Methods of monitoring chromatographic purification of proteins

Abstract

The present invention relates broadly to methods for the isolation of proteins from plasma or fractions thereof using continuous chromatography and Raman spectroscopic analysis.

Description

[0001] METHODS OF MONITORING CHROMATOGRAPHIC PURIFICATION

[0002] RELATED APPLICATION

[0003] This application claims priority to European Patent Application No. 24206222.2 filed on 11 October 2024, the entire contents of which is hereby incorporated by reference.

[0004] TECHNICAL FIELD

[0005] The present invention relates broadly to methods for the isolation of proteins from plasma or fractions thereof using continuous chromatography and Raman spectroscopic analysis.

[0006] BACKGROUND

[0007] Human blood plasma contains over 100 proteins that cover a wide range of essential functions (e.g., defence against pathogens, blood coagulation or mass transport). Therapeutic proteins purified from human blood plasma play an important role in the treatment of life-threatening diseases. In order to obtain plasma proteins for therapeutic purposes, these have to be isolated from a variety of plasma donations. The collection of human blood plasma can be performed, for example, by plasmapheresis, where whole blood is extracted from the donor and separated by physical separation procedures. During this process, cellular components are returned to the donor while the plasma is collected in a reservoir.

[0008] Existing methods of purifying proteins such as IgG and albumin from collected plasma (and fractions thereof) include chromatography and non-chromatography purification methods. Major obstacles of existing methods are the high cost and time involved in purification of a desired protein, the requirement to co-purify other proteins from the same plasma or plasma fractions (e.g. albumin & coagulation factors) and the need to ensure that the product is of a suitable quality (e.g. purity and stability) for therapeutic use.

[0009] Process automation and Process Analytical Technology (PAT) is becoming increasingly important in the pharmaceutical industry. PAT enables real-time monitoring, providing high-value approaches to process understanding, control and optimization for efficient continuous manufacturing processes. The establishment of PAT in the human plasma protein purification industry is currently at an early stage but has the potential to offer substantial advantages in this area. PAT offers the potential for purification processes, including those for the isolation of proteins from human plasma, to be controlled and optimized in real-time, and in so doing, provide greater efficiency (including downstream efficiency), improved final product yield and quality, with reduced cost and waste.

[0010] New and / or improved methods for optimising purification processes for human plasma, such as continuous chromatographic processes are highly desirable. However, due to the heterogeneity of plasma-derived product solutions and suspensions, the quantification of key chemical components, such as quantification of different proteins, excipients and / or other components, is complex and to date, has only been achieved by use of off-line analytical methods that require sampling effort and analysis lead times of commonly several days. Therefore, there is an increasing need for new analytical processes to monitor and optimise purification processes for plasma protein purification in real time.

[0011] SUMMARY

[0012] The present inventors have undertaken research and development into methods of monitoring the chromatographic purification of compositions comprising protein, using Raman spectroscopic analysis. The present inventors have surprisingly found that Raman spectroscopic analysis can be used to monitor continuous chromatographic purification of compositions comprising protein (e.g. plasma), despite the complexity of such compositions arising from the presence of a variety of other components. They have found that such analysis can be used to determine operational changes in the chromatographic purification process.

[0013] The present disclosure provides for a method of isolating a protein from plasma, or a fraction thereof, using continuous chromatography, the method comprising:

[0014] (a) loading an amount of plasma, or fraction thereof, comprising the protein onto a chromatographic medium capable of separating the protein from at least a portion of the amount of plasma or fraction thereof;

[0015] (b) eluting the protein from the chromatographic medium; and

[0016] (c) collecting the protein; wherein, Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium and / or which is in the amount of plasma, or fraction thereof, being loaded onto the chromatographic medium. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 depicts exemplary Raman spectra of training data from Example 1 obtained: (A) after preprocessing (Flow Cell); (B) after preprocessing (BioOptics), and (C) comparison of (A) and (B).

