may bind the IgG-binding affinity ligands on the solid support. Highly stable and active IgG preparations can be obtained by this method, as the IgGs are maintained close to physiological pH throughout the method, reducing the risk of aggregation and / or denaturation. The phrase “delipidation is performed at a pH” as used herein may refer to the pH of the starting material, e.g. prior to the addition of divalent cation source and prior to the addition of polyanion. Suitably, when the delipidation is performed at a pH of 7 or more, for example from about 7 to about 8, or from about 7 to about 7.5 the delipidation agent may be dextran sulfate. Suitably, when the delipidation is performed at a pH of 7 or more, for example from about 7 to about 8, or from about 7 to about 7.5 the delipidation agent may be dextran sulfate, and the starting material may be a fraction of plasma (such as cryoprecipitate supernatant or F. I+II+III). In one embodiment wherein the delipidation is performed ata pH of from about 6.5 to about 8.5, the method comprises use of dextran sulfate as the polyanionic compound for the delipidation step and an IgG-binding affinity ligand in the chromatography column as part of a single or multiple (e.g. dual, optionally wherein the two columns are eluted concurrently) affinity chromatography process, wherein the IgG-binding affinity ligand is VHH. In this embodiment, the VHH may be the VHH of FcXP resin, optionally wherein the chromatographic matrix is CaptureSelect™ FcXP Affinity resin.

[0093] The methods of the invention may comprise the step of contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation. The IgG preparation comprises one or more of lgG1, lgG2, lgG3 and lgG4. For example, the IgG preparation may comprise lgG1, lgG2 and lgG4. For example, the IgG preparation may comprise all lgG1, lgG2, lgG3 and lgG4. The IgG preparation maycomprise lgG3, optionally wherein lgG3 is the dominant IgG subclass, for example wherein lgG3 is highly enriched relative to starting IgG. Whether the IgG preparation comprises one or more of lgG1, lgG2, lgG3 and lgG4, may depend upon the IgG-binding affinity ligand with which the delipidated sample is contacted with. As it will be appreciated by the person skilled in the art and described in more detail herein below, certain IgG-binding affinity ligands may bind all 4 subclasses of IgG (i.e. all of lgG1, lgG2, lgG3 and lgG4), whereas other IgG-binding affinity ligands may bind only, one, two, or three subclasses of IgG.

[0094] Suitably, the methods of the invention may comprise the step of contacting the delipidated sample with one chromatographic solid support comprising at least one IgG-binding affinity ligand to obtain an IgG preparation. Suitably, the one chromatographic solid support comprises one IgG-binding affinity ligand. Suitably, the one chromatographic solid support comprises one IgG-binding affinity ligand which binds all 4 subclasses of IgG (i.e. all of lgG1, lgG2, lgG3 and lgG4). Suitably, the one IgG-binding affinity ligand is VHH. In such an embodiment, the VHH may be the VHH of FcXP resin, optionally wherein the chromatographic resin is CaptureSelect™ FcXP Affinity with a matrix, for example wherein the matrix is an Agarose matrix or a POROS-based matrix.

[0095] In a particularly preferred embodiment, the present invention provides a method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0096] (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; and

[0097] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising an IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and I gG4, wherein the IgG-binding affinity ligand is VHH, preferably wherein the VHH is the VHH of FcXP resin. Such an embodiment may be described as a single affinity chromatography method. This is because a single type of chromatographic solid support is used.

[0098] In a particularly preferred embodiment, the invention provides a method of producing an IgG preparation from an IgG-containing blood-derived starting material, the method comprising: (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; and

[0099] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising an IgG-binding affinity ligand to obtain an IgG preparation, wherein the IgG-binding affinity ligand is VHH, preferably wherein the VHH is the VHH of FcXP resin. Such anembodiment may be described as a single affinity chromatography method. This is because a single type of chromatographic solid support is used.

[0100] Suitably, the method of the invention may comprise contacting the delipidated sample with two or more (for example three or four) chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation. More suitably, the method of the invention may comprise contacting the delipidated sample with two chromatographic solid supports.

[0101] In an embodiment where the method comprises contacting the delipidated sample with two or more chromatographic solid supports, some or all of the chromatographic solid supports may be connected. In this context “connected” includes directly or indirectly joining the chromatographic solid supports such that they effectively form for example a single affinity chromatography column with different affinity binding zones. The zones with different affinity binding may optionally be separated by valves. As the delipidated sample passes through each of the zones (for example two zones), it can be said the delipidated sample is contacted with two different chromatographic solid supports.

[0102] In a suitable embodiment, a first chromatographic solid support comprising Protein A (for example MabSelect PrismA™ Protein A chromatography resin) and a second chromatographic solid support comprising VHH (for example CaptureSelect FcXP) may be connected. Suitably the second chromatographic solid support (for example comprising CaptureSelect FcXP) may be connected downstream of the first chromatographic solid support (e.g. comprising Protein A). By downstream it is meant that the delipidated sample will first be contacted with the first solid support.

[0103] Suitably, the first and second chromatographic solid supports may be (or may be present in) chromatography columns. Chromatography columns used in the present invention include commercially available affinity chromatography columns and chromatography columns that are loaded with one or more chromatographic solid support comprising one or more ligands for affinity chromatography. Suitably, the columns may be of the same diameter in order to maintain the same linear flow rate across the different zones.

[0104] Suitably, the connected two or more chromatographic solid supports may be equilibrated prior to being contacted with the delipidated sample. Methods for equilibrating chromatographic solid supports (for example chromatography affinity columns) are known in the art. Merely by way of example, the connected two or more chromatographic solid supports may beequilibrated with 20 mM Tris-HCI, 150 mM NaCI pH 7.5 buffer (1X TBS buffer). Suitably, the connected two or more chromatographic solid supports may be equilibrated with 20 mM Tris-HCI, 150 mM NaCI pH 7.5 buffer (1X TBS buffer). It will be appreciated that this equilibration approach may also be applied to chromatographic solid supports that are not connected.

[0105] In the context of the present disclosure the term “contacting” means bringing the delipidated sample into sufficient proximity to the ligand to allow the ligand to bind an IgG in the delipidated sample. Suitably, the step of “contacting” may occur within an affinity chromatography column. Accordingly, the methods described herein may be affinity chromatography methods.

[0106] Affinity chromatography is a chromatographic method, i.e. a method that uses a dynamic separation technique which separates a target protein (e.g., IgG) from other molecules in the mixture and allows it to be isolated. Typically, in a chromatography method, a mobile phase (liquid or gas) transports a sample containing the target molecule of interest across or through a stationary phase (normally solid) medium. The term “affinity chromatography” as used herein, refers to a mode of chromatography where a target protein (e.g., IgG) to be separated is isolated by its interaction (affinity) with a molecule (e.g., IgG binding affinity ligand) which specifically interacts with the target protein, wherein the molecule is stationary due to be being bound to the chromatographic solid support. The chromatographic solid support may be a chromatographic resin. Generally, chromatographic solid supports are inert. Suitably, chromatographic solid support may comprise agarose, poly(styrene-divinylbenzene), polyacrylamide, polymethacrylate, cellulose, POROS™ and / or UNOsphere™ beads. Other examples of chromatographic solid supports will be known to those skilled in the art. Suitably, the chromatographic solid support may be a chromatography column. The term “affinity chromatography column” 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 to which the ligand may be attached. Alternatively, or in addition, the affinity chromatography may be performed using a batch chromatography process. Alternatively, or in addition, the affinity chromatography may be performed using a process where the affinity ligand is bound to a membrane.

[0107] The methods of the present disclosure aim to provide an IgG preparation. Accordingly, in the context of the present disclosure, the ligands are ones that may bind, for example specifically bind, an IgG. Different IgG affinity ligands are known in the art, some of which are described herein below. IgG affinity ligands may bind to different parts of the IgG.

[0108] The IgG is made up of four chains: two light and two heavy chains. Each light chain has two domains and each heavy chain contains one variable region and three constant domains Theantigen binding site is located in the Fab region (Fragment antigen binding) which contains a variable light (VL) and a variable heavy (VH) chain domain as well as constant light (CL) and constant heavy (CH1) chain domains. The CH2 and CH3 domain region of the heavy chain is called Fc (Fragment crystallizable). The IgG molecule can be considered as a heterotetramer having two heavy chains that are held together by disulfide bonds (-S-S-) at the hinge region and two light chains. The number of hinge disulfide bonds varies among the immunoglobulin subclasses. The native Fc region or Fc domain is homodimeric. The Fc domain begins in the hinge region just upstream of a papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a portion of the hinge domain (e.g., top, middle, and or lower hinge region), a CH2 domain, and a CH3 domain. As used herein, the term "CH1 domain" refers to the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, in humans extending from approximately Ell positions 118-215. The CH1 domain is adjacent to the VH (variable heavy) domain and the amino terminus of the hinge region of the immunoglobulin heavy chain molecule and does not form part of the immunoglobulin heavy chain Fc region. The term "hinge region" as used herein refers to the portion of a heavy chain molecule that joins the CH1 domain with the CH2 domain. In human IgG this hinge region typically contains approximately 25 amino acid residues and is flexible (although the lgG3 hinge region is longer and can contain up to 62 amino acid residues), thus allowing the two N-terminal antigenbinding regions to move independently. The hinge region is divided into three distinct domains: the upper, middle, and lower hinge domains. The term "CH2 domain" as used herein refers to the portion of a heavy chain immunoglobulin molecule that in humans extends from approximately Ell positions 231-340. As used herein, the term "CH3 domain" includes the portion of a heavy chain immunoglobulin molecule that in humans extends approximately 110 residues from the C-terminus of the CH2 domain, e.g., from approximately positions 341 to 446 (Ell numbering system). Whilst the Ell numbering herein is provided based on human IgG, the skilled person will be able to determine corresponding residues in IgG of other species without any undue burden.

[0109] Suitably, the IgG affinity binding ligand may have affinity to (in other words bind to) the Fc region of IgG (for example CH2 domain of IgG, CH3 domain of IgG, or the CH2-CH3 interface of IgG), VH domain of IgG, CH1 domain, or the CL domain of IgG. Suitably the IgG-binding affinity ligand has affinity for the CH2 region, CH3 region or CH2-CH3 interface. Preferably the IgG-binding affinity ligand has affinity for the CH3 region.

[0110] Suitably, the IgG-binding affinity ligand may be Protein A. The term "Protein A" as used herein means natural forms, recombinant forms, modified (for example truncated) forms, engineeredforms, and derivatives of the 42 kDa Staphylococcus aureus cell wall Protein A. Protein A binds to the Fc domains of I gG 1 , lgG2 and I gG4, but not to some allotypes of lgG3. Specifically, Protein A binds to the CH2-CH3 interface. Accordingly, it can be said that Protein A is an IgG affinity ligand that has affinity to the Fc region of IgG, wherein the Fc region is the CH2-CH3 interface of IgG. MabSelect PrismA™ Protein A chromatography resin is an example of a resin comprising an engineered Protein A ligand. In the context of the present disclosure, where reference is made to Protein A, it will be therefore appreciated that this may be MabSelect PrismA™ Protein A chromatography resin.

