Methods and systems for plasma processing

Abstract

The present disclosure relates to methods for directing collected plasma in a protein purification process. The method includes testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor. Collected plasma units are separated based on the respective determined concentration level and directed to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level. The present disclosure also relates to a test kit for determining a concentration level of a protein of interest in a plasma unit collected from a donor.

Description

METHODS AND SYSTEMS FOR PLASMA PROCESSINGRelated Application DataThis application claims priority from US Provisional Patent Application No. 63 / 737905 filed on 23 December 2024 and entitled “Methods and Systems for Plasma Processing”, the entire contents of which are hereby incorporated by reference.Sequence ListingThis present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.Field

[0001] The present disclosure relates to methods and systems for protein purification and, in particular, to a method and system for processing of plasma for a protein purification process.Background

[0002] Fractionation of therapeutic proteins from donated plasma yields important therapeutic products that can be used to treat critical health conditions such as primary immunodeficiency, immune thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, Kawasaki disease, and others.

[0003] Plasma is obtained from donors as either recovered plasma or source plasma. Recovered plasma is obtained as a by-product of whole blood donations. Source plasma is obtained by a process called plasmapheresis, which involves extracorporeal separation of plasma from whole blood and return of other blood components to the donor.

[0004] A shortage in plasma supply can affect the global production of plasma protein products. Currently, plasma supply is forecast to increase at a slower rate than global demand for proteins such as Ig and albumin.

[0005] In some instances, co-purification of one or more proteins from the same protein mixture is required. For example, during plasma fractionation, several blood plasma fractions containing one or more plasma proteins (e.g., immunoglobulin G (IgG), albumin and coagulation factors) may each be obtained from the same blood plasma sample by a series of processing steps to isolate each individual protein. Each of these plasma fractions can then be processed for therapeutic use.

[0006] Collection and purification of plasma protein products (including raw materials) comprises the majority of the total production costs. To date, several methods of plasma fractionation have been developed. Traditionally, manufacturing processes for isolating human immunoglobulin were generally based on the Cohn, Kistler-Cohn, or the Kistler-Nitschmann process. These processes utilise large bioreactors and hazardous chemicals to selectively precipitate out various proteins from pooled plasma obtained from multiple donors. While effective, these processes have high footprint requirements and generate a large amount of waste by-products that may be expensive to dispose of. Further, these processes may result in poor yield of some plasma protein fractions due to additional processing steps required to remove protein aggregates. Advances in chromatography methods such as affinity chromatography have provided alternative approaches for purifying specific proteins out of a complex mixed input, such as human IgG from pooled donated plasma, with comparable quality. However, affinity chromatography resins and materials are currently significantly more expensive than the established Cohn, Kistler-Cohn, or the Kistler-Nitschmann based methods, when compared to similar plasma input volumes.

[0007] It will therefore be apparent to the skilled person that there is a need in the art for improved methods of fractionating proteins from plasma.Summary

[0008] The present disclosure is based on the inventors’ identification of a method of determining a concentration of IgG in donor plasma and directing plasma samples to a selected purification process based on whether the determined concentration is above or below a threshold. It will be apparent to the skilled person from the disclosure herein that the methods of the disclosure permit high IgG plasma samples to be maintained separate from low IgG plasma samples and directed separately to respective purification processes.

[0009] According to one aspect of the present disclosure, there is provided a method for directing collected plasma in a protein purification process, the method including:testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor;directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest;wherein collected plasma determined as having a concentration level of the protein of interest greater than a threshold concentration level is directed to a first protein purification processing pathway and wherein collected plasma determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway.

[0010] The method may include further including processing the collected plasma unit via the first or second purification processing pathway, to produce an isolated protein of interest from the collected plasma unit.

[0011] The method may include pooling the collected plasma unit from the donor with plasma units collected from other donors. Collected plasma determined as having a concentration level of the protein of interest greater than the threshold concentrationlevel may be pooled separately from collected plasma determined as having a concentration level of the protein of interest less than the threshold concentration level.

[0012] The blood, serum or plasma sample may be obtained during collection of the plasma unit. The plasma unit may be collected by a plasmapheresis procedure. The blood, serum or plasma sample is obtained during the plasmapheresis procedure.

[0013] The blood, serum or plasma sample may be obtained prior to collection of the plasma unit.

[0014] The blood, serum or plasma sample may be obtained after collection of the plasma unit. In some examples, a plasma sample may be obtained from the collected plasma unit after plasmapheresis and prior to any pooling of the collected plasma unit with plasma units collected from other donors.

[0015] In some examples, a blood sample may be obtained by a finger-prick test.

[0016] The first purification processing pathway may achieve a higher yield of the protein of interest than the second purification processing pathway.

[0017] The first and / or second purification processing pathway may include a process configured to isolate the protein of interest (for example, a first protein of interest). The first and / or second purification processing pathway may include a process configured to isolate one or more different proteins of interest (e.g. a second, third, fourth and / or further protein of interest).

[0018] The first purification processing pathway may include a process configured to isolate the protein of interest (e.g. a first protein of interest). The second purification processing pathway may include a process configured to isolate one or more different proteins of interest (e.g. a second, third, fourth and / or further protein of interest). In some examples, the second purification processing pathway may not include a process configured to isolate the first protein of interest.

[0019] The first and / or second purification processing pathway may include an affinity-based chromatography process and / or a precipitation process (e.g. an alcohol-based or non-alcohol-based precipitation process).

[0020] The first and / or second purification processing pathway may include an alcohol (e.g. ethanol) or non-alcohol-based process for isolating the protein of interest and / or one or more different proteins of interest. In some examples, the non-alcohol-based process for isolating the protein of interest and / or one or more different proteins of interest is an affinity -based chromatography process. In some examples, the non-alcohol-based process for isolating the protein of interest and / or one or more different proteins of interest is a non-alcohol precipitation process (e.g. a salt, heat, acid or hydrophillic polymer based precipitation process).

[0021] In some examples, the first and / or second purification processing pathway may include an affinity-based chromatography process with an affinity -based chromatography medium which binds a first protein of interest or one or more different proteins of interest. In some examples, the first purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium which binds a first protein of interest and one or more different proteins of interest are purified in the first processing pathway using a non-alcohol-based precipitation process. In some examples, the first purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium which binds the first protein of interest and one or more different proteins of interest are purified in the first processing pathway using an alcohol based (e.g. ethanol) precipitation process.

[0022] In some examples, the second purification processing pathway may include an alcohol-based process which purifies the first protein of interest and one or more different proteins of interest. In some examples, the second purification processing pathway may include an affinity-based chromatography process which purifies one or more different proteins of interest. In another example, a first protein of interest is purified by a first processing pathway which comprises an affinity chromatography stepwhich purifies the first protein of interest and one or more additional proteins of interest are purified in a second processing pathway.

[0023] In some examples, the first and / or second purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium, wherein the medium comprises a ligand that binds to the Fc domain of a human IgG molecule. In some examples, the first purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium, wherein the medium comprises a ligand that binds to the Fc domain of a human IgG molecule. In some examples, the second purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium, wherein the medium comprises a ligand that binds to the Fc domain of a human IgG molecule.

[0024] According to another aspect of the present disclosure, there is provided a method for directing collected plasma in a protein purification process, the method including:(a) testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor;(b) directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest;wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first protein purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway,wherein the first protein purification processing pathway includes an affinitybased chromatography process, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises aligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.

[0025] In some examples, the ligand binds to the Fc domain of one or more or all subclasses of a human IgG molecule. In some examples, the ligand is capable of specifically binding to a CH3 domain of human IgG. For example, the ligand binds to or specifically binds to a CH3 domain of at least one subclass of human IgG. In some examples, the ligand is an antibody fragment. For example, the antibody fragment is a camelid-derived single domain [VHH] antibody fragment or a VHH antigen-binding protein. In some examples, the chromatography medium comprises a ligand comprising a VHH antibody fragment conjugated to a matrix. For example, an agarose-based matrix.

[0026] In some examples, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising an amino acid sequence set forth in SEQ ID NO: 1 or a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: 1. In some examples, the VHH antigen-binding protein comprises an amino acid sequence set forth in SEQ ID NO: 1. In some examples, the VHH antigen-binding protein comprises a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: 1.

[0027] In some examples, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising a framework region comprising an amino acid sequence set forth in SEQ ID NO: 1 or a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: 1. In some examples, the framework region comprises an amino acid sequence set forth in SEQ ID NO: 1. In another example, the framework region comprises a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: 1.

[0028] In some examples, the affinity-based chromatography medium comprises an VHH antigen-binding protein comprising an amino acid sequence that comprises 4framework regions, FR1, FR2, FR3 and FR4, and 3 complementarity determining regions, CDR1, CDR2 and CDR3, that are operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues;the CDR2 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3; and,the CDR3 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4; and,wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID NO: 1; and wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO: 1.

[0029] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising an amino acid sequence that comprises 4 framework regions, FR1, FR2, FR3 and FR4, and 3 complementarity determining regions, CDR1, CDR2 and CDR3, that are operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues;the CDR2 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3; and,the CDR3 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4; and, wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID NO: 1, andwherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO: 1 and wherein the antigenbinding protein specifically binds to the Fc domain of a human IgG molecule and does not bind to an IgG molecule of murine origin or bovine origin.

[0030] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues.

[0031] In one example, the affinity -based chromatography medium comprises a VHH antigen-binding protein comprising a CDR2 comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3.

[0032] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising a CDR3 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4; and, wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID NO: 1.

[0033] In one example, each of the framework regions of the VHH antigen -binding protein has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO: 1.

[0034] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising an amino acid sequence that comprises 4 framework regions, FR1, FR2, FR3 and FR4, and 3 complementarity determining regions, CDR1, CDR2 and CDR3, that are operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues;the CDR2 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3; and,the CDR3 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4; and, wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID NO: 1, andwherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO: 1 and wherein the antigen binding protein specifically binds to the Fc domain of a human IgG molecule and does not bind to an IgG molecule of murine origin or bovine origin.

[0035] In one example, the affinity -based chromatography medium comprises a VHH antigen-binding protein comprising a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues.

[0036] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising a CDR2 comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3.

[0037] In one example, the affinity-based chromatography medium comprises a VHH antigen-binding protein comprising a CDR3 comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4.

[0038] In some examples, the first and / or second purification processing pathway may include an affinity-based chromatography process with an affinity -based chromatography medium, wherein the medium comprises a Protein A ligand or variant or fragment thereof. In one example, the first purification processing pathway may include an affinity-based chromatography process with an affinity-based chromatography medium, wherein the medium comprises a Protein A ligand or variant or fragment thereof. In another example, the second purification processing pathway may include an affinity-based chromatography process with an affinity -based chromatography medium, wherein the medium comprises a Protein A ligand or variant or fragment thereof.

[0039] In some examples, the first and / or second purification processing pathway may include an affinity -based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that binds to or specifically binds to at least one subclass of human IgG. In some examples, the first and / or second purification processing pathway may include an affinity-based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that binds to or specifically binds to a CH3 domain of at least one subclass of human IgG. For example, the first and / or second purification processing pathway may include an affinity-based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that is a camelid-derived single domain [VHH] antibody fragment or a VHH antigen-binding protein.

[0040] In some examples, the first purification processing pathway may include an affinity-based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that binds to or specifically binds to at least one subclass of human IgG. In some examples, the first purification processing pathway may include an affinity -based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that binds to or specifically binds to a CH3 domain of at least one subclass of human IgG. For example, the first purification processing pathway may include an affinity-based chromatography process with a first chromatography medium comprising a Protein A ligand or variant or fragment thereof and a second chromatography medium comprising a ligand that is a camelid-derived single domain [VHH] antibody fragment or a VHH antigen-binding protein.