[0018] Figure 2 depicts predicted vs true IgG content for the validation of Model No. 2 in Example 1 for (A) Run 1; (B) Run 2; and (C) Run 3.

[0019] Figure 3 depicts predicted vs true IgG content for the BioSMB feasibility study for Model No. 2 in Example 1.

[0020] Figure 4 depicts predicted vs true IgG content for the exposure time study conducted in Example 1 : (A) 5 s exposure time; (B) 2 s exposure time.

[0021] Figure 5 depicts the true IgG and total protein detected in flowthrough for the 4CV feasibility study of Run 2 described in Example 2.

[0022] Figure 6 depicts predicted vs true IgG content for the 4CV feasibility evaluation of the PLS model in Example 2: (A) Run 1; (B) Run 2.

[0023] Figure 7 depicts the true IgG and total protein detected in flowthrough for the 12CV feasibility study of Run 1 described in Example 2.

[0024] Figure 8 depicts predicted vs true IgG content for the 12CV feasibility evaluation of the PLS model in Example 2: (A) Run 1; (B) Run 2.

[0025] Figure 9 depicts predicted vs true IgG content for Run 2 of the 12CV feasibility evaluation in Example 2, with optimised PLS model, where the model was (A) calibrated with spectra data from 12CV Run 2; (B)-(C) validation against 12CV Run 1 data.

[0026] Figure 10 depicts Raman spectra in Example 2 from mixtures of CPP and FT together with the spectra of CPP spiked with IgG (A) coloured according to IgG value; and (B) coloured according to total protein value. Figure 11 depicts (A) calibration of the PLS model of Example 2 based on total protein at line data from 12CV Run 2, and (B)-(C) validation using 12CV Run 1 data thereof.

[0027] Figure 12 depicts predicted vs true IgG content for PLS Models No. (A) 1; (B) 2; (C) 3; and (D) 4 of Example 3.

[0028] Figure 13 (A) tabulates and (B) depicts the continuous chromatography method for the feasibility study of Example 1.

[0029] DETAILED DESCRIPTION

[0030] The present disclosure describes the following various non-limiting embodiments, which relate to investigations undertaken to develop methods for purifying proteins from plasma that comprise monitoring by Raman spectroscopy.

[0031] General terms

[0032] In the following description, reference is made to the accompanying drawings which form a part hereof, and which is shown, by way of illustration, several embodiments. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present disclosure.

[0033] With regard to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0034] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0035] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0036] Throughout this disclosure, 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. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0037] Those skilled in the art will appreciate that the disclosure herein 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 examples, steps, features, methods, processes, compounds, and compositions, referred to or indicated in this specification, individually or collectively, and any and all combinations thereof.

[0038] 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.

[0039] Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item).

[0040] As used herein, the phrase “at least one of’, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination. As used herein, the term “about”, unless stated to the contrary, typically refers to a range of up to + / - 10% of the designated value, and includes smaller ranges therein, for example + / - 5% or + / - 1% of the designated value.

[0041] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0042] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, 4.75, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0043] 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.

[0044] Specific terms

[0045] The term “purify” or “purifying” or “purification” shall be taken to mean the removal, whether completely or partially, of at least one impurity present in the plasma, or fraction thereof, which thereby improves the level of purity of protein in solution.