[0111] As it will be appreciated by a person of skill in the art, by contacting the delipidated sample with a (first) chromatographic solid support comprising Protein A as the IgG-binding affinity ligand, it may be possible to obtain an IgG preparation comprising lgG1, lgG2, and lgG4. Accordingly, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to further contact the delipidated sample with a second chromatographic solid support which comprises an IgG-binding affinity ligand that binds lgG3. Protein G is an example of such an IgG-binding affinity ligand. Other suitable IgG-binding affinity ligands are also described below.

[0112] Suitably, the (first) chromatographic solid support comprising Protein A as the IgG-binding affinity ligand may be MabSelect PrismA™ Protein A chromatography resin.

[0113] The term "Protein G", as used herein refers to natural forms, recombinant forms, modified (for example truncated) forms, engineered forms and derivatives of a 65-kDa (G148 Protein G) or a 58 kDa (C40 protein G) cell surface protein that is expressed by group G streptococcal bacteria and for example Staphylococcus aureus. Protein G binds to the Fc domains of lgG1, I gG2, lgG3 and lgG4. Specifically, Protein G is an IgG affinity ligand that has affinity to the Fc region of IgG, wherein the Fc region is the CH2-CH3 interface of IgG.

[0114] By contacting the delipidated sample with a chromatographic solid support comprising Protein G as the IgG-binding affinity ligand, it may be possible to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4. Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises an IgG-binding affinity ligand that binds lgG1, lgG2, lgG3 and lgG4. Protein G is an example of such an IgG-binding affinity ligand. However, Protein G is a relatively expensive IgG-binding affinity ligand, and chromatographic solid supports comprising Protein G have a relatively low IgG-binding capacity. Accordingly, in another embodiment it may be desirable to contact the delipidatedsample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4, and that exhibits a relatively high IgG-binding capacity and stability to cleaning with NaOH), and a second chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein G. Although Protein G binds lgG1, lgG2, lgG3 and lgG4, by contacting the sample firstly with Protein A, lgG1, lgG2 and lgG4 may be removed from the sample, thereby allowing Protein G present on the second chromatographic solid support to only bind lgG3. This tandem protocol reduces the amount of Protein G required, thus reducing the cost.

[0115] In another example, the IgG-binding affinity ligand may be a single domain antibody fragment, such as camelid heavy chain antibody fragment (VHH). “VHH” refers to a class of small camelid antibodies that can be generated to have affinities for different epitopes, including the Fc region or the CH1 region of IgG. Suitably, in the context of the present disclosure, VHH may bind to the Fc region of IgG. An example of a commercially available resin that comprises VHH that binds to the Fc region of IgG is FcXL (also known as the CaptureSelect™ FcXL Affinity resin (Thermo Fisher Scientific, Waltham, Mass., USA)) or FcXP (also known as the CaptureSelect™ FcXP Affinity resin (Thermo Fisher Scientific, Waltham, Mass., USA)). FcXL and FcXP bind lgG1, lgG2, lgG3 and lgG4. Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises an IgG-binding affinity ligand that binds I gG 1 , 1 gG2, lgG3 and lgG4. FcXL and FcXP are examples of resins that comprise such an IgG-binding affinity ligand. However, like Protein G, FcXL and FcXP are expensive IgG-binding affinity ligand resins and are reported to have a limited stability to cleaning with NaOH. Furthermore, using VHH as an IgG-binding affinity ligand may be associated with additional advantages relative to Protein G, such as higher IgG-binding capacity, increased IgG purity, and / or more favorable operating characteristics. Accordingly, in another embodiment it may be desirable to contact the delipidated sample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4), and a second chromatographic solid support which comprises an IgG-binding affinity ligand such as VHH (e.g. the second chromatographic solid support may comprise FcXL and / or FcXP). Although VHH (e.g. the VHH of FcXL and / or FcXP resin) bind I gG 1 , I gG2, lgG3 and I gG4, by contacting the sample firstly with Protein A, I gG 1 , lgG2 and lgG4 may be removed from the sample, thereby allowing the VHH (e.g. the VHH of FcXL and / or FcXP resin) present on the second chromatographic solid support to only bind lgG3. This tandem protocol reduces the amount of VHH required. It therefore reduces the amount of e.g. FcXL and / or FcXP resin that is required.FcXL and FcXP are commercially available resins that contain affinity ligands made from recombinant camelid single-domain antibody fragments (VHH). These VHH affinity ligands are fragments of approximately 12 to 15 kDa, comprising the three complementarity-determining regions (CDRs) that form the antigen-binding domain. The ligand in both FcXL and FcXP matrices recognizes the CH3 domain of all human IgG subclasses (lgG1, lgG2, lgG3, and lgG4). The primary distinction between FcXP and FcXL is that the matrix in FcXP features increased binding capacity, but both variants target the Fc region of IgGs.

[0116] Suitably, the one or more chromatographic solid supports may comprise or consist of MAbsorbent® A2P HF. Suitably, the one or more chromatographic solid supports may comprise or consist of MAbsorbent® HF LL. Suitably, the one or more chromatographic solid supports may comprise or consist of LigaTrap IgG. Suitably the one or more chromatographic solid supports may comprise or consist of IgSelect.

[0117] Suitably, in an embodiment where the first chromatographic solid support comprises an IgG-binding affinity ligand such as Protein A and the second chromatographic solid support comprises an IgG-binding affinity ligand such as VHH, the first chromatographic solid support may comprise MabSelect PrismA™ protein A chromatography resin, and the second chromatographic solid support may comprise FcXP or FcXL, preferably FcXP. As explained elsewhere herein, this combination of first and second chromatographic solid supports has a number of surprising advantages, such as increased IgG yield and purity, reduced processing time and reduced cost. The present inventors surprisingly found that the dual-affinity chromatography on connected columns of MabSelect PrismA and CaptureSelect FcXP resins provided the major source of IgG purification, increasing the purity of IgG from ~ 13% to ~ 96% in one step. The inventors have surprisingly found that FcXP affinity columns can be loaded with delipidated material for a large number of cycles (including NaOH cleaning) without substantial loss of IgG recovery.

[0118] In another example, the IgG-binding affinity ligand may have affinity to the CH1 domain. By way of example CaptureSelect™ lgG-CH1 is such an affinity ligand. CaptureSelect™ IgG-CH1 may bind lgG1, lgG2, lgG3 and lgG4. Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises CaptureSelect™ lgG-CH1. However, like Protein G, CaptureSelect™ lgG-CH1 is an expensive IgG-binding affinity ligand. Accordingly, in another embodiment it may be desirable to contact the delipidated sample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4), and asecond chromatographic solid support which comprises an IgG-binding affinity ligand such as CaptureSelect™ lgG-CH1. Although CaptureSelect™ lgG-CH1 binds lgG1, lgG2, lgG3 and lgG4, by contacting the sample firstly with Protein A, lgG1, lgG2 and lgG4 may be removed from the sample, thereby allowing CaptureSelect™ lgG-CH1 present on the second chromatographic solid support to only bind lgG3. This tandem protocol reduces the amount of CaptureSelect™ lgG-CH1 required.

[0119] Suitably, in an embodiment where the first chromatographic solid support comprises an IgG-binding affinity ligand such as Protein A and the second chromatographic solid support comprises an IgG-binding affinity ligand such as CaptureSelect™ lgG-CH1, the first chromatographic solid support may comprise MabSelect PrismA™ protein A chromatography resin.

[0120] In another example, the IgG-binding affinity ligand may be FcRn. FcRn is an IgG-binding affinity ligand that binds the Fc region of IgG. “FcRn” also known as “neonatal Fc receptor” refers to a heterodimer consisting of an a-chain and a p2-microglobulin. In vivo, FcRn is critical to protect IgG from lysosomal degradation and to regulate the IgG half-life in circulation. FcRn binds IgG (including lgG1, lgG2, lgG3 and lgG4). Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises an IgG-binding affinity ligand that binds lgG1, lgG2, lgG3 and lgG4. FcRn is an example of such an IgG-binding affinity ligand. However, like Protein G, FcRn is an expensive IgG-binding affinity ligand. Accordingly, in another embodiment it may be desirable to contact the delipidated sample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4), and a second chromatographic solid support which comprises an IgG-binding affinity ligand such as FcRn. Although FcRn binds lgG1, lgG2, lgG3 and lgG4, by contacting the sample firstly with Protein A, lgG1, lgG2 and lgG4 may be removed from the sample, thereby allowing FcRn present on the second chromatographic solid support to only bind lgG3. This tandem protocol reduces the amount of FcRn required.

[0121] In another example, the IgG-binding affinity ligand may have affinity for the CL region of IgG. Suitably, the IgG-binding affinity ligand having affinity for the CL region of IgG may be selected from the group consisting of CaptureSelect™ KappaXP (Thermo Fisher Scientific, Waltham, Mass., USA), CaptureSelect™ KappaXL (Thermo Fisher Scientific, Waltham, Mass., USA), CaptureSelect™ LambdaXP (Thermo Fisher Scientific, Waltham, Mass., USA), and CaptureSelect™ LambdaXL (Thermo Fisher Scientific, Waltham, Mass., USA). The CL regionis defined elsewhere herein. In one embodiment, the IgG-binding affinity ligand may have affinity for the CL region derived from a kappa light chain (e.g., a human kappa light chain), and / or the lambda light chain region (e.g., a human lambda light chain). CaptureSelect™ KappaXP and CaptureSelect™ KappaXL have affinity to the CL region derived from the kappa chain. CaptureSelect™ LambdaXP and CaptureSelect™ LambdaXL have affinity to the CL region derived from the lambda chain. These affinity ligands bind lgG1, lgG2, lgG3 and lgG4 which contain kappa or lambda LCs. Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises an IgG-binding affinity ligand that binds lgG1, lgG2, lgG3, lgG4, as well as other types of antibodies including IgA, IgM, IgE and IgD. CaptureSelect™ KappaXP, CaptureSelect™ KappaXL, CaptureSelect™ LambdaXP and CaptureSelect™ LambdaXL are examples of such an IgG-binding affinity ligand. However, these are expensive IgG-binding affinity ligand and in order to capture all IgG molecules use of ligands for binding kappa and lambda is needed. Furthermore, their capacities, durability and / or lifespans may not be as high as other ligands. Accordingly, in another embodiment it may be desirable to contact the delipidated sample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4), and a second chromatographic solid support which comprises an IgG-binding affinity ligand such as one selected from the group consisting of CaptureSelect™ KappaXP, CaptureSelect™ KappaXL, CaptureSelect™ LambdaXP and CaptureSelect™ LambdaXL, thereby allowing the second chromatographic solid support to only bind lgG3 as well as other immunoglobulins containing kappa or lambda LCs. This tandem protocol reduces the amount of CaptureSelect™ KappaXP, CaptureSelect™ KappaXL, CaptureSelect™ LambdaXP and CaptureSelect™ LambdaXL required. Optionally, prior to contacting the sample with the second chromatographic solid support, it may be desirable to contact the sample with further chromatographic solid support capable of binding IgE, IgD and / or IgM. Optionally, where the second chromatographic solid support comprises a kappa-selective IgG-binding affinity ligand (for example CaptureSelect™ KappaXP) a lambda-selective IgG-binding affinity ligand (for example CaptureSelect™ LambdaXP) may also be used together with the kappa-selective IgG-binding affinity ligand.