[0041] In some examples, the affinity -based chromatography process may be performed substantially continuously or as a batch process. In some examples, theaffinity-based chromatography process may be performed substantially continuously such that a flow rate of the feed during the chromatography cycle is substantially constant. Substantially continuous loading may comprise no idle time, or minimised idle time. In some examples, substantially continuous loading may include interruptions to the flow of the feed a time taken to allow connection of the feed source to the next separation unit (for example, the time taken to operate a switching valve). In other embodiments, the loading and / or flow rate may be discontinuous. For example, there may be an idle time between successive cycles and / or cycle segments.

[0042] In some examples, the first and / or second purification processing pathway may include a precipitation process. In one example, the first purification processing pathway may include a precipitation process. In another example, the second purification processing pathway may include a precipitation process. For example, an alcohol-based or non-alcohol based precipitation process. In some examples, the first and / or second purification processing pathways do not include an affinity-based chromatography step comprising an affinity-based chromatography medium which binds the protein of interest. In some examples, the alcohol-based precipitation process comprises an ethanol-based precipitation process. For example, the ethanol-based precipitation process is a Cohn, a Kistler-Cohn or a Kistler-Nitschmann fractionation processes, or any modified or improved forms of the foregoing. In some examples, the non-alcohol based precipitation process comprises the use of a non-alcohol based precipitation agent such as, for example, ammonium sulphate, citrate, or octanoic acid. In other examples, the non-alcohol based precipitation process comprises the step of heating the plasma to a temperature where the protein of interest remains stable while the proteins which are not of interest denature and are subsequently removed, e.g. as described in WO publication number WO18165766.

[0043] Exemplary purification processes include the following, each of which is hereby incorporated by reference. Exemplary affinity purification processes are disclosed for example in CSL Behring WO publications, including WO23194944, WO23007445, W025008781 describing affinity chromatography plasma purification processes, including immunoglobulin and albumin purification. Further exemplarypurification processes include those disclosed in for example WO publications WO21099529, WO19219890 and WO22248648 relating to improved IgG yield processes. Additional exemplary methods include for example affinity-based IgG purification processes described in e.g. WO publication WO23170553, WO15136217 and WO 16073401 and ethanol fractionation processes based on Cohn’s principles, as characterized in WO publication Nos. WO11149472, WO11011753, WO11150284, WO13126904, W016012803 and WO18019898. An exemplary octanoic acid fractionation process is described in for example European application No, 893450 and WO publication Nos. WO15137531; WO05082937 and WO11131786. An exemplary salt precipitation method is described in, for example, WO22146856. Other exemplary purification processes include that disclosed in for example WO22258590.

[0044] In one example, the ethanol-based precipitation process is an improved ethanol-based precipitation process. For example, a high yielding ethanol-based precipitation process. In one example, the non-alcohol-based precipitation process is an improved non-alcohol based precipitation process. For example, a high yielding nonalcohol based precipitation process. In one example, the alcohol-based precipitation process is an improved alcohol based precipitation process. For example, a high yielding alcohol based precipitation process. The skilled person will appreciate that a purification process may be classified as high-yielding or low-yielding based on the concentration of the target protein (e.g., POI) relative to a conventional or known fractionation method, such as the Cohn ethanol fractionation process.

[0045] The skilled person will appreciate that the first and / or second purification processing pathway steps include one or more additional purification steps to isolate the protein of interest. In one example, the additional purification steps further comprise purifying the protein of interest preparation with one or more steps selected from a group consisting of precipitation, viral filtration, isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof. Additional purification steps will be apparent to the skilled person and / or described herein. Testing the sample of blood, serum or plasma may include testing using a lateral flow assay. The lateral flow assay may include aconjugate configured to label the protein of interest in the blood, serum or plasma sample. The lateral flow assay may comprise a control line and at least one test line. A binding ligand specific to the protein of interest may be fixed at the at least one test line.

[0046] The lateral flow assay may be configured such that the test line is activated when the protein of interest concentration of the blood, serum or plasma sample is equal to and / or above a predetermined concentration level. An intensity of the activated test line may be indicative of the concentration of protein of interest in the blood, serum or plasma sample.

[0047] The at least one test line may comprise a single test line. The predetermined concentration level in the sample may be correlated with the threshold concentration level in the collected plasma unit.

[0048] The lateral flow assay may include a control line and a plurality of test lines. Activation of one or more of the test lines may be indicative of one of a corresponding one or more discrete predetermined concentration levels of the protein of interest in the collected plasma unit. An intensity of one or more of the test lines may be indicative of a concentration level of the protein of interest in the collected plasma unit. In some examples, the intensity of two or more test lines, when read together, may be indicative of a concentration level of the protein of interest in the collected plasma unit.

[0049] In some examples, activation and / or intensity of one or more test lines may be indicative of a respective concentration level of one or more different proteins in the collected plasma unit.

[0050] In some examples, the threshold concentration level for a protein of interest may be determined at least partially based on a standard concentration distribution curve in plasma for the protein of interest. For example, the threshold concentration level may be determined relative to a median value of the concentration distribution curve for the protein of interest in plasma. In some examples, the thresholdconcentration level may be determined as equal to the median value of the concentration distribution curve for the protein of interest in plasma. In some examples, the threshold concentration level may be determined as a percentage above the median value of the standard concentration distribution curve for the protein of interest in plasma, such as at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more above the median value. In some examples, the threshold concentration level may alternatively or additionally be determined as a value below the median value of the standard concentration distribution curve for the protein of interest in plasma, such as at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50% about 60%, about 70%, about 80%, about 90%, or more below the median value.

[0051] In some examples, the threshold may be selected based on a percentile of the standard distribution curve of the protein of interest in plasma, such as above the 50th, 60th, 70th, 80th, or 90th percentile. In some further examples, the threshold may be selected based on a percentile below the median of the standard distribution curve, such as at or below the 50th, 40th, 30th, 20th, or 10th percentile. In some examples, protein concentration levels may be categorized relative to the threshold as low, medium, or high, wherein low levels may correspond to concentrations below the threshold, high levels may correspond to concentrations above the threshold, and medium levels may correspond to concentrations at or around the threshold, including concentrations within a defined range above and / or below the threshold. . In some examples, the boundaries between low, medium, and high concentration levels may be overlapping or non-exclusive, and may be selected to optimize sensitivity, specificity, or clinical utility for a given application.

[0052] In some examples, a low concentration level of the protein of interest may correspond to a plasma concentration below the lower limit of the reference range for that protein. In some examples, a high concentration level of the protein of interest may correspond to a plasma concentration above the upper limit of the reference range for that protein. In some examples, a medium concentration level of the protein of interest may correspond to a plasma concentration within the reference range, includingconcentrations within a central portion of the reference range, or within a defined subrange around a median or mean value of the reference range.

[0053] In some examples, the standard distribution curve for the concentration levels of a protein of interest in plasma may be defined relative to an established clinical reference range for that protein. Such reference ranges may be based on population-derived normal ranges, laboratory reference intervals, or clinically accepted ranges, and may vary depending on assay methodology, calibration standards, demographic factors, and clinical context. This approach may be used in addition to or instead of fixed concentration ranges for specific proteins (for example, such as IgG or other protein of interest.

[0054] In some examples, the protein of interest is immunoglobulin G (IgG), and medium IgG levels may correspond to plasma IgG concentrations within a clinically accepted reference range for IgG, while low IgG levels may correspond to concentrations below the lower limit of the reference range and high IgG levels may correspond to concentrations above the upper limit of the reference range. In some examples, medium IgG levels may further correspond to concentrations within a midportion of the IgG reference range, such as between approximately the 25th and 75th percentile of the reference range, or within a defined percentage above and / or below a median IgG concentration.

[0055] In some examples, such as when the protein of interest (POI) is IgG, the threshold concentration level may be in a range of between approximately 2g / L and lOg / L. In some examples, such as when the protein of interest (POI) is IgG, the threshold concentration level may be in a range of between approximately 2g / L and 16g / L. In some examples, such as when the protein of interest (POI) is IgG, the threshold concentration level may be in a range of between approximately 7g / L and 16g / L. In some examples, such as when the protein of interest (POI) is IgG, the threshold concentration level may be selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, 7.5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, lO.Og / L, 10.50 g / L, 11.0 g / L, 11.5 g / L, 12g / L, 12.5g / L, 13.0g / L, 13.5g / L,14.0g / L, 14.5 g / L, 15.0g / L, 15.5g / L, or 16.0g / L or any value in-between. In some examples, such as when the protein of interest is IgG, the thresholds concentration for IgG may be set at or around the lower limit, upper limit, or median of the IgG reference range, depending on the intended purification processing pathway. In some examples, such as when the protein of interest (POI) is IgG, the threshold value of the protein of interest concentration in plasma may be 3.0g / L. In some examples, such as when the protein of interest (POI) is IgG, the threshold value of the protein of interest concentration in plasma may be about 3.0g / L.

[0056] In some examples, such as when the protein of interest (POI) is albumin, the threshold concentration level may be in a range of between approximately 25g / L and 60g / L. In some examples, such as when the protein of interest (POI) is albumin, the threshold concentration level may be selected from 25.0g / L, 25.5g / L, 26.0g / L, 26.5g / L, 27.0g / L, 27.5g / L, 28.0g / L, 28.5g / L, 29.0g / L, 29.5g / L, 30.0g / L, 30.5 g / L, 31.0 g / L, 31.5 g / L, 32g / L, 32.5g / L, 33.0g / L, 33.5g / L, 34.0g / L, 34.5 g / L, 35.0g / L, 35.5g / L, 36.0g / L, 36.5g / L, 37.0g / L, 37.5g / L, 38.0g / L, 38.5g / L, 39.0g / L, 39.5g / L, 40.0g / L, 40.5 g / L, 41.0 g / L, 41.5 g / L, 42g / L, 42.5g / L, 43.0g / L, 43.5g / L, 44.0g / L, 44.5 g / L, 45.0g / L, 45.5g / L, 46.0g / L, 46.5g / L, 47.0g / L, 47.5g / L, 48.0g / L, 48.5g / L, 49.0g / L, 49.5g / L, or 50.0g / L, 50.5 g / L, 51.0 g / L, 51.5 g / L, 52g / L, 52.5g / L, 53.0g / L, 53.5g / L, 54.0g / L, 54.5 g / L, 55.0g / L, 55.5g / L, 56.0g / L, 56.5g / L, 57.0g / L, 57.5g / L, 58.0g / L, 58.5g / L, 59.0g / L, 59.5g / L, 60.0g / L or any value in-between. In some examples, such as when the protein of interest is albumin, the thresholds concentration for albumin may be set at or around the lower limit, upper limit, or median of the albumin reference range, depending on the intended purification processing pathway. In some examples, such as when the protein of interest (POI) is albumin, the threshold value of the protein of interest concentration in plasma may be at or around 35g / L.

[0057] In some examples, the concentration levels of the protein of interest may be classified as low, medium, or high relative to the respective reference range for that protein using analogous criteria. For example, medium albumin levels may correspond to plasma albumin concentrations within an established albumin reference range; medium Cl esterase inhibitor levels may correspond to concentrations within areference range associated with normal complement regulation; medium factor VIII or factor XI levels may correspond to activity or concentration levels within a normal coagulation reference range; medium alpha- 1 antitrypsin levels may correspond to concentrations within a reference range associated with normal protease inhibition; and medium prothrombin levels may correspond to concentrations or activity levels within a reference range associated with normal thrombin generation. The person skilled in the art will appreciate that any standard criteria for providing a respective reference range for a protein of interest can be used to determine the concentration classification and relevant threshold for a protein of interest and the criteria is not limited to those specifically described herein.

[0058] According to another aspect of the present disclosure, there is provided a method of separating collected plasma for a protein purification process, the method including:testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in collected plasma unit from each donor;separating the collected plasma units based on the respective determined concentration level; anddirecting the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level.