[0046] The term “isolating” shall be taken to refer to any process which provides, from a composition comprising a component, that component in a form or composition with improved absolute purity, relative purity (i.e. purity with respect to a particular impurity or impurities), yield, and / or concentration. In some cases, “isolating” may comprise the removal or transfer, whether completely or partially, of a component (e.g. a protein) from a composition comprising that component. It will be understood that “isolating” does not necessarily entail the creation, obtaining, or collection of an absolutely pure form (i.e. free from all impurities), or a purer form, of the “isolated” component. A component that is isolated from a composition, whether completely or in part, need not be obtained or collected in a form or composition in which other components which may have been present in the initial composition are absent. This is to say, that a component may be removed or transferred from one composition, and ultimately obtained or collected as a part of the same or a different composition. An “isolated” component may also be obtained or collected in an enriched-state (i.e. higher concentration) as a part of a composition that comprises the same or substantially similar components to those of the composition from which the component was isolated from, or as a part of a different composition. Enrichment may involve, although need not entail, an increase in purity of the component. In some embodiments, isolating of a component (e.g protein) results in purification of the component (e.g. protein). This is to say, that in some embodiments, a method of isolating a protein is a method of purifying the protein. An increase in purity may be absolute (i.e. the component is of greater purity with respect to all components present in the relevant compositions before and after the purification process). An increase in purity may be relative (i.e. with an increase with respect to a particular impurity or impurities present in the relevant compositions before and after the purification process). Isolating may comprise an increase in both absolute and relative purity.

[0047] The term “impurity” or “impurities” shall be taken to include one or more components in the plasma, or fraction thereof, other than the protein that is desired to be isolated. For example, impurities may include plasma lipids, plasma proteins, proteases (e.g. serine proteases, kallikrein, plasmin and FXa), protease inhibitors (e.g. serine protease inhibitors, Cl esterase inhibitor, alpha- 1- antitrypsin, anti-thrombin, P- antithrombin, a-antithrombin, a-2-macroglobulin, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), a-1 esterase inhibitor proteins), transport proteins (e.g., transferrin, ceruloplasmin, haptoglobin, hemoglobulin and hemopexin), IgA and IgM, factor VIII, fibrinogen, von Willebrand factor, clotting factors and activated forms thereof (e.g. FX / FXa, FIX / FIXa, FVII / FVIIa and thrombin), cofactors and activated forms thereof (e.g., FV / Va, FVIII / VIIIa, FXIII / XIIIa), prothrombin, prothrombin complex, contact system factors (e.g. FXI / FXIa, FXII / FXIIa and plasma kallikrein), PKA, prothrombin complex factors and activated forms thereof (e.g. FXI / FXIa, FII / FIIa, FVII / FVIIa, FIX / FIXa, FX / FXa), proteins S, protein C, antithrombin III, a RhD immunoglobulin, protease inhibitors including, anti angiogenic proteins (e.g., latent-antithrombin), highly glycosylated proteins (e.g., a-l-acid glycoprotein, antichymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein and C- reactive protein), components of the complement pathway, inhibitors of any component of the complement pathway and other proteins (e.g., histidine-rich glycoprotein, mannan binding lectin, C4-binding protein, GC-globulin, plasminogen, blood factors such as erythropoietin, interferon, tumor factors, tPA, and yCSF) and platelet membrane microparticles. Albumin (a-globulins and / or P-globulins) or immunoglobulin G (e.g. IgG) may also be considered impurities, where they are not a protein that is desired to be isolated. Reference herein to a factor (e.g., Factor X or FX, includes reference to the zymogen (or inactivated) form as well as the activated form (e.g., Factor Xa or FXa) and vice versa.

[0048] 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.

[0049] The term “plasma” shall refer to the straw-coloured / 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.

[0050] 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 cryosupernatant (also known as cryo-poor plasma). For example, plasma may be fractionated by precipitation, such as ethanol, ammonium sulphate or octanoic acid fractionation. Ethanol precipitation may be used to produce IgG-containing Oncley fractions, Cohn fractions, 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 (I+)II+III 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 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, for example, ion exchange chromatography, hydrophobic interaction chromatography, or isoagglutinin affinity chromatography.

[0051] The term “cryo-precipitate” or “cryo-precipitates” refers to proteins in plasma that precipitate out of solution when a unit of frozen plasma is slowly thawed in the cold. Cryo-precipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII and platelet membrane microparticles.

[0052] The term “cryo-poor plasma” (also known as cryosupernatant) shall be taken to mean plasma removed of cryo-precipitates.

[0053] The term “cryo-rich plasma” shall be taken to mean plasma comprising components typically found in cryo-precipitates.