[0122] In another example, the IgG-binding affinity ligand may have affinity for the VH region of IgG.

[0123] In another example, the IgG-binding affinity ligand may have affinity to the variable light (VL) region of IgG. An example of such a ligand is Protein L. The term, "Protein L” refers to a protein having a molecular weight of 90 to 100 kDa derived from finegoldia magna strain. The Protein L may bind to all antibodies including IgG, IgM, IgE and IgD, as well asfragments thereof, for example, variants of antibodies including single chain variable fragment (scFv) and Fab fragment, etc., of the antibody.

[0124] By contacting the delipidated sample with a chromatographic solid support comprising Protein L as the IgG-binding affinity ligand, it may be possible to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4. Accordingly, in one embodiment, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4 it may be desirable to contact the delipidated sample with a (e.g. a single) chromatographic solid support which comprises an IgG-binding affinity ligand that binds lgG1, lgG2, lgG3 and lgG4. Protein L is an example of such an IgG-binding affinity ligand. However, Protein L is an expensive IgG-binding affinity ligand and binds weakly to lambda light chains of immunoglobulins. Accordingly, in another embodiment it may be desirable to contact the delipidated sample with a first chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein A (that binds lgG1, lgG2, and lgG4), and a second chromatographic solid support which comprises an IgG-binding affinity ligand such as Protein L. Although Protein L binds lgG1, lgG2, lgG3 and lgG4, by contacting the sample firstly with Protein A, lgG1, lgG2 and lgG4 may be removed from the sample, thereby allowing Protein L present on the second chromatographic solid support to only bind lgG3. This tandem protocol reduces the amount of Protein L required. Protein L, in addition to binding IgG, may also bind IgM, IgE and IgD. Accordingly, to remove IgM, IgE, and / or IgD, it may be it may be desirable to contact the sample with further chromatographic solid support capable of binding IgE, IgD and / or IgM, prior to contacting the sample with the second chromatographic solid support comprising Protein L.

[0125] In the above mentioned embodiments, the delipidated sample is contacted with a first chromatographic solid support comprising Protein A as the first IgG-binding affinity ligand and a second chromatographic solid support comprising Protein G, FcRn, FcXP, FcXL, KappaXP, KappaXL, LambdaXP, LambdaXL, and / or Protein L, as the second IgG-binding affinity ligand. However, in another example, the delipidated sample may be contacted with a first chromatographic solid support comprising CaptureSelect™ lgG3 resin. In such an example, the second IgG-binding affinity ligand that the delipidated sample may be contacted with may be Protein A, or Protein G.

[0126] By contacting the delipidated sample with a chromatographic solid support comprising CaptureSelect lgG3TMas the IgG-binding affinity ligand, it may be possible to obtain an IgG preparation comprising I gG3, as CaptureSelect lgG3TMhas affinity for lgG3. The delipidated sample may be then contacted with a second IgG-binding affinity ligand such as Protein A(that binds lgG1, lgG2, and lgG4) to obtain an IgG preparation comprising lgG1, lgG2 and lgG4. lgG3 and lgG1, lgG2 and lgG4 preparations may then be combined to obtain a preparation comprising lgG1, lgG2, lgG3 and lgG4.

[0127] The above-mentioned examples provide methods that may provide preparations comprising lgG1, lgG2, lgG3 and lgG4. These preparations may be obtained upon combining the preparation obtained from each of the affinity chromatography resins. However, as it will be appreciated by the skilled person, by not combining said preparations it may be possible to obtain a first IgG preparation comprising lgG1, lgG2, and lgG4 and a second IgG preparation comprising lgG3. Accordingly, the methods described herein may be used to obtain IgG preparation comprising one or more IgG subclasses selected from the group consisting of I gG 1 , 1 gG2 , lgG3 and lgG4. For example, the methods described herein may be used to obtain an IgG preparation comprising lgG1, lgG2, and lgG4. For example, the methods described herein may be used to obtain an IgG preparation comprising lgG3. For example, the methods described herein may be used to obtain an IgG preparation comprising lgG1, lgG2, lgG3, and lgG4.

[0128] As mentioned elsewhere herein, the method described herein may provide an effective way of reducing viral contamination in the starting material. The term “viral contamination” refers to virus particles that may be present in the starting material. Suitably, the method may reduce the number of viral particles by at least 1 log 10, at least 2 log 10, at least 3 log 10, at least 4 Iog10, at least 5 Iog10, at least 6 Iog10, or more compared to the starting material.

[0129] A method of the present disclosure that may be particularly effective at reducing viral contamination may comprise contacting the delipidated sample with a first and second solid support, wherein the first solid support comprises MabSelect PrismA and the second solid support comprises CaptureSelect FcXP resins. Suitably, the delipidation step may be with dextran sulfate or ammonium polyphosphate. Suitably, methods of the disclosure may reduce the number of viral particles by at least 1 log 10, at least 2 log 10, at least 3 log 10, at least 4 Iog10, at least 5 Iog10, at least 6 Iog10, or more compared to the starting material.

[0130] Suitably methods of the invention may include a step of cleaning affinity chromatography columns with 0.1 M NaOH between runs using a contact time of 15 min to 3 h (e.g. 1 h) and a column temperature of 2-8 °C.

[0131] A method of preparing an IgG preparation with an optional delipidation stepThe present inventors have identified a combination of affinity chromatography columns that when used in a dual affinity chromatography set-up have an unusually high IgG yield of 99.6%, whereas in the art, other methods typically have an IgG yield of up to 92%-96%. Whilst the difference in percentage may at first glance appear small, due to the costly nature of affinity chromatography, the present method provides an important and noticeable advantage, due to the limited supply and high cost of human plasma.

[0132] In another aspect, the present invention provides a method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0133] (a) contacting the IgG-containing starting material with a first chromatographic solid support comprising a first IgG-binding affinity ligand, wherein the first IgG-binding affinity ligand is Protein A, to obtain a first IgG bound fraction and a first flow-through; (b) contacting the first flow-through with a second chromatographic solid support comprising a second IgG-binding affinity ligand, wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), to obtain a second IgG bound fraction and a second flow-through; and

[0134] (c) eluting the first IgG bound fraction and the second IgG bound fraction to obtain an IgG preparation.

[0135] Suitably, prior to step (a) the IgG-containing starting material has undergone delipidation, optionally wherein the delipidation is with a polyanionic compound.

[0136] It will be appreciated the considerations set out in this disclosure relating to a method of preparing an IgG preparation with a delipidation step apply equally to a method of preparing an IgG preparation without a delipidation step unless required otherwise by the context. Therefore, the options and definitions provided elsewhere herein (e.g. IgG preparation, starting material, contacting, chromatographic solid supports, IgG-binding affinity ligands, bound fractions, flow-through, elution etc) also apply to this aspect. Further, the methods of the present aspect have an optional delipidation step, it will be appreciated that in embodiments where delipidation is present (i.e. the method of (a)-(c) described above wherein prior to step (a) the IgG-containing starting material has undergone delipidation) the options and definitions provided elsewhere herein (e.g. IgG preparation, starting material, contacting, chromatographic solid supports, IgG-binding affinity ligands, bound fractions, flow-through, elution, delipidation, polyanionic compound, etc.) also apply to this aspect. The present inventors identified that a method of producing an IgG preparation without a delipidation step as described herein may provide a surprisingly pure IgG preparation and / or reduce the total affinity resin (MabSelect PrismA™ plus CaptureSelect™ FcXP) required with a single IgG affinity resin, such as CaptureSelect™ FcXP by half.In such a method, the delipidation step is optional. It will be appreciated therefore, that the method may be performed without a delipidation step. Alternatively, the method may be performed with a delipidation step prior to step (a) of said method.

[0137] Suitably, in such a method the Protein A may be any Protein A as described herein. For example, the Protein A may be an engineered Protein A (for example MabSelect PrismA™ Protein A chromatography resin, also referred to herein as “MabSelect PrismA™ Protein A”). Suitably the Protein A may be Purolite DurA Cycle A50 (ECOLAB), Amsphere A+ (JSR Life Sciences), TOYOPEARL Super A and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience), MabCaptureC High Capacity (Thermo Scientific), and KanCapA 3G (Kaneka). By the same token, the VHH may be any VHH as described herein above. Suitably, the VHH may bind to the Fc region of IgG. An example of such a VHH is the VHH of FcXL or FcXP resin. More suitably, the VHH may be the VHH of FcXP resin. Suitably, the VHH may bind to the CH1 domain of IgG. An example of such a VHH is CH1-XL, for example the VHH ofCaptureSelect™ CH1-XL.

[0138] In a suitable embodiment, the Protein A may be MabSelect PrismA™ Protein A and the VHH may be the VHH of FcXP resin.

[0139] In some embodiments a first, more cost-effective solid support comprising at least one IgG-binding affinity ligand is used to purify a large portion of IgG (for example lgG1, lgG2 and lgG4), followed by a second column, that may purify I gG3, making it possible to use a reduced volume of resin in the second column. This may apply, for example, when the resin in the first column is Protein A and the resin in the second column is a Protein G or a VHH resin such as FcXP or a synthetic mimic of Protein A which binds lgG3. In these embodiments, the volume ratio of the resin in the first column to the second column may be 55:45 to 95:5, preferably 60:40 to 90:10, more preferably 65:35 to 85:15, for example, 70:30 to 80:20.

[0140] As it will be appreciated by the person of skill in the art, Protein A binds I gG 1 , lgG2 and lgG4. Accordingly, the first flow-through obtained upon completion of the step (a) may be substantially free of lgG1, lgG2 and lgG4, but comprise lgG3. Further, since VHH may bind to lgG1, lgG2, lgG3, and lgG4 the second flow-through may be substantially IgG free. Upon eluting the first IgG bound fraction a first eluted IgG bound fraction comprising lgG1, lgG2 and lgG4 may be obtained. Upon elution of the second bound fraction a second eluted IgG bound fraction comprising I gG3, and optionally one or more of I gG 1 , lgG2 and lgG4 may be obtained.

[0141] Suitably, the first and second eluted IgG bound fractions may be combined, to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4.As discussed above, elution buffers may be of low pH. In some embodiments, the elution buffer is of pH 4.0 or less, pH 3.5 or less, pH 3.0 or less, pH 2.5 or less, or pH 2.0 or less. In some embodiments, the elution buffer is of pH 1.5-4.0, pH 1.5-3.5, pH 1.5-3.0, pH 1.5-2.5, or pH 1.5-2.0. In embodiments where a first chromatographic solid support and a second chromatographic solid support are used, an elution buffer may be added to the first chromatographic solid support resulting in a first eluate wherein the first eluate comprises the first IgG bound fraction, and the first eluate passes directly onto the second chromatographic solid support resulting in an IgG preparation comprising the first IgG bound fraction and the second IgG bound fraction. In such a way, the first chromatographic solid support and the second chromatographic solid support may be eluted concurrently. In some embodiments the composition of the elution buffer is such (e.g. it is of a sufficiently low pH) that the first IgG bound fraction substantially does not bind to the second chromatographic solid support during the concurrent elution process.