[0059] Collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level may be directed to a first purification processing pathway. Collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level may be directed to a second, different purification processing pathway.

[0060] According to another aspect of the present disclosure, there is provided a method of separating collected plasma for a protein purification process, the method including:testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in collected plasma unit from each donor;separating the collected plasma units based on the respective determined concentration level; anddirecting the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level,wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a second, different purification processing pathway, wherein the first protein purification processing pathway includes an affinitybased chromatography process, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.

[0061] In some examples, the protein of interest is immunoglobulin G (IgG) and collected plasma units determined as having a concentration level of IgG greater than a threshold concentration level of IgG are directed to a first purification processing pathway and collected plasma units determined as having a concentration level of IgG lower than the threshold concentration level are directed to a second purification processing pathway different from the first purification processing pathway, wherein the first purification processing pathway includes a first process configured to isolate IgG, wherein the second purification processing pathway includes a second process configured to isolate IgG, wherein the first purification processing pathway achieves a higher yield of IgG than the second purification processing pathway (for example, the first process configured to isolate IgG may be different from the second process configured to isolate IgG and the first process configured to isolate IgG may achieve ahigher yield of IgG than the second process configured to isolate IgG). In some examples both the first and second purification processing pathways include steps configured to isolate one or more additional proteins of interest, e.g. albumin.

[0062] Collected plasma units determined as having a concentration level of the protein of interest greater than the threshold concentration level may be pooled prior to direction to the first purification processing pathway. Collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level may be pooled separately prior to direction to the second purification processing pathway.

[0063] The method may further include collecting the plasma units from the plurality of donors.

[0064] The method may further include processing the separated collected plasma units to produce isolated protein of interest from the collected plasma unit.

[0065] According to another aspect of the present disclosure, there is provided a test kit for determining a concentration level of a protein of interest in a plasma unit collected from a donor, the test kit comprising:a lateral flow assay configured to test a sample of blood, serum or plasma from the donor; wherein the lateral flow assay includes:a conjugate configured to label the protein of interest in the blood, serum or plasma sample;a control line; andat least one test line, wherein a binding ligand specific to the protein of interest is fixed at the at least one test line.

[0066] The test kit may comprise an automated reader configured to detect activation and / or intensity of the control line and the at least one test line to determine a concentration of the protein of interest in the blood, serum or plasma sample.

[0067] The automated reader may be configured to correlate the determined concentration of the protein of interest in the blood, serum or plasma sample with a concentration of the protein of interest in a plasma unit collected from the donor.

[0068] The automated reader may be configured to determine whether the collected plasma has a concentration level of the protein of interest greater than a threshold concentration level or less than the threshold concentration level.

[0069] The lateral flow assay may be configured to activate at least one test line when the concentration of protein of interest concentration of the blood, serum or plasma sample is equal to a predetermined concentration level.

[0070] The predetermined concentration level may be indicative of a threshold concentration level of protein of interest in the plasma unit collected from the donor.

[0071] The lateral flow assay may include a control line and a plurality of test lines.

[0072] Activation of one or more of the test lines may be indicative of one of a corresponding plurality of discrete predetermined concentration levels of the protein of interest in the collected plasma unit.

[0073] Activation and / or intensity of one or more of the test lines may be indicative of a respective concentration level of one or more different proteins in the collected plasma unit. In some examples, the intensity of two or more test lines, when read together, may be indicative of a concentration level of the protein of interest in the collected plasma unit.

[0074] In some examples, the protein-of-interest is a plasma protein. In some examples, the protein-of-interest is a plasma protein selected from the group consisting of immunoglobulin (Ig), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor,a prekallikrein activator (PKA), a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an anti-thrombin III, alpha- 1 acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof, a complement component, an oligomeric form or degradation product of any of the foregoing and combinations thereof.

[0075] In some examples, the protein-of-interest is an immunoglobulin. For example, the immunoglobulin is a normal polyvalent human immunoglobulin. For example, the immunoglobulin is IgG. In another example, the immunoglobulin is IgA. In a further example, the immunoglobulin is IgM.

[0076] In another example, the immunoglobulin is a hyperimmune immunoglobulin, for example an immunoglobulin used in a hyperimmune Ig preparation which contains pathogen-specific neutralising antibodies to hepatitis B, tetanus, varicella-zoster, rabies or Covidl9. In another example the hyperimmune Ig is Rh (D) (anti-D) immunoglobulin.

[0077] In some examples, the protein-of-interest is an apolipoprotein Al. In some examples, the protein-of-interest is an albumin. For example, a-globulins and / or [3-globulins. In one example, the protein-of-interest is a protease. For example, the protease is a serine protease and / or a plasmin. In some examples, the protein-of-interest is plasminogen. In some examples, the protein-of-interest is an alpha- 1- antitrypsin. In some examples, the protein-of-interest is a fibrinogen. In some examples, the protein-of-interest is a von Willebrand factor.

[0078] In one example, the protein-of-interest is a coagulation factor and / or activated form thereof. For example, the coagulation factor is factor Xa, factor VII, factor VIII, a factor IX, factor XII, factor XIII and / or factor XI. In one example, the coagulation factor is factor Xa. In another example, the coagulation factor is factor VII. In a further example, the coagulation factor is factor VIII. In one example, the coagulation factor is factor IX. In another example, the coagulation factor is factor XI. In a further example, the coagulation factor is factor XII. In one example, the coagulation factor is factorXIII. In some examples, the protein-of-interest is an activated clotting factor. For example, the activated clotting factor is selected from a group consisting of FXa, FIXa, FVIIa and thrombin. For example, the activated clotting factor is FXa. For example, the activated clotting factor is FIXa. For example, the activated clotting factor is FVIIa. For example, the activated clotting factor is thrombin.

[0079] In one example, the protein-of-interest is a complement component. For example, the complement component is complement component Iq (Clq), Clr and / or Cis. In one example, the complement component is Clq. In another example, the complement component is Clr. In a further example, the complement component is Cis. In one example, the protein-of-interest is a contact system factor. For example, the contact system factor protein is selected from a group consisting of FXIa, FXIIa and kallikrein. For example, the contact system factor protein is FXIa. For example, the contact system factor protein is FXII. For example, the contact system factor protein is kallikrein.

[0080] In some examples, the protein-of-interest is a Prekallikrein activator (PKA). In some examples, the protein-of-interest is a prothrombin complex. In some examples, the protein-of-interest is a Cl esterase inhibitor. In some examples, the protein-of-interest is a protein C. In some examples, the protein-of-interest is an anti-thrombin III. In some examples, the protein-of-interest is alpha acid glycoprotein. In some examples, the protein-of-interest is haptoglobin. In some examples, the protein-of-interest is hemopexin. In some examples, the protein-of-interest is transferrin. In some examples, the protein-of-interest is Factor H.

[0081] In some examples, the protein-of-interest is a protease inhibitor. In one example, the protease inhibitor is a serine protease inhibitor. For example, the serine protease inhibitor is selected from the group consisting of a Cl inhibitor, an alpha- 1-antitrypsin and an anti-thrombin.

[0082] In one example, the protein-of-interest is an oligomeric form or degradation product of any of the foregoing plasma proteins.Brief Description of Drawings

[0083] Embodiments will now be described, by way of example only, with reference to the accompanying drawings in which:

[0084] Figure l is a flow diagram illustrating a method for directing plasma in a protein purification process, according to one example of the present disclosure.

[0085] Figure 2 is a flow diagram illustrating typical steps in collecting plasma from a donor;

[0086] Figure 3 is a flow diagram illustrating a simplified conventional protein purification process for fractionation of plasma to produce isolated plasma protein products;

[0087] Figure 4 is a flow diagram illustrating a simplified conventional protein purification process for fractionation of plasma to produce isolated plasma protein products, including a chain of processing steps;

[0088] Figure 5 is a flow diagram illustrating separating plasma protein purification processing pathways according to one embodiment of the present disclosure;

[0089] Figure 6 shows a captured image of 15 lateral flow assay (LFA) dipsticks used to determine a protein concentration in 15 samples of plasma;

[0090] Figure 7 is a graph showing the detected LFA test line signal strength (AU) for the LFAs of Figure 6, plotted against nephelometer measured IgG concentration (g / L);

[0091] Figure 8 is a graph showing IgG concentration (g / L) measured by nephelometric analysis for the 15 plasma samples tested with the LFA dipsticks of Figure 6, divided into two groups based on the cut-off indicated in Figure 7;

[0092] Figure 9 shows a captured image of 24 lateral flow assay (LFA) dipsticks used to determine a protein concentration in 24 respective samples of plasma;

[0093] Figure 10 is a graph showing IgG concentration (g / L) measured by nephelometric analysis, plotted against the expected IgG (g / L) for the the 24 samples tested with the LFA dipsticks of Figure 9;

[0094] Figure 11 shows an example reference chart from a Protia ImmuneCheck IgG LFA test kit;

[0095] Figure 12 is a graph showing a frequency distribution with the results of testing 40 plasma samples determined using a Protia ImmuneCheck IgG LFA test kit and visual inspection of the test result; and

[0096] Figure 13 shows the visual inspection results of Figure 12 plotted against IgG concentration (g / L) measured by nephelometric analysis.Detailed Description

[0097] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0098] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0099] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only.Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0100] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

[0101] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0102] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0103] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0104] As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

[0105] Furthermore, as used herein the singular forms of “a”, “and” and “the” include plural references unless the context clearly dictates otherwise.Selected Definitions

[0106] As used herein, the term “isolate”, “isolated”, “isolating” or “isolation” shall be taken to mean the separation of, whether completely or partially, a protein (e.g., aplasma protein such as IgG) present in a protein mixture (e.g., plasma or a plasma fraction).

[0107] The term “plasma unit” shall be taken to mean a volume of plasma collected during a donation procedure. The donation procedure can include plasmapheresis or whole blood donation. The exact volume of plasma in each plasma unit may vary depending on factors such as the donor’s body weight and whether the plasma unit is obtained by plasmapheresis or whole blood donation.

[0108] The term “plasma” shall refer to the straw-col oured / pale yellow component of blood obtained from one or more blood donor(s). Methods of obtaining plasma from a donor will be apparent to a skilled person and / or described herein. For example, plasma is obtained by removing red blood cells from donated blood. For example, plasma is obtained by plasmapheresis.

[0109] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0110] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0111] As used herein, the term “protein mixture” shall be understood to refer to a solution containing two or more proteins or a complex mix of proteins, including the protein-of-interest.