[0054] The term “clarified” or “clarifying” shall be taken to mean a process of passing a plasma or fraction thereof through a centrifuge, separator, and / or suitable filter (e.g. depth filter, filter cake, and / or 1.2 and 0.45 / 0.22 pm membrane filter) to remove one or more impurities prior to use in a method described herein.

[0055] The term “dissociation constant” shall refer to the pKa of a buffer. pKa = - logio(Ka), wherein Ka is the acid dissociation constant of the buffering agent of the buffer. For example, a wash buffer of 20 mM sodium dihydrogen phosphate, 40 mM sodium chloride at a pH of 7.4 comprises sodium dihydrogen phosphate as the buffering agent. Phosphoric acid has three dissociation constants (pKal : 2.16, pKa2: 7.21, pKa3: 12.32).

[0056] The term “elution” (and related terms “eluted” or “eluting”) shall refer generally to the output from the chromatographic medium, irrespective of the cycle or phase of the chromatographic purification. It will be understood therefore, for example, that a sample “eluting from the chromatographic medium”, may include breakthrough during loading of the column, washings, equilibration waste, and the output from a protein elution step (i.e., where bound protein is released from the chromatographic medium by means of passing an elution buffer through the column).

[0057] The term “specifically binds”, “specifically binding” or “binds specifically” shall be taken to mean that a protein of the disclosure reacts or associates more frequently, 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 CH3 domain of human IgG 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. The term “ligand” shall be taken to mean a molecule immobilised to a matrix of the affinity chromatography resin which specifically binds to the protein of interest (e.g., the CH3 domain of human IgG). For example, the ligand is a camelid-derived single domain [VHH] antibody fragment.

[0058] The term “enriched preparation” shall be taken to include an eluate, solution or pharmaceutical composition described herein. For example, the enriched preparation of the present disclosure comprises IgG at greater purity compared to IgG in the plasma or fraction thereof (i.e., prior to a method of the disclosure).

[0059] The term “camelid-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, CaptureSelect IgG-CHl affinity resin, and CaptureSelect FcXP agarose affinity resin. 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).

[0060] The term “matrix” shall be taken to mean a support to which the ligand is immobilised. Exemplary matrices are cross-linked poly(styrene-divinylbenzene) matrix and agarose-based matrix.

[0061] The term “load” or “loading” as used herein, refers to the application of a sample (e.g., protein mixture) or solution (e.g., buffer) to the separation unit until the entire sample is applied to or contained within the unit.

[0062] The term “dynamic binding capacity” or “DBC” of a chromatography resin shall be taken to refer to the maximum amount of protein that the resin will bind under operating conditions before significant breakthrough of unbound protein (e.g., IgG) occurs.

[0063] As used herein, the term “washing” refers to the process of flowing a solution (e.g., a buffer) through the separation unit after loading so as to remove substances (e.g., proteins) not adsorbed or bound on the chromatography media, for example to remove substances or proteins from which the protein-of-interest is to be separated. Multiple washes are possible using different solutions.

[0064] The term “per mL of resin” shall be taken to refer to per mL of wet packed volume of resin. The term “bed height” shall be taken to mean the height at which the chromatography resin is packed into a column. It will be apparent to the skilled person that reference to “total bed height” refers to the bed height of all columns in the continuous chromatography set-up.

[0065] The term “non-loading phase” shall be taken to mean a phase other than the loading phase of the continuous chromatography method. For example, a non-loading phase can refer to an equilibration phase, a wash phase, an elution phase, a strip phase and / or a re-equilibration phase.

[0066] The term “cycle” shall be taken to mean one round of equilibrating, loading, binding, elution, stripping, sanitising, and / or regenerating performed on the resin.

[0067] The term “purity” shall refer to the portion of the protein of interest (e.g., IgG) relative to the total protein content expressed as a percentage.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] The term “plasma protein product” shall refer to a preparation, composition and / or protein product comprising a plasma protein (e.g. IgG or albumin) derived from the purification of the plasma or fraction thereof. Typically, the plasma protein is the predominant protein in the plasma protein product.