[0142] In a suitable embodiment, a first chromatographic solid support comprising Protein A (for example MabSelect PrismA™ Protein A chromatography resin) and a second chromatographic solid support comprising VHH (for example CaptureSelect FcXP) may be connected. Suitably the second chromatographic solid support (for example comprising CaptureSelect FcXP) may be connected downstream of the first chromatographic solid support (e.g. comprising Protein A). By downstream it is meant that the delipidated sample will first be contacted with the first solid support.

[0143] Suitably, the second chromatographic solid support may be a synthetic Protein A mimic which binds lgG3.

[0144] Suitably, the IgG preparation (for example obtained in step (c)) may be purified by subjecting the IgG preparation to flow-through anion-exchange chromatography. Examples of flow-through anion-exchange chromatography are mentioned elsewhere herein.

[0145] Suitably according to any of the methods described herein, between the delipidation step and the chromatographic step or first chromatographic step, the sample is treated with a viral inactivation surfactant. In such an embodiment the final product may be associated with improved IgG yield. Additionally, the final product may be associated with reduced virion activity. Suitably the viral inactivation surfactant may be a deviron surfactant. Preferably the viral inactivation surfactant is selected from N,N-dimethyltetradecylamin-N-oxide, Alcohols C11-15-secondary ethoxylated, and 4-(1,1,3,3-tetramethylbutyl)phenol ethoxylated. Thesepreferred viral inactivation surfactants are also known by their respective CAS numbers of 3332-27-2, 68131-40-8 and 9036-19-5. These preferred viral inactivation surfactants are also known as Deviron C16 Emprove Evolve Detergent, Deviron 13-S9 Emprove Expert Detergent, and Triton X-100 Emprove Expert Detergent respectively. Most preferably the viral inactivation surfactant is alcohols C11-15-secondary ethoxylated, also known as CAS 68131-40-8, also known as Deviron 13-S9 Emprove Expert Detergent. The inventors believe that the improved results from using a viral inactivation surfactant in this way may arise from a lower lipid content being present in the delipidated samples, and so less detergent and milder incubation conditions are required.

[0146] According to any of the methods described herein it is preferred that the starting material (i.e. the IgG-containing starting material or the IgG-containing blood-derived starting material) is not serum.

[0147] According to any of the methods described herein it is preferred that the starting material (i.e. the IgG-containing starting material or the IgG-containing blood-derived starting material) is plasma, plasma cryoprecipitate supernatant or fraction l+ll+lll from the Cohn method.

[0148] According to any of the methods described herein it is highly preferred that the polyanionic compound used in the delipidation is dextran sulfate or dextran sulfate salt, most preferably dextran sulfate. Dextran sulfate or dextran sulfate salt is associated with improved removal of phospholipids. Dextran sulfate or dextran sulfate salt is associated with high yields of IgG.

[0149] According to any of the methods described herein it is highly preferred that the polyanionic compound used in the delipidation is dextran sulfate or dextran sulfate salt wherein the concentration of dextran sulfate or dextran sulfate salt is from about 0.01 wt% to 0.50 wt%, preferably wherein the concentration of dextran sulfate or dextran sulfate salt is from about 0.01 wt% to 0.10 wt%. In a preferable example, the concentration of dextran sulfate or dextran sulfate salt is from about 0.03 wt% to 0.10 wt%. Preferably, the concentration of dextran sulfate or dextran sulfate salt is from about 0.01 wt% to 0.05 wt%, or from about 0.03 wt% to 0.05 wt%.. The concentration of dextran sulfate or dextran sulfate salt may be about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.10 wt%, about 0.20 wt%, about 0.30 wt%, about 0.30 wt%, about 0.40 wt%, about 0.50 wt%, or any range there inbetween. In one example, the concentration of dextran sulfate or dextran sulfate salt may be about 0.01 wt%. In another example, the concentration of dextran sulfate or dextran sulfate salt may be about 0.03 wt%. In another example, the concentration of dextran sulfate or dextran sulfate salt maybe about 0.05 wt%. In another example, the concentration of dextran sulfate or dextran sulfate salt may be about 0.10 wt%. In another example, the concentration of dextran sulfate or dextran sulfate salt may be about 0.50 wt%. Where dextran sulfate or dextran sulfate salt is used in delipidation it is preferred that the divalent cation source is CaCh, preferably wherein the concentration of CaCh is 20-80 mM, more preferably the concentration of CaCh is 50 mM. These embodiments are associated with especially high yields.

[0150] According to any of the methods described herein it is preferred that there is no IgG precipitation step between the delipidation and the contacting the delipidated sample with one or more chromatographic solid supports. Similarly it is preferred that there is no IgG precipitation step between the delipidation and the contacting the IgG-containing starting material with a first chromatographic solid. The IgG remaining in solution throughout the delipidation process is advantageous for IgG yield because precipitation of IgG followed by redissolving the IgG commonly results in loss of IgG material. Further, the IgG remaining in solution throughout the delipidation process is advantageous for IgG purity and quality, as precipitation of IgG may be associated with some irreversible changes to the IgG structure that adversely impact the product. It could be envisioned that, in an alternative, IgG is precipitated between the steps of delipidation and contacting the delipidated sample with a chromatographic support, for example where the precipitation is caused by ammonium sulfate. Precipitation in this manner would be expected to result in a loss of yield of IgG.

[0151] According to any of the methods described herein it is preferred that the delipidation involves using a filter press to remove lipid impurities, more preferably wherein the IgG remains in solution throughout the delipidation process. Used herein the term filter press refers to any filtration device which is suitable for the separation of lipid impurities as a solid, pseudo-solid or similar aggregate from the fluid sample. The IgG remaining in solution throughout the delipidation means substantially all (for example, greater than 95%) of the total IgG content of the sample is dissolved in the aqueous phase at all points of delipidation, such as on addition of the polyanionic compound and throughout filtration by the filter press. It is preferred that a filter press is used during the filtration process as it is possible for the IgG to remain in solution throughout filtration. As discussed above, the IgG remaining in solution throughout the delipidation process may be advantageous to the quality, purity and yield of the final product.

[0152] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims. Preferred, suitable, and optional features of any one particular aspect or embodiment of thepresent invention are also preferred, suitable, and optional features of any other aspect or embodiment described herein.

[0153] All documents filed herewith are hereby incorporated by reference into the present specification.

[0154] Analytical Methodology

[0155] Purified IgG was characterized by the Grifols Bioanalytics Department with the same assays used to monitor quality attributes of Gamunex-C®. Individual plasma proteins were quantified by immunonephelometry using a Siemens BNII Instrument and Siemens reagents. Total protein was determined by Biuret using a using a Beckman ALI480 chemistry analyzer and reagents and standards sourced from Beckman Coulter. IgG purity was defined as the concentration of IgG (measured by immunonephelometry) divided by the total protein concentration in each sample, multiplied by 100. IgG subclass analysis was performed by immunonephelometry on a Siemens BNII instrument with Siemens reagents. Lipids (cholesterol, triglycerides, phospholipids and fatty acids were quantified using a Beckman ALI480 chemistry analyzer using reagents and standards purchased from Beckman Coulter and Wako Diagnostics. Size distribution of IgG was determined by size-exclusion HPLC in an Agilent HPLC system equipped with a 7.5 mm X 60 cm TSKgel G3000SW column and a 7.5 mm X 7.5 cm TSKgel Guard SW column (TOSOH Bioscience, LLC), using a mobile phase buffer of 50 mM sodium phosphate, 0.15 M NaCI, 0.05% sodium azide (pH 6.5) at a flowrate of 0.8 mL / min. Hepatitis B titer was determined with a MONOLISA™ Anti-HBs EIA kit (BioRad Laboratories, Inc.). Isoagglutinin titer (anti-A and anti-B) was determined using microtiter plate-based direct spin haemagglutination with papain-treated group A, B, and O D-negative red blood cells (European Pharmacopoeia 2.6.20 Method B). Non-activated partial thromboplastin time (NAPTT) procoagulant activity (clotting time, sec) was based on the European Pharmacopoeia (EP) monograph <2.6.22> Activated Coagulation Factors; this method varies from EP <2.6.22> by using a sample neutralization that allows for lower dilution of samples than specified by the EP. Activated coagulation in test samples is reported in both clotting time (seconds) and in international units (mIU / mL FXIa Equivalence Units, EU) relative to an in-house standard that is traceable to the WHO international standard for Factor Xia, NIBSC 13 / 100. Non-specific complement activation was assessed by comparing C5a generation by purified IgG relative to serum. Prekallikrein activator (PKA) was is measured per European Pharmacopoeia (EP) monograph <2.6.15> Monograph Prekallikrein Activator. Turbidity measurements were made according to USEPA Method 180.1.

[0156] ExamplesExample 1.

[0157] Removal of lipids from protein solution prior to affinity chromatography

[0158] Lipid removal from cryoprecipitate supernatant was investigated using various reagents. In this example, dextran sulfate (range 0.05% w / w to 0.15% w / w, MW > 36 kDa) and polyphosphate (range 0.05% w / w to 0.15% w / w, MW > 10 kDa) were used in the presence of calcium chloride (25 mM to 75 mM) and were both found to be effective in removing cholesterol, triglycerides, phospholipids, fatty acids and apolipoprotein B, as well as to clarify cryoprecipitate supernatant at 2-8°C and 18-25°C (Tables 1 and 2). Importantly, both delipidating agents allowed for high recoveries of IgG in the resulting supernatants.

[0159] The order of addition of dextran sulfate and calcium chloride to cryoprecipitate supernatant was not critical. However, delipidation occurred more effectively when polyphosphate was added to cryoprecipitate several minutes prior to calcium chloride. On the other hand, delipidation occurred to a similar extent at2-8°C and 18-25°C, and at incubation times ranging from 1 to 4 h. Surprisingly, polyphosphate was found to be superior to dextran sulfate in removing the fibrinogen present in this plasma fraction (Table 1). Fibrinogen removal with polyphosphate was highly robust and more effective at 2-8°C than at 18-25°C (Table 2). Recoveries of IgG were high in all tested conditions. At room temperature and in the presence of 0.1% w / w dextran sulfate, all tested divalent cation sources were effective for the delipidation step (see e.g. Table 3). Specifically, in these tests, magnesium chloride was comparable to calcium chloride in removing lipids and apolipoprotein B but appeared to cause some loss of IgG (Table 3). Under the same conditions, manganese chloride was less effective in removing lipids and apolipoprotein B but removed more fibrinogen than either calcium chloride or magnesium chloride; IgG recovery was complete with manganese chloride (Table 3). The results in Tables 1 and 2 indicated that both dextran sulfate and polyphosphate were effective at removing triglycerides and phospholipids from cryoprecipitate supernatant, and that dextran sulfate was more effective at removing phospholipids from cryoprecipitate supernatant, and at low temperatures dextran sulfate was especially effective at removing triglycerides and phospholipids from cryoprecipitate supernatant.