[0112] The term “specifically binds”, “specifically binding” or “binds specifically” shall be taken to mean that a protein of the disclosure reacts or associates morefrequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a ligand capable of specifically binding to a constant domain of human IgG, such as CHI, CH2, CH3, CH4 or combinations thereof, with materially greater affinity (e.g., 1.5 fold or 2 fold or 5 fold or 10 fold or 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other antigens. Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

[0113] The term “plasma fraction” or “fraction thereof’ shall refer to plasma which has been fractionated to isolate one or more desirable protein components from the plasma. For example, plasma may be fractionated to isolate cryo-precipitates (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold) and cryosupematant (also known as cryo-poor plasma). For example, plasma may be fractionated by non-alcohol (non-volatile) based methods or processes such as chromatography (e.g., affinity chromatography as described in for example, International patent publication Nos. WO23007445; WO23194944; W025008781 and WO23170553) and modifications thereof, precipitation based methods such as salt precipitation, e.g. ammonium sulphate, citrate or acetate precipitation (e.g. such as described in EP patent 1928915 and US patent 11732004), hydrophilic polymer polyethylene glycol precipitation (such as described in e.g. patent publication WO0248176), acid precipitation, or by ethanol precipitation to produce for example, IgG-containing Oncley fractions, Cohn fractions, ammonium sulphate precipitates, or Precipitates A (KN A), B (KN B), and the Precipitate of Supernatant B (KN B+l) from plasma as described in US patent 3,301,842. Plasma fractions include II+III precipitate produced according to Cohn methods such as Method 6, Cohn et. al. J. Am; Chem. Soc., 68 (3), 459-475 (1946), Method 9, Oncley et al. J. Am; Chem. Soc., 71, 541-550 (1946), or the 1+11+111 precipitate, Method 10, Cohn et.al. J. Am; Chem. Soc., 72, 465-474 (1950); as well as the method of Deutsch et.al. J. Biol. Chem. 164, 109-118 (1946) or the Precipitate-A, B and the Precipitate of Supernatant B of Nitschmann and Kistler Vox Sang. 7, 414-424 (1962); Helv. Chim. Acta 37, 866-873 (1954). For example, the plasma may be fractionated by ethanol precipitation as described in patent publicationsW011149472, WO11011753, WO11150284, and US Patent 11,325,964. For example, the plasma may be fractionated by octanoic acid fractionation as described in European application 893450. Typically, Cohn Fractions, and Kistler / Nitschmann Precipitate’s A (KN A), B (KN B) and the Precipitate of Supernatant B (KN B+l) exist as a suspended paste. Other purification techniques including chromatography may be used. It will be appreciated by persons skilled in the art that the presently disclosed methods for directing and / or separating collected plasma samples and related test kits may be used in combination with a variety of plasma fractionation processes, not limited to those specifically described herein.

[0114] The term “cryo-precipitate” or “cryo-precipitates” refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold. Cryo-precipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII and platelet membrane microparticles.

[0115] The term “cryo-poor plasma” shall be taken to mean plasma removed of cryoprecipitates.

[0116] The term “cryo-rich plasma” shall be taken to mean plasma comprising components typically found in cryo-precipitates.

[0117] The term “immunoglobulin G (IgG)”, also known as “gamma globulin” or “immune globulin”, shall be taken to mean antibody of isotype G. There are several subclasses of IgG, for example, IgGl, IgG2, IgG3 and IgG4.

[0118] The term “chromatography medium” shall be taken to mean a solid or semi solid phase for use in chromatography. In one example, a chromatography medium is made up of a porous or non-porous support to which a plurality of ligands are attached, examples of which are described herein.

[0119] The term “ligand” as used herein shall be taken to mean a molecule immobilised on a matrix of a chromatography medium that determines the binding properties of the matrix.

[0120] The term “matrix” shall be taken to mean a support to which the ligand is immobilised.

[0121] The term “cam elid-derived single domain [VHH] antibody fragment” shall be taken to mean a VHH domain of a camelidae antibody. The camelidae antibody is an antibody produced from camels and llamas and has no CHI domain normally present in human immunoglobulins and only one VHH domain. Exemplary affinity chromatography resins comprising the camelid-derived [VHH] antibody fragment include CaptureSelect® antibody affinity chromatography resins (Thermo Fisher). For example, CaptureSelect® FcXL affinity resin, POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher), CaptureSelect IgG-CHl affinity resin, and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography resins include IgSelect® affinity resin (Cytiva), HiTrap® IgSelect® affinity resin (Cytiva), Pierce® Protein G agarose affinity resin (Thermo Fisher), and Protein G sepharose 4 fast flow affinity resin (Cytiva), CaptureSelectTM KappaXL affinity resin, CaptureSelectTM IgG-Fc (ms) affinity resin, CaptureSelectTM LC-lambda (Hu) affinity resin, CaptureSelectTM kapp XP affinity resin, CaptureSelectTM IgG3 affinity resin.

[0122] As used herein, “octanoic acid” refers to octanoic acid and is used interchangeably with “caprylic acid” and includes the corresponding caprylate form, (the conjugate base or salt thereof), unless the context indicates otherwise.

[0123] The term “industrial or commercial scale” or “large scale” or “manufacturing scale” shall refer to the amount of product that would be produced in a batch that was designed for clinical testing, formulation, sale and / or distribution to the public. For example, industrial scale refers to large scale purification of IgG from the plasma or fraction thereof to produce the plasma protein product.

[0124] The terms “plasma protein product” shall refer to a preparation, composition and / or protein product comprising a plasma protein (e.g. IgG) derived from the purification of the protein mixture (e.g., plasma or fraction thereof). Typically, the plasma protein is the predominant protein in the plasma protein product.

[0125] The term “pharmaceutical composition” shall be taken to mean a formulation of the protein product (e.g., the plasma protein product) with compounds generally accepted in the art for the delivery to mammals. Exemplary compounds include all pharmaceutically acceptable carriers, diluents or excipients thereof. Pharmaceutical compositions containing plasma protein products may also be known as plasma-derived medicinal products (PDMPs).Plasma collection

[0126] The concentration of proteins in donated plasma can vary widely. For example, where the protein of interest (POI) is IgG, typical plasma IgG concentration may range from 2g / L to 13g / L in a typical healthy adult population. Several factors, including lifestyle, age, gender, race, weight and frequency of plasma donation can all contribute to changes in IgG concentration, making it difficult to predict the concentration of a particular donated plasma unit.

[0127] The present disclosure provides a method measuring donor POI (e.g. IgG) levels prior to pooling of plasma samples from multiple donors. Being able to quantify donor plasma POI levels prior to pooling allows diversion of POI rich plasma directly to preferred purification processes, in order to provide a faster and more cost-efficient POI purification process and minimise the cost of POI generated per litre of plasma. Due to the numerous factors contributing to variation in donated plasma POI concentration, obtaining a measurement indicative of POI concentration for each collected plasma unit provides an accurate method for separating the collected plasma units based on POI concentration.

[0128] Serum proteins have high correlation between venous serum and finger-tip blood / serum concentration, which suggests that a finger prick blood sample can be used for fast identification of donor POI levels prior to the start of the apheresis process, which may minimise interruption to the normal donation process.

[0129] Some purification processes may achieve higher yields of a POI (such as IgG), but may be more expensive. For example, affinity resins used in affinity chromatography can have relatively low binding capacity and chromatography purification from an average size batch can reach volumes of several hundred litres, being a huge capital investment in the amount of resin used, the infrastructure to handle and pack the chromatography columns, along with the running costs. To minimize the cost per unit of the POI generated per litre of plasma through an affinity purification method, it is preferable to only load plasma with the highest concentrations (above the threshold concentration level) of the POI to such processes, while plasma having a lower concentration of the POI (below the threshold concentration level) may be directed to less expensive processes, such as precipitation processes (e.g. an ethanol-based precipitation process or a non-alcohol precipitation process). In some examples, the first processing pathway includes an affinity -based chromatography process configured to extract the POI and the second processing pathway includes a precipitation process configured to extract the POI.

[0130] The present disclosure provides methods and apparatus for a protein purification process. An example method 10 according to the present disclosure is illustrated by the flowchart of Figure 1. The method 10 includes testing a sample of blood, serum or plasma from a donor at 130. Based on the test, a concentration level of a protein of interest in plasma collected from the donor is determined at 140. The collected plasma unit is then directed to a selected protein purification process, based on the determined concentration level of the protein of interest. Specifically, the determined concentration level is compared to a threshold concentration level at decision point 150 and, based on the comparison, the collected plasma unit is directed either to a first protein purification process, as indicated at 152, or directed to a different, second protein purification process, as indicated at 154. As indicated by thedotted lines at 110 and 120, the method 10 optionally includes the steps of obtaining the blood, serum or plasma sample from the donor and collecting the plasma from the donor.

[0131] It will be appreciated that the steps of method 10 need not be performed in the order as illustrated in the example flow diagram of Figure 1, but may be performed in an alternative order in other examples. For example, the sample may be obtained at step 110 and tested at step 130 prior to or after the collection of plasma from the donor at step 120. In some examples, the sample may be obtained directly from the donor. For example, the sample may be blood, serum or plasma drawn from the donor, or obtained via a finger-prick. In some examples, the sample may be obtained and / or tested during collection of plasma from the donor at step 120. In some examples, the sample may be obtained from collected plasma, e.g. a collected plasma unit, after collection from the donor.

[0132] Figure 2 shows a flow diagram of a typical steps involved in step 120 of collecting plasma from a donor at item 120 of method 10. The plasma collection 120 may include several stages, such as a donor suitability assessment 122, plasmapheresis 124 and post-production 126. Opportunities for obtaining and / or testing the sample of blood, serum or plasma exist within each of these stages.

[0133] The donor suitability assessment 122 stage may include initial steps of donor intake and profile creation, as well as performing a complete health assessment. As plasma may be donated as frequently as twice weekly, these checks may not be performed at every donor visit but may instead only be performed on a donor’s first visit, or may be refreshed at routine intervals, such as annually. At each visit, donors will generally be asked to fill in a qualitative health questionnaire and be subjected to a vitals check and / or screening tests overseen by a health professional. Generally, the check / tests includes at least a haematocrit suitability test. In the method 10 according to the present disclosure, step 110 of obtaining the sample may be performed before, during, or after such screening tests. For example, in the method 10, the screening testsmay include collection of a blood sample by phlebotomy or finger-prick to determine a concentration level of a protein of interest in the donor’s plasma.

[0134] The plasma collection stage 124 may include preparation of the plasmapheresis device and the donor for collection of the donated plasma unit. Once prepared, the plasmapheresis procedure is initiated and allowed to run until completion (for example, until collection of a predetermined amount of plasma), at which point the plasmapheresis procedure is terminated. The method according to the present disclosure may comprise the step 110 of obtaining a sample at any of these points. In some examples, a sample may be obtained directly from the donor during preparing the donor for connection to the plasmapheresis device, prior to performing plasmapheresis. For example, a blood sample may be collected by phlebotomy or by finger-prick. In some examples, a sample may be obtained during the plasmapheresis procedure, for example by diverting a small sample of the donor’s blood, plasma or serum extracorporeally. In some examples, the sample may be collected during or after termination of the plasmapheresis procedure.

[0135] In other examples, the plasma unit may be collected from a whole blood donation following separation of the plasma from other blood components. In such cases, the sample may be obtained directly from the donor before, during or after collection of the whole blood. Alternatively or additionally, the sample may be obtained from the collected whole blood donation, or from the plasma unit once separated from the whole blood donation.

[0136] The post-production phase 126 may involve steps of finishing and / or testing of the collected plasma unit, as shown in Figure 2. Generally, collected plasma units are frozen prior to storage. Obtaining a sample 110 according to the method 10 of the present disclosure may be performed prior to freezing and storage of the collected plasma unit. For example, a sample may be obtained from the collected plasma unit during finishing and / or testing operations, prior to freezing.

[0137] The collected plasma units may be held in inventory for a predefined period, prior to being provided (e.g. shipped) to a manufacturing facility for processing to produce plasma protein products.

[0138] In some examples, the method may include labelling the plasma units, based on the determined protein of interest concentration. For example, the plasma units may be labelled with a marking, stamp, sticker, tag or other suitable label. In some examples, the label plasma may include a scannable component, such as a barcode or “quick-reference” (QR) code. Incorporation of a QR code or barcode ID may allow quick and easy tagging of the collected plasma unit with the determined concentration of the protein of interest (e.g. IgG).