[0072] 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.

[0073] The term “pharmaceutical composition” shall be taken to mean a formulation of a plasma-derived protein with compounds generally accepted in the art for the delivery of proteins to mammals. Exemplary compounds include all pharmaceutically acceptable carriers, diluents or excipients thereof. The term “treat” or “treatment” or “treating” shall be taken to mean administering a therapeutically effective amount of IgG such that one or more symptoms or characteristics of the condition is reduced in the subject or that the subject is no longer clinically diagnosed with the condition.

[0074] The term “preventing”, “prevent” or “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a specified condition in a subject. A subject may be predisposed to or at risk of developing a condition but has not yet been diagnosed with the condition.

[0075] The term “delaying progression of’ includes reducing or slowing down the progression of a condition in a subject and / or at least one symptom of the condition.

[0076] The term “condition” shall be taken to mean a state of being or health status of a subj ect in need of treatment with a plasma-derived protein. Exemplary conditions include but are not limited to primary immunodeficiency disease (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP).

[0077] The term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and nonhuman primates. For example, the subject is a human.

[0078] The term (coefficient of determination) “R2” indicates the percentage of variance explained by the prediction model. The higher the coefficient, the better the correlation between the reference data and spectral data.

[0079] The term “false positive (FP)” indicates the number of actual negatives that were misclassified as positive.

[0080] The term “true positive (TP)” indicates the number of actual positives that were correctly classified as positive.

[0081] The term “false negative (FN)” indicates the number of actual positives that were misclassified as negative.

[0082] The term “true negative (TN)” indicates the number of actual negatives that were correctly classified as negative.

[0083] The term “sensitivity (recall)” is the ratio between the number of correctly classified positives and the total number of actual positives: number of true positives TP Sensitivity = - - - - - — - = total acutal positives (TP + FN)

[0084] The term “specificity” is the ratio between the number of correctly classified negatives and total number of actual negatives: number of true negatives TN

[0085] Specificity — - — - total acutal negatives (TN + FP) The term “false positive rate” is the ratio between the number of true negatives wrongly classified as positive (false positive) and the total number of actual negatives:

[0086] The term “false negative rate” is the ratio between the number of true positives wrongly classified as negative (false negative) and the total number of actual positives: number of false negatives FN

[0087] False negative rate = - - - - - = - - - total acutal positives FN + TP)

[0088] The term “accuracy” is the ratio of all correct classifications and the total number of all classifications made:

[0089] The term “precision” is the ratio between true positives and all values classified as positive including the false positives.

[0090] The term “confusion matrix” describes a specific table layout that allows visualization of the performance of a classification and contains true positives, true negatives, false positives, and false negatives.

[0091] The term, “bias” is the Systematic averaged deviation between the reference values and the predicted values.

[0092] SF-iCXc i ~ ? i) predicted value

[0093] Bias — —;- - - — yc = Reference method value n n - Number of samples

[0094] The term “cross validation” or “cross-validation” (also referred to as internal validation) describes the procedure where individual leave-out samples (defined by the user) are removed from the calibration or training set. Using the remaining samples, a chemometric model is established and used to predict the previously extracted sample. A comparison of the predicted with the actual values determined by the reference method shows how well the model predicts the samples.

[0095] The term “k-fold cross validation” is an iterative validation procedure performed based on the parameter k, meaning k iterations are done during the cross-validation process. The training dataset is split into k subgroups. Then, for each iteration k-1 subgroups are used for the training of the model whereas the remaining subgroup functions as validation dataset. The root mean square error of cross-validation is obtained from the average root mean square error of all iteration of the cross validation. The root mean square error of prediction is obtained from the original test dataset and therefore represents the model performance on a dataset outside the training dataset. The root mean square error of calibration is obtained based on the complete training dataset. For PLS models a rank must be selected which is the number of multivariate factors used to explain the variance of the dataset. Increasing the rank results in increased complexity of the model which could lead to an overfitting of the model to the training data making it unable to perform precise predictions on new data. Therefore, the best rank is as small as possible but also leads to a small root mean square error and a large R2.