[0160] In conclusion, all tested polyanions, including dextran sulfate and polyphosphate, in combination with a divalent cation, such as calcium, were effective in reducing the lipid content of complex protein solutions, such as cryoprecipitate supernatant and other plasma fractions, reducing turbidity below 25 NTUs. In addition, all tested polyanions, including dextran sulfate and polyphosphate, were effective at removing fibrinogen from the cryoprecipitate supernatant. Last, polyphosphate was more effective than dextran sulfate in removing fibrinogen from this plasma fraction. As reported by vendors of affinity chromatography resins, removal of lipids and fibrinogen should result in prolongation of useful lifetime of affinity chromatography resins.Table 1

[0161] Delipidation of cryoprecipitate supernatant with 0.1% dextran sulfate

[0162] (MW > 40 kDa) or 0.1% ammonium polyphosphate (MW > 10 kDa), in the presence of 50 mM calcium chloride, at 2-8°C. _

[0163] Percent of anah / te remaining soluble

[0164] Analyte Dextran sulfate* Ammonium polyphosphatet Cholesterol 24 25

[0165] Triglycerides 11 17

[0166] Fatty acids 84 84 Phospholipids 44 49

[0167] Fibrinogen 40 < 6 Apolipoprotein B < 3 < 2

[0168] Total protein 93 ND*

[0169] IgG 96 97

[0170] FXI:Ag 46 <3

[0171]

[0172] Turbidity (NTU)#16 21

[0173] *Calcium chloride was added either prior to or after dextran sulfate.

[0174] ’Ammonium polyphosphate was added 10 to 30 min prior to calcium chloride. *ND, not determined.

[0175] #Turbidity of cryoprecipitate supernatant was 321-335 NTU prior to delipidation.

[0176]

[0177]

[0178]

[0179] Table 2

[0180] Delipidation of cryoprecipitate supernatant with 0.1% dextran sulfate

[0181] (MW > 40 kDa) or 0.1% ammonium polyphosphate (MW > 10 kDa), in the presence of 50 mM calcium chloride, at 18- 25°C.

[0182] Percent of analyte remaining soluble Analyte Dextran sulfate* Ammonium polyphosphate! Cholesterol 28 25 Triglycerides 12 10

[0183] Fatty acids ND ND Phospholipids 37,6 49 Fibrinogen 44 52 Apolipoprotein B <3 <3

[0184] Total protein ND ND

[0185] IgG 97 100

[0186] FXI:Ag ND 21

[0187]

[0188] Turbidity (NTU)#10,1 19,7

[0189]

[0190] *Calcium chloride was added either prior to or after dextran sulfate.

[0191] ■•■Ammonium polyphosphate was added 10 to 30 min prior to calcium chloride.JND, not determined.

[0192] Table 3

[0193] Effect of different divalent cation salts (50 mM) on

[0194] delipidation of cryoprecipitate supernatant by 0.1%

[0195] dextran sulfate (> 500 kDa) at 18-25°C

[0196] Percent of analyte remaining soluble

[0197] CaCI2MgCI2MnCI2

[0198] Cholesterol 25,6 26,4 59,3

[0199] Triglycerides 12,9 12,7 50,8

[0200] Fatty acids 84,4 84,7 70,4

[0201] Apolipoprotein

[0202] B < 3,2 1,6 4,7

[0203] Fibrinogen 48,2 57,0 27,1

[0204] IgG 99,0 95,8 101

[0205]

[0206] Example 2. Delipidation by dextran sulfate at acidic pH

[0207] To delipidate a plasma fraction, paste (i.e. precipitated plasma fraction) was resolubilized as follows: (1) in WFI at pH 4.2; (2) in 150 mM NaCI / 10 mM citric acid (pH 7.4); and (3) in WFI at pH 8.5. Delipidation was subsequently performed at 2-8°C with 50 mM CaCh plus 0.1% w / w dextran sulfate, followed by centrifugation at 12,000 x g for 30 min at 2°C.

[0208] Table 4: Comparison of delipidation at acidic, neutral, and basic pH

[0209] pH 4.2 pH 7.4 pH 8.5

[0210] Starting

[0211] Turbidity

[0212] plasma >800 564 >800

[0213] (NTU)

[0214] fraction

[0215] Delipidated IgG (mg / mL) 2.9 6.83 6.34

[0216] plasma

[0217] Turbidity

[0218] fraction 89.1 10.4 8.4

[0219] (NTU)

[0220] supernatant

[0221] IgG recovery 47% 92% 93%

[0222]

[0223] Attempted delipidation at acidic pH (pH 4.2) showed 47-48% IgG recovery, which is substantially lower compared to >90% IgG recovery obtained with delipidation performed at pH >7 (Table 4). This was confirmed by turbidity (NTUs) results, which showed high turbidity values (89.1 NTU) and therefore poor delipidation occurring at pH 4.2. By contrast, high delipidation occurred at pH >7 with turbidity values <10.4 NTUs. Together, these results demonstrate that delipidation with dextran sulfate can be achieved with different plasma fractions at neutral pH.

[0224] Example 3. Capture of IgG using a single IgG-affinity column

[0225] CaptureSelect™ FcXP was used to demonstrate the effectiveness of this method when using delipidated cryoprecipitate supernatant as column load material.

[0226] In this example, delipidated cryoprecipitate supernatant was loaded directly onto a column containing CaptureSelect™ FcXP resin (ThermoFisher Scientific), a camelid antibody-based IgG-affinity resin, at a loading rate of 30 mg of IgG / mL of resin. The column was equilibrated with 1X TBS (TBS buffer refers to Tris-buffered saline, pH 7.5). Cryoprecipitate supernatant was delipidated with dextran sulfate at RT as explained in Example 1. After loading, the column was washed with 1X TBS, until the absorbance at 280 nm decreased to baseline. IgG was then eluted with 20 mM acetic acid, pH 4.0 (Figure 2).Analyses of the column load material and the eluate showed a purification of IgG from 10- 15% in the delipidated cryoprecipitate supernatant up to >95% in the column eluate fraction (Table 5; IgG purity is defined as the ratio of IgG concentration with respect to total protein concentration, multiplied by 100). IgG purity calculated based on total protein was -100% at eluate; IgG purity based on the major impurity proteins (IgA, IgM) was 99.3%. IgG recovery was >95%. Most impurity proteins, including IgA and IgM, did not bind to this resin and appeared in the column flow-through fraction. The ratios of IgA to IgG and IgM to IgG were both reduced 30- to 40-fold,. Importantly, the proportion of IgG subclasses in the eluate closely resembled that in human plasma (Table 5).

[0227] Table 5

[0228] Capture of IgG from delipidated cryoprecipitate supernatant on a column CaptureSelectTM FcXP IgG-Affinity resin. After loading, the column was washed until absorbance (280 nm) decreased to baseline and was then eluted with low-pH buffer.

[0229] Analyte total concentration

[0230] (mg / mL)

[0231] Analyte Column Load Column Eluate

[0232] IgG 5,44 11,64

[0233] IgA 1,09 0,08

[0234] IgM 0,46 0,03

[0235] IgG Purity* 13,7 100

[0236] IgG Percent Total IgG

[0237] Subclass

[0238] lgG1 65.5 65.7

[0239] lgG2 24.8 25.3

[0240] lgG3 4.0 3.6

[0241] lgG4 5.7 5.5

[0242]

[0243] IgG purity = {[lgG] / [total protein]} X 100.

[0244] Example 4: Capture of IgG using two tandem IgG-affinity columns

[0245] A Protein A column was connected to a smaller CaptureSelect™ FcXP column to prove the effectiveness of this method using delipidated cryoprecipitate supernatant as load material. In this example, delipidated cryoprecipitate supernatant was loaded directly onto a column containing MabSelect PrismA™ resin (Cytiva), at a loading rate of 70 mg of IgG / mL of resin. The outlet from the Protein A column was connected directly to a column containing CaptureSelect™ FcXP resin; this latter column contained 20% of the bed volume of the ProteinA column. Both columns were equilibrated with 1XTBS buffer (Tris-buffered saline, pH 7.5). After loading, the columns were washed first with 1X TBS buffer until baseline absorbance at 280 nm was reached. IgG was then eluted concurrently from the two connected columns with 50 mM acetate buffer, pH 3.0 (Figure 3).

[0246] SDS-PAGE analysis of the column load material and the low-pH eluate showed that these tandem affinity columns purified IgG to a high extent from delipidated cryoprecipitate supernatant (Figure 4).. IgG recovery from this dual-affinity chromatography step was >95%; less than 1.2% of loaded IgG was found in the column flow-through fraction. Most impurity proteins did not bind and appeared in the flow-through fraction. While the ratio of IgA to IgG was reduced 11 -fold in the column eluate, the ratio of IgM to IgG was reduced 4-fold. The proportion of IgG subclasses in the eluate from these tandem affinity columns was similar to that in human plasma (Table 6) in which the lgG3 recovery is over 80%.

[0247] The use of dual-affinity columns allowed a complete capture of IgG from the complex load material using at least half the amount of total affinity resin (MabSelect PrismA™ plus CaptureSelect™ FcXP), which is required with a single IgG affinity resin, such as CaptureSelect™ FcXP.

[0248] Table 6

[0249] Capture of IgG from delipidated cryoprecipitate supernatant on connected columns of MabSelect PrismA™ and CaptureSelect™ FcXP IgG-affinity resins. After loading, the columns were washed until absorbance at 280 nm decreased to baseline and then were eluted in series with low-pH buffer.

[0250] Analyte total concentration

[0251] (mg / mL)

[0252] Analyte Column Load Column Eluate

[0253] IgG 6,71 20,3

[0254] IgA 1,36 0,37

[0255] IgM 0,41 0,31

[0256] IgG Percent Total IgG

[0257] Subclass

[0258] lgG1 63,7 65,0

[0259] lgG2 26,8 25,6

[0260] lgG3 3,5 2,9

[0261] lgG4 6,1 5,8

[0262]

[0263] Example 5: Polishing step with Fractogel® TMAE flow-through chromatography

[0264] To further reduce IgA and IgM present in the eluate from dual-affinity chromatography, a polishing step with Fractogel® EMD TMAE Hicap (M) resin (EMD Millipore) was developed. In this example, a 20-cm column was equilibrated with 20 mM sodium acetate buffer, pH 5.35. Pooled eluate from the dual-affinity columns was diluted with twice the volume of 10 mM acetate buffer, pH 5.35 and then adjusted to pH 5.35 prior to being loaded onto this anion- exchange column. The operating pH was chosen both to optimize IgG recovery and to avoid formation of turbidity observed when the pH of IgG solutions was adjusted closer to pH 6. The flow-through fraction contained highly purified IgG (Figure 5).

[0265] Analyses of the column load material and the flow-through fraction showed that this flow- through mode chromatography reduced the content of both IgA and IgM below the level of detection, as per immunonephelometry (Table 7). IgG recovery from this polishing step was 95.1%. SDS-PAGE showed that the resulting IgG exhibited a purity similar to that of commercial IgG for clinical use (Figure 4).