[0139] Depending on the determined concentration, the donated plasma units can be separated and accurately directed to downstream processes where the protein of interest is purified (for example, via affinity chromatography and / or alcohol-based precipitation methods). For example, the donated plasma units may be separated into a “low protein-of-interesf ’ group having a determined protein of interest concentration below the threshold concentration level, and a “high protein-of-interesf ’ group having a determined protein of interest concentration above the threshold concentration level. The “high protein-of-interesf ’ group may be directed to a first protein purification processing pathway and the “low protein-of-interesf ’ group may be directed to a second protein purification processing pathway, different from the first protein purification processing pathway.Protein purification

[0140] Figure 3 is a flow diagram illustrating a simplified conventional protein purification process for fractionation of plasma to produce isolated plasma protein products. Typically, for industrial-scale fractionation of proteins from plasma, frozen units of collected plasma are opened and the plasma is pooled for thawing, as indicated at 160. The pooled plasma is subjected to one or more purification processes 170 to isolate plasma-derived proteins 180. The purification process(es) may include, forexample, precipitation and / or chromatography processes. Each purification process may also product an eluate or supernatant fraction 190 of the plasma, which is depleted of the plasma isolated during that process. The process will usually include dedicated viral reduction steps, before and / or after the purification steps, to ensure that no infectious agents are present in the final products.

[0141] Conventional plasma fractionation processes follow a chain or a “backbone” of successive processing steps, as illustrated in Figure 4, which separates the plasma into multiple fractions to successively isolate a plurality of desired plasma proteins. For example, after pooling of the plasma 160, the process shown in Figure 4 includes purification process A (171), followed by a purification process B (172) and optionally by a third (173) or further purification processes, which are configured to isolate respective first, second and third plasma proteins 181, 182, 183. The supernatant and / or eluate fraction from each purification process, e.g. 191, 192, feeds into the subsequent purification process. It may be necessary to perform the purification processes 171, 172, 173 in a predetermined order to allow isolation of all three (or more) plasma-derived proteins 181, 182, 183 from the same pool of plasma. However, each purification process (and / or viral reduction step) may cause yield losses of the other plasma-derived proteins. For example, purification process A (171) may result in yield loss of plasma-derived protein 2. Where plasma-derived protein 2 is of particular interest (for example, for reason of being of higher value or in greater demand), this yield loss may be particularly undesirable.

[0142] Generally, plasma pooling involves combining of 10,000 to 50,000 donations in order to achieve the volumes required for industrial processing. In conventional processing procedures, the plasma donations are pooled indiscriminately. Once the plasma donations are pooled, although traceability is maintained through to the final product, the ability to separate out and / or divert specific plasma donations, or groups of plasma donations, to different processing pathways is lost. As can be appreciated from Figure 4, all plasma may be directed through the same processing pathway.Alternatively, where portions of the pooled plasma are directed to different pathways, this is generally not based on any measured characteristic of the plasma. Rather, theplasma may donations may be divided before or after pooling based on processing volume requirements.

[0143] By contrast, the method 10 according to the present disclosure involves determining the protein concentration level of each of the collected plasma units, as indicated at 140 of Figure 1. Based on the determined concentration level being above or below a threshold concentration level, as indicated by decision point 150, the collected plasma units are then directed to either a first purification processing pathway at 152 or directed to a second purification processing pathway at 154. In some examples, the method 10 may include directing the collected plasma units to a third, fourth, or further processing pathways, based on a determined concentration level relative to a second, third or further threshold concentration level of the protein of interest and / or a threshold concentration level of a second, third or further protein of interest.

[0144] In some examples, the method 10 may include processing the collected plasma units to produce isolated protein of interest from the collected plasma units. One example of a method 10 including further processing of the collected plasma units is illustrated in Figure 5.

[0145] Figure 5 is a flow diagram illustrating a simplified example of a method 10 for fractionation of plasma to produce isolated plasma protein products, including separating and directing the collected plasma units based on the determined concentration level of the protein of interest. Further, the method 10 may include additional steps not illustrated. For example, one or both of the first and / or second processing pathways may include a common step of cryo-precipitation following pooling of the plasma. Cryoprecipitation produces a cryoprecipitate fraction and a cryopoor plasma (or cryo-supernatant) fraction. The cryoprecipitate fraction and / or the cryo-poor plasma fraction may be subject to further fractionation steps to isolate one or more proteins of interest.

[0146] The flow diagram of Figure 5 may follow on from the method as shown in Figure 1, and includes the steps of determining the protein concentration level of a collected plasma unit 140, comparing the determined protein concentration to a threshold concentration level at decision point 150 and, based on the comparison, the collected plasma unit is directed either to a first protein purification processing pathway, as indicated at 152 and the subsequent chain of processing steps, or directed to a different, second protein purification process, as indicated at 154 and the subsequent chain of processing steps. For example, it may be desirable to direct plasma having a concentration of the protein of interest above the concentration threshold level (“high POI plasma”) to a processing pathway including a purification process, or sequence of purification processes, which is optimised for isolation of the protein of interest. For example, the first purification processing pathway may be optimised to maximise yield (e.g. by minimising yield loss) of the protein of interest. Further it may be desirable to direct plasma having a concentration of the protein of interest below the concentration threshold level (“low POI plasma”) to a processing pathway including a purification process, or sequence of purification processes, which achieves isolation of the protein of interest at a lower cost, and / or which is optimised for isolation of a one or more different proteins of interest.

[0147] In the example illustrated in Figure 5, the first and second processing pathways include respective steps for pooling collected plasma units identified as having a POI concentration below the threshold concentration level at 161 and for pooling collected plasma units identified as having a POI concentration above the threshold concentration level at 162. As such the “high POI” and “low POI” plasma is maintained separate from each other.

[0148] The first processing pathway includes purification process B (172) and, optionally, purification process C (173), while the second processing pathway includes purification process A (171) and, optionally, purification process D (174). It will be appreciated that the flow diagram of Figure 5 is a simplified example only, and that the method 10 is therefore not limited to the number or configuration of purification processes shown, and that the method 10 may include a greater or lesser number ofpurification processes and / or a different configuration of purification processes within the purification pathways. The purification processes 171, 172, 173, 174 may include an affinity-based chromatography process and a precipitation process. A precipitation process may comprise a non-alcohol or an alcohol -based precipitation process. For example, the purification process may include precipitation, such as ethanol, ammonium sulphate, citrate, and octanoic acid fractionation.

[0149] In some examples, plasma units identified as having a protein-of-interest (POI) concentration above the threshold concentration level may be directed to a purification pathway that includes an affinity -based chromatography process. Affinity chromatography may be selected for such “high POI” plasma units because it enables comparatively higher yield and / or purity of the POI (for example, IgG) compared to other methods.

[0150] In some examples, plasma units identified as having a POI concentration below the threshold concentration level (“low-POI” plasma) may be processed using methods comprising ethanol precipitation steps (for example, Cohn, Kistler-Cohn or Kistler-Nitschmann fractionation processes). Ethanol-based methods may provide a cost-effective approach for isolating proteins, while still allowing recovery of additional plasma proteins of interest from plasma with lower POI concentrations.

[0151] Any of the purification processes 171, 172, 173, 174 may be the same, or may be different to any of the other purification processes 171, 172, 173, 174. Any of the purification processes 171, 172, 173, 174 may be configured to isolate the same protein-of-interest (e.g. a first protein of interest) from the plasma as another of the purification processes 171, 172, 173, 174, or may be configured to isolate a different protein-of-interest (e.g. a second, third, fourth or further protein of interest) from the plasma. That is, any of the plasma-derived proteins 181, 182, 183, 184 may be the same as another of the plasma-derived proteins 181, 182, 183, 184 and may be the protein of interest (e.g. a first protein of interest) or a different protein of interest (e.g. a second, third, fourth or further protein of interest).

[0152] One or more of the purification processes 171, 172, 173, 174 may produce a protein of interest enriched solution. In some examples, the protein of interest (e.g. IgG) may be further purified from the protein-of-interest enriched solution by one or more further purification steps selected from the group consisting of: precipitation, such as ethanol, ammonium sulphate, citrate and octanoic acid fractionation; membrane or resin chromatography (for example, ion exchange chromatography, hydrophobic interaction chromatography, isoagglutinin affinity chromatography); viral inactivation; viral filtration and ultrafiltration / diafiltration. In some examples, the method may further comprise precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation. In certain examples, the method further comprises octanoic acid fractionation.

[0153] In one example, plasma-derived protein 2 (182) is a protein of interest (POI), for example a first protein of interest. It will be appreciated that, in other examples, another plasma-derived protein may be the protein of interest, or there may be a plurality of proteins of interest (e.g. first, second, third, fourth and / or further proteins of interest). In this example, the first processing pathway may be optimised to maximise yield of plasma-derived protein 2 (182). For example, the method 10 may enable isolation of both plasma derived protein 1 and plasma-derived protein 2 (181, 182), whilst achieving a higher yield of POI plasma-derived protein 2 (182), being the protein of interest, compared to the conventional sequential processing pathway illustrated in Figure 4. This may be, in part, due to avoiding yield losses caused by subjecting the pooled high-concentration plasma to purification process A prior to purification process B.

[0154] In some examples, both the first and second processing pathways may include a purification process configured to isolate the POI. The first and second pathways may include the same purification process, or different purification processes configured to isolate the POI. The first processing pathway may achieve a higher yield of the POI than the second processing pathway. For example, the first purification processing pathway may be configured to isolate the POI (e.g. a first POI) and the second purification processing pathway may be configured to isolate one or more differentPOIs (e.g. second, third, fourth and / or further POIs). The first processing pathway may be optimised to maximise yield of the POI, while the second processing pathway may be optimised to maximise yield of the one or more different POIs. Nevertheless, the second processing pathway may also include one or more processes also configured to isolate the POI. The method 10 allows separating and directing of collected plasma units containing a relatively high concentration of POI (above threshold) to the higher yielding first processing pathway, while enabling isolation of additional POI from collected plasma units containing a relatively low concentration of POI (above threshold) via the second processing pathway.

[0155] It will be appreciated that the method of the present disclosure is applicable to other proteins of interest and / or to more than one protein of interest.Determining protein concentration

[0156] Referring again to Figure 1, the method 10 includes testing the sample of plasma, serum or blood, as indicated at 130 to determine the concentration level of the protein of interest in the collected plasma unit at 140. The test at 130 may be configured to indicate when a concentration of protein of interest concentration of the blood, serum or plasma sample is equal and / or above a predetermined concentration level. The predetermined concentration level in the blood, serum or plasma sample may be correlated to the protein of interest concentration in the collected plasma unit. In some examples, the predetermined concentration level in the blood, serum or plasma sample may be indicative of a protein of interest concentration in the collected plasma unit equal to the threshold concentration level. Where the test is performed on a sample of plasma (e.g. a sample taken directly from the collected plasma unit) the predetermined concentration level may be the same as the threshold concentration level. However, where the test is performed on a sample of donor blood or serum, the predetermined concentration level may be correlated with the threshold concentration level.

[0157] Where the POI is IgG, the threshold concentration level of protein of interest in the collected plasma unit may be in a range of between approximately 2g / L and lOg / L. For example, the threshold concentration level may be selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, 7.5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, or lO.Og / L.

[0158] The testing 130 of the sample may be performed using a number of different methods. In examples described in further detail below, the testing 130 is configured to determine the concentration level of IgG in the collected plasma unit. However, it will be appreciated that the testing 130 may be configured to determine the concentration level of a different protein of interest and / or multiple proteins of interest in the collected plasma unit.

[0159] In some examples, the testing 130 may be configured to provide a quantitative, or semi-quantitative, determination of the concentration of the protein of interest in the sample of blood, serum or plasma. In some examples, the testing 130 may be configured to provide a qualitative determination of the concentration of the protein of interest in the sample of blood, serum or plasma. For example, the testing 130 may be configured to determine whether the concentration of the protein of interest in the sample of blood, serum or plasma is above or below a predetermined concentration level indicative of the threshold concentration level in the collected plasma unit. The testing 130 may be used to categorise the plasma units into two or more groups. For example, the testing 130 may be used to separate the plasma units into a “low protein-of-interesf ’ group having a determined protein of interest concentration below the threshold concentration level, and a “high protein-of-interesf ’ group having a determined protein of interest concentration above the threshold concentration level.