[0266] Table 7

[0267]

[0268] Polishing step for IgG purified on dual-affinity columns,

[0269] using column of Fractogel® EMD TMAE Hicap (M) resin in

[0270] flow-through mode. _

[0271] Analyte Concentration (mg / mL)

[0272] Analyte Column Load Column Flow-through

[0273] IgG 5,67 ' 5,34

[0274] IgA 0,12 < 0,01

[0275] IgM 0,11 < 0,009

[0276]

[0277] Example 6. Polishing step with POROS XQ flow-through chromatography

[0278] To further reduce IgA and IgM present in the eluate from single or dual-affinity chromatography, a polishing step with POROS XQ resin (ThermoFisher) was developed. In this example, POROS XQ column was equilibrated with 17 mM sodium acetate buffer (pH 6.2). Pooled eluate from the single affinity column FcXP was adjusted to pH 6.2 by ultra / diafiltration (LIF / DF) using 17 mM sodium acetate, pH 6.2, as diafiltration buffer. The flow-through fraction contained a high amount of purified IgG (Figure 6).

[0279] Analyses of the column load material and the flow-through fraction showed that this flow- through mode chromatography reduced the content of both IgA and IgM to a level comparable to that of commercial IgG approved for clinical use in humans (Table 8a and 8b). IgG recovery was >95%. Together, the analytical data uncovered that the combination of dual-affinity chromatography and anion exchange chromatography, as well as single-affinity chromatography and anion exchange chromatography resulted in a final composition of purified IgG of sufficient purity for clinical use.Table 8a. Polishing step for IgG purified on single affinity column eluate, using column of POROS XQ resin in flow through mode

[0280] Analyte Concentration (mg / mL)

[0281] Column Flow- Analyte Column Load through

[0282] IgG 3,81 3,30

[0283] IgA 0,0147 <0,0034

[0284] IgM 0,0043 <0,0023

[0285]

[0286] Table 8b. Polishing step for IgG purified by dual-affinity chromatography, using column of POROS XQ resin in flow through mode

[0287] Analyte Concentration (mg / mL)

[0288] Column Flow- Analyte Column Load through

[0289] IgG 7,57 5,61

[0290] IgA 0,124 <0,0034

[0291] IgM 0,060 <0,0023

[0292]

[0293] Example 7. Concentration, formulation and analysis of purified IgG

[0294] To allow more conclusive analyses of the quality attributes of purified materials, samples were concentrated after chromatography steps. In this example, IgG in the flow-through fraction from the Fractogel® EMD TMAE Hicap (M) column was concentrated up to ~5%. Sample was concentrated with a Millipore Labscale Tangential Flow Filtration (TFF) system equipped with three Pellicon XL 50 kDa filters. Sample was next formulated by diafiltration with 0.2 M glycine-HCI, pH 4.2 in this same TFF system. Recovery of IgG across this step was nearly 100%, with a minimal loss (-2.5%) due to hold-up volume within the instrument. There was no measurable IgG in the permeate from this operation. Following formulation, the IgG sample was stored at 5°C.

[0295] Measured recoveries of IgG in each of the four process steps (delipidation, single or dual affinity chromatography, anion-exchange chromatography, and concentration plus formulation) allowed calculation of overall recovery of IgG. Product IgG recoveries for the four process steps resulted in an overall IgG recovery up to -90%. This high IgG yield was attained by minimizing the number of process steps and by achieving >95% recovery of IgG in each process step. Affinity chromatography provided the main source of IgG purification in this process, requiring only a single further polishing step to achieve adequate final purity. Thehigh IgG yield obtained from cryoprecipitate supernatant greatly exceeds IgG yields (typically, no greater than 66%) achievable with Cohn cold-alcohol-based IgG production processes. In conclusion, these results demonstrate that the methods of the invention produce compositions of clinical grade IgG in high yield and in a short time, compared with traditional fractionation and precipitation methods.

[0296] Example 8. Comparison of properties of IgG purified using single-affinity or dual-affinity chromatography methods

[0297] After product purification by either single-affinity or dual-affinity chromatography method and subsequent polishing by flow-through anion-exchange chromatography, the IgG compositions were concentrated to 5% and were extensively analyzed for critical quality attributes (Table 9). In both cases, the attributes of the composition of purified IgG were found to be comparable to those of a commercial IgG formulations approved for clinical use in humans.

[0298] Table 9

[0299] Comparison of affinity-purified IgG with purified IgG

[0300] Process (IgG at 5%)

[0301] Single-affinity*+ Dual-affinity1+ Dual-affinity1+ Property Poros XQ Poros XQ Fractogel IgG (mg / mL) 56,3 50,7 53,6 Total protein (mg / mL) 55,1 50,8 51,6 IgG purity (IgG / total protein) 100 100 100 IgG aggregates (%) BDL BDL 0,12 IgG monomer (%) 96,6 97,5 97,4 IgG dimer (%) 3,3 2,5 2,6 IgG post-monomer (%) BDL BDL < 0,1 igGl (%) 68,5 68,0 67,4 igG2 (%) 25,9 26,3 26,6 igG3 (%) 3,3 2,8 2,4 igG4 (%) 2,3 3,0 3,6 Non-specific complement activation 1,2

[0302] ND ND

[0303] (C5a ratio to serum)

[0304] C3 (mg / mL) <0,005 <0,005 <0,005 Hepatitis B (IIJ / mL) ND ND 3,4 Cholesterol (mg / mL) <10 ND < 70 Triglycerides (mg / mL) 10 ND 44 Fatty acids (mEq / L) ND ND < 0.1

[0305]

[0306] Albumin (mg / mL) 0,007 0,039 0,030 IgA (mg / mL) <0,003 0,005 0,020 IgM (mg / mL) 0,003 <0,002 <0,009 Transferrin (mg / mL) <0,002 0,003 0,003 Anti-A titer (1 :X) 32 32 32 Anti-B titer (1 :X) 32 16 32 NAPTT (clot time in sec) 282 298 284 NAPTT - FXIa EU ND ND < 1 Fc Fragment integrity (%) 106 114 114 PKA (IU / mL) <2 <2 < 3

[0307] *lgG purified with sing e affinity column of CaptureSelect™ FcXP.

[0308] ■•■IgG purified with dual-affinity colun nns of MabSelect PrismA™ and CaptureSelect™ FcXP. Assays for IgG (immunonephelometr y) and total protein (Biuret assay) yielded a value >100%.

[0309] ND: Not Determined

[0310] BD L: Below Detection Limit

[0311]

[0312] Example 9. Lifetime of CaptureSelect FcXP resin

[0313] FcXP ligand from CaptureSelect FcXP resins is reported to be sensitive to standard alkaline sanitization. In this example, the lifetime of CaptureSelect FcXP resin was investigated under conditions developed for manufacture of commercial IgG approved for clinical use in humans. A CaptureSelect FcXP column was loaded repetitively with delipidated cryoprecipitate supernatant at a loading ratio of 30 g IgG / L resin (Figure 7). After each chromatography cycle, the resin was washed and sanitized according to manufacturer’s instructions, with 0.1 M NaOH to advance product viral clearance.

[0314] IgG recovery from one CaptureSelect FcXP column was measured over consecutive cycles of loading the column with delipidated cryoprecipitate supernatant (30 g IgG / L resin), with 0.1 M NaOH wash after each run. Periodically, between loadings with cryoprecipitate supernatant, this column was overloaded with purified IgG (80 g IgG / L resin) to determine residual IgG-binding capacity. A second column was exposed solely to 0.1 M NaOH for the time indicated and overloaded periodically with purified IgG (80 g IgG / L resin).

[0315] Under the tested conditions, CaptureSelect FcXP resin could be loaded repetitively with delipidated cryoprecipitate and exposed to 0.1 M NaOH for at least 54 h without detectable decrease in IgG recovery. If sanitization of this resin is limited to 15 min of exposure to 0.1 M NaOH, >200 chromatography runs can be anticipated to be performed for each batch of resin without diminishment of IgG recovery. These results demonstrated that repetitive exposure ofCaptureSelect FcXP resin to delipidated cryoprecipitate supernatant did not diminish the binding capacity of this resin for purified IgG any more than did exposure only to NaOH. Together, these results uncover that delipidation of the starting material prior to affinity chromatography provides the suitable degree of purity to the column load to preserve the resin beyond 50 h of contact time with NaOH, providing a solution regarding industrial reusability and a cost-effective technology.

[0316] Example 10.

[0317] IgG prepared by the method of the invention, which is depicted in Figure 1, has been found to unexpectedly exhibit superior physicochemical and stability properties, compared with IgG prepared by the standard cold-ethanol Cohn process. As shown in Table 10, 10% IgG prepared by the method of the invention is much less prone to gelation at elevated temperatures, compared with 10% IgG prepared by the cold-ethanol process.

[0318] In addition to this increased stability, it has surprisingly been found that a IgG product prepared by the method of the present invention passes much more readily through a 20-nm filter than IgG prepared by the cold-ethanol method (Table 10). As it is shown in Table 10, there is a significant improvement in the capacity expressed as protein filtration ratio (IgG Kg / m2of 20nm filter), i.e. the Vmax increased by 55% when using the IgG product obtained by the method of the invention, reaching 42 kg / m2of protein. This increase is also reflected in the V75, which increased by 50%. Further, as shown in Table 10, other productivity parameters are also improved with a positive impact on filtration process time.

[0319] These results indicate a significant improvement in the capacity of the filtration system, which is especially beneficial for the IgG process. On the other hand, protein recovery remained high at >98%, ensuring the process's reliability and effectiveness.Tablflffdiii ii 20 Nttttenm pore szeanoraon perormance o processnermeae 10

[0320] ldii R Ptteevanrocess conons

[0321] ii Kl bhti i Ettnecquvaenno cases

[0322] / ( k)i CIG2tapacyggm Commercial

[0323] t parameers %

[0324] Parameter (Cohn Invention IgG increase

[0325] process) IVIg

[0326] Nanofiltration

[0327] Planova 35N + Planova 20N

[0328] tandem

[0329] Material

[0330] Characterization 30 UAs at pH

[0331] 30 UAs at pH 4.2 (protein 4.2

[0332] concentration, pH)

[0333] and prefiltration

[0334] pore size

[0335] Prefiltration

[0336] Prefiltration 0.1 um 0.1 um

[0337] Transmembrane

[0338] Pressure at 20nm

[0339] end filter (bar) and 0.9-1.0 bar /

[0340] 1.0-1.1 bar / 25ec Temperature 25SC

[0341] (bar)

[0342] Qi / Qfinal*100 25% 70% punctual flux (at 12

[0343] 171 77% 303

[0344] Kg IgG / m2)

[0345] Vmax Theoreticala27 kg / m255% 42 kg / m2

[0346] V75 Theoreticalb14 kg / m250% 21 kg / m2

[0347] V75 Experimentalc12 kg / m2NP Not achieved6

[0348] Thermal stability of 10% IgG product by Gelation is No gelation was observed with the NP

[0349] accelerated degradation at 572Cdobserved final product

[0350]

[0351] aVmax: maximum volume of protein fluid that can be processed by the filter before it becomes clogged. Obtained from 1 / slope from t vs t / kg protein filtrated

[0352] bV75theoretical: Vmax / 2

[0353] cV75experimental: experimental filtration ratio observed (kg of IgG filtrated) when 75% decay in flow rate is achieved

[0354] dAfter material incubation for 4 h at 57°C

[0355] eflow rate decay to 75% not achieved as it has only dropped by 30%.

[0356] Planova nanofilters from Asahi Kasei.

[0357] Example 11.

[0358] The highest IgG recovery is observed when low concentrations of dextran sulfate are used for the delipidation step in the method of the invention, which is depicted in Figure 1.

[0359] Figures 8 and 9 show that the highest IgG recovery is observed at the lowest dextran sulfate concentration (0.01%), and recovery decreases as concentration increases. At 0.1%, IgG recovery remains high (90.99%), but is slightly lower than at 0.01-0.05%. This supports the conclusion that low dextran sulfate concentrations are beneficial for IgG yield. Regarding turbidity (NTU), values are relatively low (ranging from 10.5 to 16.3 NTU), suggesting minimal insoluble material during delipidation. This supports the conclusion that the IgG remains in solution during the delipidation step. Of note, the dextran sulfate concentration used in Figure 9B is 0.1%. All dextran sulfate concentrations provided here as percentages are wt%.

[0360] Embodiments of the invention

[0361] 1. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0362] (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; and

[0363] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4.

[0364] 2. A method of producing a preparation from an IgG-containing blood-derived starting material, the method comprising:

[0365] (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; and

[0366] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation.3. The method of embodiment 2, wherein delipidating is performed using a polyanionic compound.

[0367] 4. The method of embodiment 2 to 3, wherein the IgG preparation comprises one or more of lgG1, lgG2, lgG3, lgG4.

[0368] 5. The method of embodiment 4, wherein the IgG preparation comprises at least lgG1, lgG2 and lgG4.

[0369] 6. The method of embodiment 5, wherein the IgG preparation comprises lgG1, lgG2, lgG3 and lgG4.

[0370] 7. The method of embodiment 1 or any one of embodiments 3 to 6, wherein the polyanionic compound is selected from the group consisting of polyphosphate, dextran polymer, and hydroxyapatite resin; wherein the polyanionic compound is combined with a divalent cation source, optionally wherein the divalent cation source is MgCh, CaCh, MnCh, MgBr2, CaBr2, MnBr2, MgSO4, MnSO4, calcium acetate, calcium citrate, calcium gluconate, calcium lactate, magnesium acetate, magnesium citrate, magnesium gluconate, magnesium lactate, manganese acetate, manganese citrate, manganese gluconate, manganese lactate, and hydrates and / or mixtures thereof.

[0371] 8. The method of embodiment 7, wherein the dextran polymer is, dextran sulfate or dextran sulfate salt.

[0372] 9. The method of embodiment 7 or 8, wherein the divalent cation source is CaCh.

[0373] 10. The method of embodiment 9, wherein the polyanionic compound is ammonium polyphosphate.

[0374] 11. The method of embodiment 10, wherein the delipidation is performed at a temperature from about 2°C to about 25°C, from about 2°C to about 12°C, or from about 2°C to about 8°C.

[0375] 12. The method of any one of the preceding embodiments, wherein the delipidation is performed at a pH of from about 6.5 to about 8.5, or from about 7.5 to about 8.

[0376] 13. The method of any one of the preceding embodiments, wherein the starting material is plasma or serum, optionally wherein the plasma is a plasma cryoprecipitate supernatant, plasma cryoprecipitate filtrate, anti-coagulated plasma, citrated plasma or a precipitated plasma fraction, further optionally wherein the plasma fraction is fraction l+ll+lll or ll+lll from Cohn method.

[0377] 14. The method of any of the preceding embodiments, wherein the IgG-binding affinity ligand has affinity for the Fc region of IgG, optionally wherein the IgG-binding affinity ligand hasaffinity for the CH2 region, CH3 region or CH2-CH3 interface, preferably wherein the IgG-binding affinity ligand has affinity for the CH3 region.

[0378] 15. The method of embodiment 14, wherein the ligand is selected from the group consisting of Protein G, Protein A, a synthetic Protein A mimic and FcRn.

[0379] 16. The method of any one of the preceding embodiments, wherein the IgG-binding affinity ligand has affinity for the VH region of IgG.

[0380] 17. The method of any one of the preceding embodiments, wherein the IgG-binding affinity ligand has affinity for the CL region of IgG.

[0381] 18. The method of any one of the preceding embodiments, wherein the IgG-binding affinity ligand has affinity for the VL region of IgG, optionally wherein the ligand is Protein L.

[0382] 19. The method of any one of the preceding embodiments, wherein the IgG-binding affinity ligand has affinity for the CH1 region.

[0383] 20. The method of any one of the preceding embodiments, wherein the IgG is human, nonhuman, humanized, or chimeric.

[0384] 21. The method of any one of the preceding embodiments, wherein the IgG is monospecific or multispecific.

[0385] 22. The method of any one of the preceding embodiments, wherein the IgG is polyclonal or monoclonal.

[0386] 23. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0387] (a) contacting the IgG-containing starting material with a first chromatographic solid support comprising a first IgG-binding affinity ligand, wherein the first IgG-binding affinity ligand is Protein A, to obtain a first IgG bound fraction and a first flow-through; (b) contacting the first flow-through with a second chromatographic solid support comprising a second IgG-binding affinity ligand, wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), to obtain a second IgG bound fraction and a second flow-through; and

[0388] (c) eluting the first IgG bound fraction and the second IgG bound fraction to obtain an IgG preparation, wherein the IgG preparation comprises the first IgG bound fraction and the second IgG bound fraction.24. The method of embodiment 23, wherein prior to step (a) the IgG-containing starting material has undergone delipidation, optionally wherein the delipidation is with a polyanionic compound.

[0389] 25. The method of embodiment 23 or 24, wherein the IgG preparation comprises one or more of lgG1, lgG2, lgG3, lgG4.

[0390] 26. The method of embodiment 25, wherein the IgG preparation comprises at least I gG 1 , lgG2 and lgG4.

[0391] 27. The method of embodiment 26, wherein the IgG preparation comprises lgG1, lgG2, lgG3 and lgG4.

[0392] 28. The method of any one of embodiments 24 to 26, wherein the polyanionic compound is selected from the group consisting of polyphosphate, dextran polymer, and hydroxyapatite resin; wherein the polyanionic compound is combined with a divalent cation source, optionally wherein the divalent cation source is MgCh, CaCh, MnCh, MgBr2, CaBr2, MnBr2, MgSO4, MnSO4, calcium acetate, calcium citrate, calcium gluconate, calcium lactate, magnesium acetate, magnesium citrate, magnesium gluconate, magnesium lactate, manganese acetate, manganese citrate, manganese gluconate, manganese lactate, and hydrates and / or mixtures thereof.

[0393] 29. The method of embodiment 28, wherein the dextran polymer is, dextran sulfate or dextran sulfate salt.

[0394] 30. The method of embodiment 28 or 29, wherein the divalent cation source is CaCh.

[0395] 31. The method embodiment 28, wherein the polyanionic compound is ammonium polyphosphate.

[0396] 32. The method of embodiment 31, wherein the delipidation is performed at a temperature from about 2°C to about 25°C, from about 2°C to about 12°C, or from about 2°C to about 8°C.

[0397] 33. The method of any one of embodiments 23 to 32, wherein the delipidation is performed at a pH of from about 6.5 to about 8.5, or from about 7.5 to about 8.

[0398] 34. The method of any one of embodiments 23 to 33, wherein the starting material is plasma or serum, optionally wherein the plasma is a plasma cryoprecipitate supernatant, plasma cryoprecipitate filtrate, anti-coagulated plasma, citrated plasma or a precipitated plasma fraction containing IgG, further optionally wherein the starting material is a plasma fraction, wherein the plasma fraction is fraction l+ll+lll or ll+lll from Cohn method.35. The method of any one of embodiments 23 to 34, wherein Protein A is engineered protein A, optionally wherein Protein A is MabSelect PrismA™ Protein A.

[0399] 36. The method of any one of embodiments 23 to 35, wherein the VHH binds the Fc region of IgG.

[0400] 37. The method of embodiment 36, wherein the VHH is the VHH of FcXP or FcXL resin, preferably FcXP resin.

[0401] 38. The method of any one of embodiments 23 to 36, wherein the VHH binds the CH1 region.

[0402] 40. The method of any one of embodiments 23 to 38, wherein the IgG is human, non-human, humanized, or chimeric.

[0403] 41. The method of any one of embodiments 23 to 40, wherein the IgG is monospecific or multispecific.

[0404] 42. The method of any one of embodiments 23 to 41, wherein the IgG is polyclonal or monoclonal.

[0405] 43. The method of any one of embodiments 23 to 42, wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occur concurrently, preferably wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occurs by addition of an elution buffer to the first chromatographic solid support resulting in a first eluate wherein the first eluate comprises the first IgG bound fraction, and the first eluate contacts the second chromatographic solid support resulting in an IgG preparation comprising the first IgG bound fraction and the second IgG bound fraction, more preferably wherein first IgG bound fraction substantially does not bind to the second chromatographic solid support during the elution process.

[0406] 44. The method of any one of the preceding embodiments wherein the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 95:5, preferably 60:40 to 90:10, more preferably 65:35 to 85:15, most preferably 70:30 to 80:20; or wherein the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15.

[0407] 45. The method of embodiment 44, wherein the first IgG-binding affinity ligand is Protein A and wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), preferably wherein the camelid heavy chain antibody fragment (VHH) is the VHH of FcXP resin.46. The method of any one of the preceding embodiments, wherein the method further comprises the step of subjecting the IgG preparation to flow-through anion-exchange chromatography.

[0408] 47. An apparatus for performing the method of any of the preceding embodiments.

[0409] 48. An IgG preparation obtainable or obtained by the method of any of the preceding embodiments.

[0410] 49. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0411] (a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; and

[0412] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4.

[0413] 50. A method of producing an IgG preparation from an IgG-containing blood-derived starting material, the method comprising:

[0414] (a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; and

[0415] (b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation.

[0416] 51. The method of embodiment 50, wherein delipidating is performed using a polyanionic compound.

[0417] 52. The method of embodiment 50 or 51 , wherein the IgG preparation comprises one or more of lgG1, lgG2, lgG3, lgG4; optionally wherein the IgG preparation comprises at least lgG1, lgG2 and lgG4; further optionally wherein the IgG preparation comprises lgG1, lgG2, lgG3 and lgG4.