[0160] The testing 130 may include one or more method of detecting the protein of interest, such as IgG in the sample. In the examples described in further detail below, the testing 130 includes using a lateral flow assay. However, the testing may additionally or alternatively include other methods. For example, the testing 130 may include using caprylic acid-based precipitation including+A280 absorbance of IgG andestimation of remaining IgG concentration, for example using a method similar to that described in described in the 2021 white paper “Intensification of Human Plasma IgG Purification for Intravenous and Subcutaneous Administration” issued by Millipore. In another example, the testing may include a sensor utilising a modified surface plasmon resonance assays (e.g. fiber optic (FO)-based surface plasmon resonance (SPR) or localized surface plasmon resonance (LSPR) biosensors). For example, testing 130 may include a sensor similar to the “ArgusEye”, or Causeway “Titan” sensors and / or concepts described in Debnath et al 2023, Lab on a Chip. In some examples, testing may include the use of biosensor techniques based on detecting the presence of an analyte based on changes in motion of a functionalised particle such as the “Helia” sensor.

[0161] In some examples, testing 130 may include use of a modified versions of a handheld or tabletop device in combination with an assay configured to target the protein of interest, such as human IgG. The assay may include: electrode biosensors such as produced by Omini Lab® or similar; a light polarization-based assay, such as that produced by Linear Diagnostics or similar; a microfluidic immunofluorescence assay, such as that produced by LumiraDx™ or similar; an immunoassay analyzer, such as that produced by Genalyte Maverik™ or similar; or the “VeraSTAT-V” assay produced by Randox™ or similar. In some examples, the testing 130 may include use of a disposable, single “point-of-care” method. For example, the testing 130 may include use of a lateral flow assay (LFA). The lateral flow assay may be configured to target the protein of interest, for example human IgG. For example, the testing 130 may include use of a lateral flow assay such as the Human IgG Fc Dipstick assay produced by Cytodiagnostics Inc. and / or the “PROTIA” Human IgG LFA.

[0162] In some examples, the testing 130 may include use of a paper-based microfluidic device, such as described in Unal et al 2021 or similar, or a microfluidicbased assay such as that produced by Sensoreal Inc., or similar. In some examples, the testing 130 may include use of a high-throughput, batch-based assays. In some examples, the testing 130 may include use of an immunonephelometry assay. For example, the testing 130 may include use of the Siemens™ BNII IgG system, orsimilar. In some examples, the testing 130 may include use of an immunoturbidimetry assay. For example, the testing 130 may include use of the Dialab™ IgG immunoturbidimetric assay, or the Human IgG assay developed by Roche™ for Cedex® Bio & Bio HT Analyzers. In some examples, the testing 130 may include use of an enzyme linked immunosorbent assay (ELISA). For example, the testing 130 may include use of the Bio-Plex Pro Human IgG total isotyping assay produced by Bio-Rad Laboratories, or the Valita™ Titer Assay produced by Beckman Coulter Life Sciences.

[0163] In some examples, the testing 130 of the sample of blood, serum or plasma includes testing using a lateral flow assay (LFA). The LFA may include a conjugate configured to label the protein of interest in the blood, serum or plasma sample. The label may produce a measurable signal in response to the binding. For example, the label may comprise a visualization aid. In some examples, the visualization aid may comprise gold nanoparticles. In some examples, the label may comprise a fluorescent tag. However, other suitable labels may be substituted. The protein of interest (such as human IgG) may be targeted by a binding partner covalently linked to the label (e.g. the visualisation aid).

[0164] The LFA may comprise a control line and at least one test line. In some examples, the LFA may be configured to indicate a concentration of the protein of interest in the sample of blood, serum or plasma from the donor. In some examples, the LFA may be configured to indicate a concentration of the protein of interest in the plasma unit. The LFA may be configured to provide a quantitative, or semi-quantitative, determination of the concentration of the protein of interest in the sample of blood, serum or plasma. For example, the LFA may be configured to indicate an absolute value, or a value range for the protein concentration in the sample and / or in the collected plasma unit. In some examples, the LFA may be configured to provide a qualitative determination of the concentration of the protein of interest in the sample of blood, serum or plasma. For example, the LFA may be configured to categorize the sample (and thus the collected plasma unit) between two or more categories. For example, the LFA may be configured to indicate whether the sample (and the collected plasma unit to which the sample relates) belongs to a “high protein-of-interesf ’ groupor a “low protein-of-interest” group, based on the detected protein concentration in the sample.

[0165] A binding ligand specific to the protein of interest may be fixed at the at least one test line. The amount of the binding ligand fixed at the test line may be configured such that the test line is activated when the of protein of interest concentration of the blood, serum or plasma sample is equal to and / or above a predetermined concentration level. For example, the amount of the binding ligand fixed at the test line may be configured to sequester substantially all of the labelled protein of interest from the sample to activate the test line when the concentration of protein of interest concentration of the blood, serum or plasma sample is equal to the predetermined concentration level. The predetermined concentration level may be correlated with a protein of interest concentration in the collected plasma unit. For example, the predetermined concentration level may be indicative of a protein of interest concentration in the collected plasma unit equal to the threshold concentration level.

[0166] In some examples, the LFA may include a single test line. For example, the LFA dipsticks shown in Figure 6 include a control line and a single test line. The single test line may be configured such that activation of the single test line may be indicative of a predetermined concentration level. In such examples, the predetermined concentration level may be correlated with (and / or indicative of) the threshold concentration level. For example, the amount of binding ligand fixed at the single test line may be configured such the single test line is activated when the protein of interest concentration in the sample is indicative of the threshold concentration level of protein of interest concentration in a collected plasma unit.

[0167] An intensity of the activated test line may be indicative of the concentration of protein of interest in the blood, serum or plasma sample. In some examples, the intensity of the test line signal may increase with an increase in concentration of the protein of interest. In some examples, the intensity of one or more test lines may decrease with an increase in concentration of the protein of interest. For example, in theLFA illustrated in Figure 6, the intensity of the test line signal increases with increasing concentration of IgG in the sample.

[0168] In some examples, the LFA may include a control line and a plurality of test lines. In some such examples, the amount of binding ligand fixed at each of the plurality of test lines may be configured such that activation of one or more of the plurality of test lines is indicative of one of a corresponding plurality of discrete predetermined concentration levels of the protein of interest in the collected plasma. Each of the test lines may be indicative of a respective predetermined concentration level of one of a plurality of different proteins in the collected plasma.

[0169] An intensity of one or more of the activated test lines may be indicative of the concentration of protein of interest in the blood, serum or plasma sample. In some examples, the intensity of one or more signals from the test lines may increase with an increase in concentration of the protein of interest. In some examples, the intensity of one or more test lines may decrease with an increase in concentration of the protein of interest. For example, in the LFA illustrated in Figure 11, the LFA includes a control line C, a first test line T1 and a second test line T2. In this example, the intensity of the first test line T1 at first increases with increasing concentration of IgG in the sample (as indicated by the bar below the image), before decreasing, while the intensity of the second test line T2 continuously decreases with increasing concentration of IgG in the sample.

[0170] In some examples, a plurality of test lines may be configured to be activated to indicate a concentration level of the protein of interest, in combination with each other. For example, the protein of interest concentration may be determined based on an intensity of the signal of two or more test lines, when read together.

[0171] In some examples, the LFA may include a first test line having a ligand that is configured to bind to the protein of interest in the sample. The LFA may include a second test line (which could alternatively be understood as a variable control line),having a second ligand that is configured to bind to conjugate that has not bound to the protein of interest in the sample.

[0172] In some examples, the testing 130 may include reading the test result after testing the sample. In some examples, reading the test result may comprise performing a visual inspection of the LFA after testing the sample. In some examples, reading the LFA test result may be computer-assisted. In some examples, the testing 130 may include capturing an image of the LFA after testing the sample. Image analysis software may be used in reading the test result of the LFA. In some examples, a test apparatus or test kit may include the LFA and a reader in combination with analysis software. The reader may be portable and / or wireless.Experimental examples

[0173] Examples of determination of a protein of interest concentration in collected plasma according to the present disclosure are described below. In the following experiments, a commercially available LFA were used to demonstrate that the method according to the present disclosure is capable of separating plasma based on IgG concentration.Example 1

[0174] This experiment was performed to establish a test method, image capture signal analysis, and signal intensity cut-off for detection of IgG in samples using an off-the-shelf LFA test. In this example, a set of 15 samples with varying IgG concentrations were prepared as set out in Table 1 below. The samples were prepared using volumes of Cryo-rich plasma (CRP), IgG depleted plasma (IDP), phosphate buffered saline (PBS) and / or Privigen®, which were pre-assessed using a Siemens™ BNII IgG nephelometric analyser (Neph) to determine their IgG concentration.Table 1 - Example 1 - Sample preparation

[0175] Following preparation, the plasma samples were tested using a Siemens™ BNII IgG nephelometric analyser (Neph) to confirm the IgG concentration of the samples, with results as indicated in Table 2, below. Samples 1-15 were allocated a dilution identifier DI -DI 5, respectively.Table 2 - Example 1 - Nephelometric analysis<<<

[0176] The samples were then diluted with a sample dilution buffer at a ratio of 1 : 1000. Following dilution, 100 pL of LFA buffer was added to individual wells of a microtiter plate, and 50 pL of the diluted samples added to a respective well. The prepared samples were then tested using LFA dipsticks (CytoDiagnostics Human IgG Fc LFA dipstick kit). The LFA dipsticks were incubated in the wells with the arrows pointing down for 20 minutes at room temperature. The LFA dipsticks were then removed from the wells and images of the dipsticks were captured with a Bio-Rad ChemiDoc imaging system and / or by placing the dipsticks in a plastic cover and scanning at high resolution in colour. An image of the dipsticks captured using the BioRad ChemiDoc imaging system is shown in Figure 6.

[0177] The captured image of the LFA dipsticks was analysed using BioRad ImageLab software. A “lane and band” feature in the image analysis software was used to identify the control line and the test line bands on each dipstick. A similarly sized band in the background region (i.e. between the control line and the test line band was used to determine the background signal.

[0178] The intensity of each test line and control line band was analysed, by determining a “signal strength” for each band (based on the intensity / darkness of the band) measured in AU and subtracting the signal from the background band. The test line band signals were adjusted by 1,000,000 AU.

[0179] Figure 7 shows the detected test line signal strength (in AU), plotted against the nephelometer measured IgG concentration. Expected IgG concentration was used where the measured IgG concentration was below the limit of detection of the nephelometric analyser (samples D1-D3). In this example, a threshold signal strength was set at 75 AU, as indicated by the dotted line in Figure 7, to differentiate between samples which were considered to have activated the test line versus those that did not.

[0180] The samples DI -DI 5 were then grouped into those that fell above and below the threshold signal strength and graphed in a column graph using Prism GraphPad as indicated in Figure 8. The difference between the groups was found to be statistically significant, as assessed using a Mann-Whitney test (t-test, nonparametric).Example 2

[0181] In this example, a set of 24 samples with randomly generated IgG concentrations above and below a proposed IgG limit range. The samples were prepared using CRP, IDP and / or Privigen® samples, which were pre-assessed using a Siemens™ BNII IgG nephelometric analyser (Neph) to determine their IgG concentration. One control sample (sample 25) was prepared using IDP only. The samples were prepared as set out in Table 3 below.Table 3 - Example 2 - Sample preparation

[0182] Following preparation, the samples were tested using a Siemens™ BNII IgG nephelometric analyser (Neph) to confirm the IgG concentration of each sample, with results as indicated in Table 4, below. Samples 1-24 were allocated a dilution identifier D1-D24, respectively.Table 4 Example 2 - Nephelometric analysis< <

[0183] The samples were then diluted with a sample dilution buffer. In this example, the dilution was performed at a ratio of 1 :6000 (as opposed to 1 : 1000) such that activation of the test line of the LFA would indicate an IgG concentration in the undiluted sample of 3g / L. Following dilution, 100 pL of LFA buffer was added to individual wells of a non-treated microtiter plate, and 50 pL of the diluted samples added to respective wells. The prepared samples were then tested using LFA dipsticks (CytoDiagnostics Human IgG Fc LFA dipstick kit). The LFA dipsticks were incubated in the wells with the arrows pointing down for 20 minutes at room temperature. The LFA dipsticks were then removed from the wells and images of the dipsticks were captured with a Bio-Rad ChemiDoc imaging system and / or by placing the dipsticks in a plastic cover and scanning at high resolution in colour. An image of the dipsticks captured using the Bio-Rad ChemiDoc imaging system is shown in Figure 9.

[0184] The captured image of the LFA dipsticks was analysed using BioRad ImageLab software, as described above for Experiment 1. A cut-off of 75 AU was used (based on the results from Example 1) to separate the samples into two categories, based on the detected intensity of the test line band.

[0185] In this example, a 2x2 table analysis was employed, comparing the result from the normalized 75 AU cut-off (LFA) on one side to the nephelometric analyser (Neph) on the other side, as shown in Table 5 below.Table 5 - Results analysis

[0186] Using the image capture and signal analysis method and 75 AU cut off established in Example 1, and assuming the IgG concentration as determined by the nephelometric analyser as the ground truth, the LFA test method categorised 24 / 25 samples correctly. One sample had a concentration of exactly 3g / L and was miscategorised using the LFA test method. It was demonstrated that the LFA test method had a sensitivity of 100% (8 out of 8 samples under 3g / L IgG correctly determined) and a specificity of 93.8% (15 out of 16 samples at or over 3g / L IgG correctly determined). The LFA test method demonstrated a positive predictive value (PPV) of 89% and a negative predictive value (NPV) of 100%.Example 3

[0187] This experiment was performed to assess the accuracy of the Protia ImmuneCheck Human IgG LFA test kit, using plasma samples at various concentrations. In this example, 40 samples were prepared, as set out in Table 6, below. The samples each had an IgG concentration prepared to a value between 2 g / L and 13 g / L, where the values were generated using a random number generator. The 40 samples were tested twice by two operators, for a total of 160 test results.Table 6 - Example 3 - Sample preparation

[0188] In addition to the 40 samples, listed above, 10 training samples were prepared with IgG concentrations within a range from 0 g / L to 20 g / L. The training sampleswere given to the test operators with IgG concentrations indicated so that they could compare the results from samples with a known IgG concentration to the concentration reference chart provided in the Protia ImmuneCheck IgG test kit.

[0189] Following preparation, the samples were tested using a Siemens™ BNII IgG nephelometric analyser (Neph) to confirm the IgG concentration of each sample. The nephelometric analysis results are plotted against the expected IgG g / L in Figure 10. The line x=y is also plotted in Figure 10 as a visualisation aid.

[0190] Following training on the Protia ImmuneCheck IgG LFA using the training samples, the operators tested the 40 test samples and determined an IgG concentration for each sample (to the closest concentration value listed on the reference card) based on visual inspection of the LFA test result.

[0191] An example reference chart from a Protia ImmuneCheck IgG LFA test kit is shown in Figure 11. The Protia ImmuneCheck IgG LFA test includes a control line (C), a competitive quantitative line (T2) and a Sandwich inspection line (Tl). A reference bar indicating concentration of the analyte (IgG), relative to the test result is shown below the test lines. When no analyte (IgG) is present, the control line (C) and the competitive quantitative line (T2) are activated, while the sandwich inspection line remains inactivated). As the concentration of IgG in the sample increases, the intensity of the competitive quantitative line (T2) decreases, while the intensity of the sandwich inspection line (Tl) increases. When there is an excess of analytic material (e.g. as shown in the test strips on the right), the intensity of the sandwich inspection line (Tl) decreases. As such, LFA tests such as the Protia ImmuneCheck IgG LFA test (or similar) allow an operator to make an estimate of the IgG concentration in a sample.

[0192] A frequency distribution showing the results of the testing is shown in Figure 12. Operator determined results were then plotted against the nephelometric analysis results, as shown in Figure 13. The line x=y is also plotted in Figure 13 as a visualisation aid.

[0193] The results demonstrated that a visual inspection of the Protia ImmuneCheck IgG LFA was able to estimate IgG to approximately + / - 2 g / L of the nephelometric analysis result. The visual inspection of IgG concentration by the operators had an overall accuracy of 40%, where accuracy was determined as a correct visual IgG inspection when compared to the closest IgG concentration value listed on the reference card for the sample.

[0194] When the same results were used to separate samples into two groups based on a threshold concentration “cut-off’ to separate samples into “high IgG” versus “low IgG” samples, visual inspection of the Protia ImmuneCheck IgG LFA test was able to correctly separate approximately 99.19% of samples for >2.5 g / L IgG, 94.12% of samples for >5 g / L IgG, 81.32% of samples for > 7.5 g / L IgG, and 74.11% of samples for >10 g / L IgG (based on the negative predictive value, NPV).Example 4

[0195] A significant portion of error may be attributable to variance in human interpretation of test and control line signals (i.e. inter and intra-operator variability). Accuracy may be improved by introduction of computer-assisted analysis of the test result, such the image capture and signal analysis method as described in relation to example 1 and example 2 above.

[0196] Some examples may include a reader in combination with analysis software to provide a quantitative assessment of IgG concentration in the sample, based on an analysis of the LFA test result.

[0197] In one example, accuracy of categorization of plasma concentration based on visual inspection of a test result was compared to accuracy based on analysis of the test result using an automated reader. The results of this comparison are shown below in Table 7. The automated reader provided a Diversion Accuracy for a cutoff of 7 g / L of 87.8%Table 7 - Example 4 - Comparison of analysis methodsClauses

[0198] The following statements describe certain examples according to the present disclosure.1 A. A method for directing collected plasma in a protein purification process, the method including:(a) testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor; (b) directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest; wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first protein purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway.2 A. The method of example 1 A, further including processing the collected plasma unit via the first or second purification processing pathway, to produce isolated protein of interest from the collected plasma unit.3 A. The method of example 1 A or example 2A, including pooling the collected plasma units from the donor with plasma units collected from other donors, wherein collected plasma units determined as having a concentration level of the protein of interest greater than the threshold concentration level are pooled separately from collectedplasma units determined as having a concentration level of the protein of interest less than the threshold concentration level.4A. The method of any one of the preceding examples, wherein the plasma unit is collected by a plasmapheresis procedure.5 A. The method of any one of the preceding examples, wherein the blood, serum or plasma sample is obtained prior to collection of the plasma unit.6A. The method of example 5A, wherein the blood sample is obtained by a fingerprick test.7A. The method of example 4A, wherein the blood, serum or plasma sample is obtained during the plasmapheresis procedure.8A. The method of example 4A, wherein the plasma sample is obtained from the collected plasma unit after plasmapheresis and prior to any pooling of the collected plasma unit with plasma units collected from other donors.9A. The method of any one of the preceding examples, wherein the first purification processing pathway achieves a higher yield of the protein of interest than the second purification processing pathway.10 A. The method of any one of the preceding examples, wherein the first and / or second purification processing pathway includes a process configured to isolate the protein of interest and a process configured to isolate one or more different proteins of interest.11 A. The method of any one of the preceding examples, wherein the first purification processing pathway includes a process configured to isolate the protein of interest and wherein the second purification processing pathway includes a process configured to isolate one or more different proteins of interest.12 A. The method of any one of the preceding examples, wherein the first and / or second purification processing pathway includes a non-alcohol based and / or an alcohol-based purification process.13 A. The method of any one of the preceding examples, wherein the first and / or second purification processing pathway includes an affinity -based chromatography process and / or a precipitation process.14A. The method of example 13A, wherein the affinity-based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG.15 A. A method for directing collected plasma in a protein purification process, the method including:testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor;directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest;wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first protein purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway,wherein the first protein purification processing pathway includes an affinitybased chromatography process, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.16A. The method of example 14A or example 15A, wherein the ligand is capable of specifically binding to a CH3 domain of human IgG.17A. The method of example 16A, wherein the ligand comprises a camelid-derived single domain [VHH] antibody fragment.18 A. The method of any one of the preceding examples, wherein testing the sample of blood, serum or plasma includes testing using a lateral flow assay.19A. The method of example 18 A, wherein the lateral flow assay includes a conjugate configured to label the protein of interest in the blood, serum or plasma sample.20 A. The method of example 18A or example 19 A, wherein the lateral flow assay comprises a control line and at least one test line, wherein a binding ligand specific to the protein of interest is fixed at the at least one test line.21 A. The method of example 20A, wherein the lateral flow assay is configured such that the test line is activated when the protein of interest concentration of the blood, serum or plasma sample is equal to and / or above a predetermined concentration level.22A. The method of example 21 A, wherein an intensity of the activated test line is indicative of the concentration of the protein of interest in the blood, serum or plasma sample.23 A. The method of example 20A, wherein the at least one test line comprises a single test line, wherein the predetermined concentration level in the sample is correlated with the threshold concentration level in the collected plasma unit.24 A. The method of example 20 A or example 21 A, wherein the lateral flow assay includes a control line and a plurality of test lines.25 A. The method of example 24A, wherein activation of one or more of the test lines is indicative of one of a corresponding one or more discrete predetermined concentration levels of the protein of interest in the collected plasma unit.26A. The method of example 24A or example 25 A, wherein an intensity of one or more of the test lines is indicative of a concentration level of the protein of interest in the collected plasma unit.27A. The method of any one of examples 24A to 26A, wherein the intensity of two or more test lines, when read together, is indicative of a concentration level of the protein of interest in the collected plasma unit.28A. The method of example 24A, wherein activation and / or intensity of one or more of the test lines is indicative of a respective concentration level of one or more different proteins in the collected plasma unit.29A. The method of any one of the preceding examples, wherein the threshold concentration level is determined at least partially based on a standard concentration distribution curve in plasma for the protein of interest.30A. The method of example 29A, wherein the threshold concentration level is determined relative to a median value of the standard concentration distribution curve for the protein of interest in plasma, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% above the median value.31 A. The method of any one of the preceding examples, wherein the threshold concentration level is in a range of between approximately 2g / L and lOg / L.32A. The method of example 31 A, wherein the threshold concentration level is selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, ,5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, or lO.Og / L.33A. The method of example 32A, wherein the threshold value of the protein of interest concentration in plasma is about 3.0g / L.34 A. The method of any one of the preceding examples, wherein the protein of interest is an immunoglobulin.35 A. The method of example 34A, wherein the immunoglobulin is IgG.36 A. A method of separating collected plasma for a protein purification process, the method including:(a) testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in collected plasma unit from each donor;(b) separating the collected plasma units based on the respective determined concentration level; and(c) directing the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level.37 A. The method of example 36 A, wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a second, different purification processing pathway.38 A. A method of separating collected plasma for a protein purification process, the method including:testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in collected plasma unit from each donor;separating the collected plasma units based on the respective determinedconcentration level; anddirecting the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level,wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a second, different purification processing pathway, wherein the first protein purification processing pathway includes an affinitybased chromatography process, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.39 A. The method of example 37A or example 38 A, wherein collected plasma units determined as having a concentration level of the protein of interest greater than the threshold concentration level are pooled prior to direction to the first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level is pooled separately prior to direction to the second purification processing pathway.40 A. The method of any one of examples 36A to 39 A, further including collecting the plasma units from the plurality of donors.41 A. The method of any one of examples 36A to 40 A, further including processing the separated collected plasma units to produce isolated protein of interest from the collected plasma unit.42A. A test kit for determining a concentration level of a protein of interest in a plasma unit collected from a donor, the test kit comprising:a lateral flow assay configured to test a sample of blood, serum or plasma from the donor; wherein the lateral flow assay includes:a conjugate configured to label the protein of interest in the blood, serum or plasma sample;a control line; andat least one test line, wherein a binding ligand specific to the protein of interest is fixed at the at least one test line; andan automated reader configured to detect activation and / or intensity of the control line and the at least one test line to determine a concentration of the protein of interest in the blood, serum or plasma sample.43 A. The test kit of example 42A, wherein the automated reader is configured to correlate the determined concentration of the protein of interest in the blood, serum or plasma sample with a concentration of the protein of interest in a plasma unit collected from the donor.44A. The test kit of example 42A or example 43 A, wherein the automated reader is configured to determine whether the collected plasma unit has a concentration level of the protein of interest greater than a threshold concentration level or less than the threshold concentration level.45A. The test kit of example 42A, wherein the lateral flow assay is configured to activate at least one test line when the concentration of the protein of interest in the blood, serum or plasma sample is equal to a predetermined concentration level.46A. The test kit of example 45A, wherein the predetermined concentration level is correlated with a threshold concentration level of the protein of interest in the plasma unit collected from the donor.47A. The test kit of example 46A, wherein the threshold concentration level is determined at least partially based on a standard concentration distribution curve in plasma for the protein of interest.48A. The test kit of example 47A, wherein the threshold concentration level is determined relative to a median value of the standard concentration distribution curve for the protein of interest in plasma, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% above the median value.49A. The test kit of any one of examples 46A to 48A, wherein the threshold concentration level is in a range of between approximately 2g / L and lOg / L.50A. The test kit of example 49A, wherein the threshold concentration level is selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, ,5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, or lO.Og / L.51 A. The test kit of example 50A, wherein the threshold value of the protein of interest concentration in plasma is 3.0g / L.52A. The test kit of any one of examples 42A to 51 A, wherein the lateral flow assay includes a control line and a plurality of test lines.53 A. The test kit of example 52A, wherein activation of one or more of the test lines is indicative of one of a corresponding plurality of discrete predetermined concentration levels of the protein of interest in the collected plasma unit.54A. The test kit of example 52A, wherein activation and / or intensity of one or more of the test lines is indicative of a respective concentration level of one or more different proteins in the collected plasma unit.55 A. The test kit of example 52 A, wherein the intensity of two or more test lines, when read together, is indicative of a concentration level of the protein of interest in the collected plasma unit.56A. The test kit of any one of examples 42A to 55A, wherein the protein of interest is an immunoglobulin.57A. The test kit of example 56A, wherein the immunoglobulin is IgG.