[0418] 53. The method of embodiment 49, 51 or 52, wherein the polyanionic compound is selected from the group consisting of polyphosphate, dextran polymer, and hydroxyapatite resin;wherein the polyanionic compound is combined with a divalent cation source, optionally wherein the divalent cation source is MgCh, CaCh, MnCh, MgBr2, CaBr2, MnBr2, MgSCU, MnSC calcium acetate, calcium citrate, calcium gluconate, calcium lactate, magnesium acetate, magnesium citrate, magnesium gluconate, magnesium lactate, manganese acetate, manganese citrate, manganese gluconate, manganese lactate, and hydrates and / or mixtures thereof; optionally:

[0419] (a) wherein the dextran polymer is, dextran sulfate or dextran sulfate salt; and / or

[0420] (b) the divalent cation source is CaCh; optionally wherein the polyanionic compound is ammonium polyphosphate; further optionally wherein the delipidation is performed at a temperature from about 2°C to about 25°C, from about 2°C to about 12°C, or from about 2°C to about 8°C.

[0421] 54. The method of any one of embodiments 49-53, wherein the delipidation is performed at a pH of from about 6.5 to about 8.5, or from about 7.5 to about 8.

[0422] 55. The method of any one of embodiments 49-54, wherein the starting material is plasma or serum, optionally wherein the plasma is a plasma cryoprecipitate supernatant, plasma cryoprecipitate filtrate, anti-coagulated plasma, citrated plasma or a precipitated plasma fraction containing IgG, further optionally wherein the starting material is a plasma fraction, wherein the plasma fraction is fraction l+ll+lll or ll+lll from Cohn method.

[0423] 56. The method of any of embodiments 49-55, wherein the IgG-binding affinity ligand has affinity for the Fc region of IgG, optionally wherein the IgG-binding affinity ligand has affinity for the CH2 region, CH3 region or CH2-CH3 interface, preferably wherein the IgG-binding affinity ligand has affinity for the CH3 region; optionally wherein the ligand is selected from the group consisting of Protein G, Protein A, a synthetic Protein A mimic and FcRn.

[0424] 57. The method of any one of embodiments 49-56, wherein the IgG-binding affinity ligand has affinity for:

[0425] (a) the VH region of IgG; and / or

[0426] (b) the CL region of IgG; and / or

[0427] (c) the VL region of IgG, optionally wherein the ligand is Protein L and / or(d) the CH1 region.

[0428] 58. The method of any one of embodiments 49-57, wherein the IgG is human, non-human, humanized, or chimeric.

[0429] 59. The method of any one of embodiments 49-58, wherein the IgG is:

[0430] (a) monospecific or multispecific; and / or

[0431] (b) polyclonal or monoclonal.

[0432] 60. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:

[0433] (a) contacting the IgG-containing starting material with a first chromatographic solid support comprising a first IgG-binding affinity ligand, wherein the first IgG-binding affinity ligand is Protein A, to obtain a first IgG bound fraction and a first flow-through; (b) contacting the first flow-through with a second chromatographic solid support comprising a second IgG-binding affinity ligand, wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), to obtain a second IgG bound fraction and a second flow-through; and

[0434] (c) eluting the first IgG bound fraction and the second IgG bound fraction to obtain an IgG preparation, wherein the IgG preparation comprises the first IgG bound fraction and the second IgG bound fraction.

[0435] 61. The method of embodiment 60, wherein prior to step (a) the IgG-containing starting material has undergone delipidation, optionally wherein the delipidation is with a polyanionic compound.

[0436] 62. The method of embodiment 60 or 61, wherein the Protein A is MabSelect PrismA™ Protein A.

[0437] 63. The method of any one of embodiments 60 to 62, wherein the VHH binds to the Fc region of IgG; optionally wherein the VHH is the VHH of FcXP or FcXL resin.64. The method of any one of embodiments 60 to 63, wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occur concurrently, preferably wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occurs by addition of an elution buffer to the first chromatographic solid support resulting in a first eluate wherein the first eluate comprises the first IgG bound fraction, and the first eluate contacts the second chromatographic solid support resulting in an IgG preparation comprising the first IgG bound fraction and the second IgG bound fraction, more preferably wherein first IgG bound fraction substantially does not bind to the second chromatographic solid support during the elution process.

[0438] 65. The method of any one of embodiments 49 to 64 wherein the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15; optionally wherein the first IgG-binding affinity ligand is Protein A and wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), preferably wherein the camelid heavy chain antibody fragment (VHH) is the VHH of FcXP resin.

[0439] 66. The method of any one of embodiments 49 to 65, wherein the method further comprises the step of subjecting the IgG preparation to flow-through anion-exchange chromatography.

[0440] 67. An apparatus for performing the method of any of embodiments 49 to 66.

[0441] 68. An IgG preparation obtainable or obtained by the method of any of embodiments 49 to

Claims

1. CLAIMS1. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:(a) delipidating the IgG-containing starting material with a polyanionic compound to obtain a delipidated sample; and(b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation comprising lgG1, lgG2, lgG3 and lgG4.

2. A method of producing an IgG preparation from an IgG-containing blood-derived starting material, the method comprising:(a) delipidating the IgG-containing blood-derived starting material to obtain a delipidated sample; and(b) contacting the delipidated sample with one or more chromatographic solid supports comprising at least one IgG-binding affinity ligand to obtain an IgG preparation.

3. The method of claim 2, wherein delipidating is performed using a polyanionic compound.

4. The method of claim 2 or 3, wherein the IgG preparation comprises one or more of lgG1, lgG2, lgG3, lgG4; optionally wherein the IgG preparation comprises at least lgG1, lgG2 and lgG4; further optionally wherein the IgG preparation comprises lgG1, lgG2, lgG3 and lgG4.

5. The method of claim 1, 3 or 4, wherein the polyanionic compound is selected from the group consisting of polyphosphate, dextran polymer, and hydroxyapatite resin; wherein the polyanionic compound is combined with a divalent cation source, optionally wherein the divalent cation source is MgCh, CaCh, MnCh, MgBr2, CaBr2, MnBr2, MgSO4, MnSO4, calcium acetate, calcium citrate, calcium gluconate, calcium lactate, magnesium acetate, magnesium citrate, magnesium gluconate, magnesium lactate, manganese acetate, manganese citrate, manganese gluconate, manganese lactate, and hydrates and / or mixtures thereof; optionally: (a) wherein the dextran polymer is, dextran sulfate or dextran sulfate salt; and / or59(b) the divalent cation source is CaCh; optionally wherein the polyanionic compound is ammonium polyphosphate; further optionally wherein the delipidation is performed at a temperature from about 2°C to about 25°C, from about 2°C to about 12°C, or from about 2°C to about 8°C.

6. The method of any one of the preceding claims, wherein the delipidation is performed at a pH of from about 6.5 to about 8.5, or from about 7.5 to about 8.

7. The method of any one of the preceding claims, wherein the starting material is plasma or serum, optionally wherein the plasma is a plasma cryoprecipitate supernatant, plasma cryoprecipitate filtrate, anti-coagulated plasma, citrated plasma or a precipitated plasma fraction containing IgG, further optionally wherein the starting material is a plasma fraction, wherein the plasma fraction is fraction l+ll+lll or ll+lll from Cohn method.

8. The method of any of the preceding claims, wherein the IgG-binding affinity ligand has affinity for the Fc region of IgG, optionally wherein the IgG-binding affinity ligand has affinity for the CH2 region, CH3 region or CH2-CH3 interface, preferably wherein the IgG-binding affinity ligand has affinity for the CH3 region; optionally wherein the ligand is selected from the group consisting of Protein G, Protein A, a synthetic Protein A mimic and FcRn.

9. The method of any one of the preceding claims, wherein the IgG-binding affinity ligand has affinity for:(a) the VH region of IgG; and / or(b) the CL region of IgG; and / or(c) the VL region of IgG, optionally wherein the ligand is Protein L and / or(d) the CH1 region.

10. The method of any one of the preceding claims, wherein the IgG is human, non-human, humanized, or chimeric.

11. The method of any one of the preceding claims, wherein the IgG is:(a) monospecific or multispecific; and / or(b) polyclonal or monoclonal.

12. A method of producing an IgG preparation from an IgG-containing starting material, the method comprising:(a) contacting the IgG-containing starting material with a first chromatographic solid support comprising a first IgG-binding affinity ligand, wherein the first IgG-binding affinity ligand is Protein A, to obtain a first IgG bound fraction and a first flow-through; (b) contacting the first flow-through with a second chromatographic solid support comprising a second IgG-binding affinity ligand, wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), to obtain a second IgG bound fraction and a second flow-through; and(c) eluting the first IgG bound fraction and the second IgG bound fraction to obtain an IgG preparation, wherein the IgG preparation comprises the first IgG bound fraction and the second IgG bound fraction;wherein prior to step (a) the IgG-containing starting material has undergone delipidation, optionally wherein the delipidation is with a polyanionic compound.

13. The method of claim 12, wherein the Protein A is MabSelect PrismA™ Protein A.

14. The method of claim 12 or 13, wherein the VHH binds to the Fc region of IgG; optionally wherein the VHH is the VHH of FcXP or FcXL resin.

15. The method of any one of claims 12 to 14, wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occur concurrently, preferably wherein the eluting of the first IgG bound fraction and the second IgG bound fraction occurs by addition of an elution buffer to the first chromatographic solid support resulting in a first eluate wherein the first eluate comprises the first IgG bound fraction, and the first eluate contacts the second chromatographic solid support resulting in an IgG preparation comprising the first IgG bound fraction and the second IgG bound fraction, more preferably wherein first IgG bound fractionsubstantially does not bind to the second chromatographic solid support during the elution process.

16. The method of any one of the preceding claims wherein the volume ratio of the first chromatographic solid support to the second chromatographic solid support is 55:45 to 99:1, optionally 60:40 to 98:1, preferably 65:35 to 95:5, more preferably 70:30 to 90:10, even more preferably 75:25 to 87:13, most preferably 80:20 to 85:15; optionally wherein the first IgG-binding affinity ligand is Protein A and wherein the second IgG-binding affinity ligand is a camelid heavy chain antibody fragment (VHH), preferably wherein the camelid heavy chain antibody fragment (VHH) is FcXP.

17. The method of any one of the preceding claims, wherein the method further comprises the step of subjecting the IgG preparation to flow-through anion-exchange chromatography.

18. The method of any one of the preceding claims wherein the polyanionic compound used in the delipidation is dextran sulfate or dextran sulfate salt, preferably dextran sulfate.

19. The method of claim 18, wherein the concentration of dextran sulfate or dextran sulfate salt is from about 0.01 wt% to about 0.50 wt%, preferably wherein the concentration of dextran sulfate or dextran sulfate salt is from about 0.01 wt% to about 0.10 wt%.

20. The method of claim 18 or 19, wherein the divalent cation source is CaCh, preferably wherein the concentration of CaCh is 20-80 mM, more preferably the concentration of CaCh is 50 mM.

21. The method of any one of the preceding claims, wherein there is no IgG precipitation step between:(i) the delipidation and the contacting the delipidated sample with one or more chromatographic solid supports; and / or(ii) the delipidation and the contacting the IgG-containing starting material with a first chromatographic solid.

22. The method of any one of the preceding claims wherein the delipidation involves using a filter press to remove lipid impurities.

23. An apparatus for performing the method of any of the preceding claims.

24. An IgG preparation obtainable or obtained by the method of any of claims 1 to 22.63

Images