[0199] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method for directing collected plasma in a protein purification process, the method including:testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor;directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest;wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first protein purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway.

2. The method of claim 1, further including processing the collected plasma unit via the first or second purification processing pathway, to produce isolated protein of interest from the collected plasma unit.

3. The method of claim 1, including pooling the collected plasma units from the donor with plasma units collected from other donors, wherein collected plasma units determined as having a concentration level of the protein of interest greater than the threshold concentration level are pooled separately from collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level.

4. The method of claim 1, wherein the plasma unit is collected by a plasmapheresis procedure.

5. The method of claim 1, wherein the blood, serum or plasma sample is obtained prior to collection of the plasma unit.

6. The method of claim 5, wherein the blood sample is obtained by a finger-prick test.

7. The method of claim 4, wherein the blood, serum or plasma sample is obtained during the plasmapheresis procedure.

8. The method of claim 4, wherein the plasma sample is obtained from the collected plasma unit after plasmapheresis and prior to any pooling of the collected plasma unit with plasma units collected from other donors.

9. The method of claim 1, wherein the first purification processing pathway achieves a higher yield of the protein of interest than the second purification processing pathway.

10. The method of claim 1, wherein the first and / or second purification processing pathway includes a process configured to isolate the protein of interest and a process configured to isolate one or more different proteins of interest.

11. The method of claim 1, wherein the first purification processing pathway includes a process configured to isolate the protein of interest and wherein the second purification processing pathway includes a process configured to isolate one or more different proteins of interest.

12. The method of claim 1, wherein the first and / or second purification processing pathway includes a non alcohol based and / or an alcohol based purification process.

13. The method of claim 1, wherein the first and / or second purification processing pathway includes an affinity-based chromatography process.

14. The method of claim 13, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG.

15. A method for directing collected plasma in a protein purification process, the method including:testing a sample of blood, serum or plasma obtained from a donor to determine a concentration level of a protein of interest in a plasma unit collected from the donor;directing the collected plasma unit to a selected protein purification processing pathway based on the determined concentration level of the protein of interest;wherein collected plasma units determined as having a concentration level ofthe protein of interest greater than a threshold concentration level are directed to a first protein purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a different, second protein purification processing pathway,wherein the first protein purification processing pathway includes an affinitybased chromatography process, wherein the affinity based chromatography process includes an affinity-based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.

16. The method of claim 15, wherein the ligand is capable of specifically binding to a CH3 domain of human IgG.

17. The method of claim 16, wherein the ligand comprises a camelid-derived single domain [VHH] antibody fragment.

18. The method of claim 1, wherein testing the sample of blood, serum or plasma includes testing using a lateral flow assay.

19. The method of claim 18, wherein the lateral flow assay includes a conjugate configured to label the protein of interest in the blood, serum or plasma sample.

20. The method of claim 18, wherein the lateral flow assay comprises a control line and at least one test line, wherein a binding ligand specific to the protein of interest is fixed at the at least one test line.

21. The method of claim 20, wherein the lateral flow assay is configured such that the test line is activated when the protein of interest concentration of the blood, serum or plasma sample is equal to and / or above a predetermined concentration level.

22. The method of claim 21, wherein an intensity of the activated test line is indicative of the concentration of the protein of interest in the blood, serum or plasma sample.

23. The method of claim 20, wherein the at least one test line comprises a single test line, wherein the predetermined concentration level in the sample is correlated with the threshold concentration level in the collected plasma unit.

24. The method of claim 20, wherein the lateral flow assay includes a control line and a plurality of test lines.

25. The method of claim 24, wherein activation of one or more of the test lines is indicative of one of a corresponding one or more discrete predetermined concentration levels of the protein of interest in the collected plasma unit.

26. The method of claim 24, wherein an intensity of one or more of the test lines is indicative of a concentration level of the protein of interest in the collected plasma unit.

27. The method of claim 24, wherein the intensity of two or more test lines, when read together, is indicative of a concentration level of the protein of interest in the collected plasma unit.

28. The method of claim 24, wherein activation and / or intensity of one or more of the test lines is indicative of a respective concentration level of one or more different proteins in the collected plasma unit.

29. The method of claim 1, wherein the threshold concentration level is determined at least partially based on a standard concentration distribution curve in plasma for the protein of interest.

30. The method of claim 29, wherein the threshold concentration level is determined relative to a median value of the standard concentration distribution curve for the protein of interest in plasma, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% above the median value.

31. The method of claim 1, wherein the threshold concentration level is in a range of between approximately 2g / L and lOg / L.

32. The method of claim 30, wherein the threshold concentration level is selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, 7.5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, or lO.Og / L.

33. The method of claim 32, wherein the threshold value of the protein of interest concentration in plasma is about 3.0g / L.

34. The method of claim 1, wherein the protein of interest is an immunoglobulin.

35. The method of claim 34, wherein the immunoglobulin is IgG.

36. A method of separating collected plasma for a protein purification process, the method including:testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in a collected plasma unit from each donor;separating the collected plasma units based on the respective determined concentration level; anddirecting the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level.

37. The method of claim 36, wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a second, different purification processing pathway.

38. A method of separating collected plasma for a protein purification process, the method including:testing a sample of blood, serum or plasma from each of a plurality of donors to determine a concentration level of the protein of interest in a collected plasma unit from each donor;separating the collected plasma units based on the respective determined concentration level; anddirecting the separated plasma units to selected, separate processing pathways based on the determined concentration level of the protein of interest being either above or below a threshold concentration level,wherein collected plasma units determined as having a concentration level of the protein of interest greater than a threshold concentration level are directed to a first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are directed to a second, different purification processing pathway, wherein the first protein purification processing pathway includes an affinity -based chromatography process, wherein the affinity based chromatography process includes an affinity -based chromatography medium, wherein the medium comprises a ligand capable of binding to IgG; andwherein the second protein purification processing pathway includes a precipitation process.

39. The method of claim 37, wherein collected plasma units determined as having a concentration level of the protein of interest greater than the threshold concentration level are pooled prior to direction to the first purification processing pathway and wherein collected plasma units determined as having a concentration level of the protein of interest less than the threshold concentration level are pooled separately prior to direction to the second purification processing pathway.

40. The method of claim 36, further including collecting the plasma units from the plurality of donors.

41. The method of claim 36, further including processing the separated collected plasma units to produce isolated protein of interest from the collected plasma unit.

42. A test kit for determining a concentration level of a protein of interest in a plasma unit collected from a donor, the test kit comprising:a lateral flow assay configured to test a sample of blood, serum or plasma from the donor; wherein the lateral flow assay includes:a conjugate configured to label the protein of interest in the blood, serum or plasma sample;a control line; andat least one test line, wherein a binding ligand specific to the protein of interest is fixed at the at least one test line; andan automated reader configured to detect activation and / or intensity of the control line and the at least one test line to determine a concentration of the protein of interest in the blood, serum or plasma sample.

43. The test kit of claim 42, wherein the automated reader is configured to correlate the determined concentration of the protein of interest in the blood, serum or plasma sample with a concentration of the protein of interest in a plasma unit collected from the donor.

44. The test kit of claim 42, wherein the automated reader is configured to determine whether the collected plasma unit has a concentration level of the protein of interest greater than a threshold concentration level or less than the threshold concentration level.

45. The test kit of claim 42, wherein the lateral flow assay is configured to activate at least one test line when the concentration of the protein of interest in the blood, serum or plasma sample is equal to a predetermined concentration level.

46. The test kit of claim 45 wherein the predetermined concentration level is correlated with a threshold concentration level of the protein of interest in the plasma unit collected from the donor.

47. The test kit of claim 46, wherein the threshold concentration level is determined at least partially based on a standard concentration distribution curve in plasma for the protein of interest.

48. The test kit of claim 47, wherein the threshold concentration level is determined relative to a median value of the standard concentration distribution curve for the protein of interest in plasma, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% above the median value.

49. The test kit of claim 46, wherein the threshold concentration level is in a range of between approximately 2g / L and lOg / L.

50. The test kit of claim 49, wherein the threshold concentration level is selected from 2g / L, 2.5g / L, 3.0g / L, 3.5g / L, 4.0g / L, 4.5 g / L, 5.0g / L, 5.5g / L, 6.0g / L, 6.5g / L, 7.0g / L, ,5g / L, 8.0g / L, 8.5g / L, 9.0g / L, 9.5g / L, or lO.Og / L.

51. The test kit of claim 50, wherein the threshold value of the protein of interest concentration in plasma is 3.0g / L.

52. The test kit of claim 42, wherein the lateral flow assay includes a control line and a plurality of test lines.

53. The test kit of claim 52, wherein activation of one or more of the test lines is indicative of one of a corresponding plurality of discrete predetermined concentration levels of the protein of interest in the collected plasma unit.

54. The test kit of claim 52, wherein activation and / or intensity of one or more of the test lines is indicative of a respective concentration level of one or more different proteins in the collected plasma unit.

55. The test kit of claim 52, wherein the intensity of two or more test lines, when read together, is indicative of a concentration level of the protein of interest in the collected plasma unit.

56. The test kit of claim 42, wherein the protein of interest is an immunoglobulin.

57. The test kit of claim 56, wherein the immunoglobulin is IgG.

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