[0096] The term “Partial Least Squares” (Regression) is a statistical technique that reduces the predictors to a smaller set of uncorrelated components and performs least squares regression on these components, instead of on the original data.

[0097] The term, “peak integration” refers to the calculation of peak areas by integrating the measured signal within specified ranges.

[0098] The term “spectral distance” refers to the calculation of the Euclidean distance between a sample spectrum and reference spectra. Spectral distance may be used to categorise a sample as one of a number of predetermined classes of sample, wherein the sample is categorized by selecting the class that results in the smallest distance between the sample spectrum and the reference spectra of that class.

[0099] The term, “RMSE” is “root mean square error” and a common metrics in regression. It relates to the average difference between the values predicted by a model and the actual values, and provides an estimation of how well the model can predict the target value (accuracy). In some embodiments, the “actual value” describes the value that is measured with an analytical test system other than Raman. The different RMSE-based performance measures used are described as follows.

[0100] The term, “RMSECV” is “root mean square error of cross validation” and is a quantitative measure for the predictive ability of the model during cross validation. The RMSECV is comparable to the RMSEP for the external validation using an independent test set of samples.

[0101] > “ ' Yc = NIRS predicted value of calibration set yo = Reference method value n = Number of samples

[0102] The term, “RMSEP” is “root mean square error of prediction” and is a quantitative measure for the predictive ability of the model during external validation using an independent test set of samples. The RMSEP is comparable to the RMSECV for cross validation.

[0103] YT = NIRS predicted value of independent test set sample

[0104] RMSEP = I - — yr = Reference method value n = Number of samples The term, “RPD” is ratio of standard deviation (SD) and standard error of prediction (SEP), i.e.:

[0105] SD SD = Standard deviation of reference values

[0106] RPD = - SEP = Standard error of prediction

[0107] SEP

[0108] The term “SD” refers to standard deviation, which may be determined by: value

[0109] The term, “SEP” is “standard error of prediction” and is the RMSEP corrected by the bias, and is determined as: Yc = NIRS predicted value of independent test set sample

[0110] BBr yc = Reference method value n = Number of samples

[0111] The term, “uncertainty” relates to how the estimated value predicted by the model might differ from the true value to understand the degree of confidence in the prediction.

[0112] The skilled person will understand that parameters including bias, RMSEC, RMSECV, RMSEP, R2, RPD value, uncertainty and / or MAPE may be referenced to determine or adjust parameters of a model, including the optimal function for a calibration curve type (e.g. on the basis of a RMSE vs. Function plot) and the rank of a PLS model (e.g. on the basis of a RMSE vs. rank plot).

[0113] The term, “calibration curve” is the regression curve. The skilled person will understand that in general the type of a calibration curve may be simple, linear, quadratic, exponential, logarithmic, or any other type of curve.

[0114] Methods of isolating a protein from plasma or a fraction thereof using continuous chromatography

[0115] The present disclosure provides for a method of isolating a protein from plasma or a fraction thereof using continuous chromatography, the method comprising: (a) loading an amount of plasma, or fraction thereof, comprising the protein onto a chromatographic medium capable of separating the protein from at least a portion of the amount of plasma or fraction thereof;

[0116] (b) eluting the protein from the chromatographic medium; and

[0117] (c) collecting the protein; wherein, Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium and / or which is in the amount of plasma or fraction thereof being loaded onto the chromatographic medium.

[0118] Raman spectroscopy monitoring

[0119] The principle of the Raman effect is based on inelastic light scattering. The scattering behavior depends on the vibrational properties of molecules. The basic vibration or energy level of individual molecules is affected by the energy transition from the Raman laser (photons). If, for example, a high-energy laser beam hits a molecule, a distinction is made between three different types of light scattering depending on the vibrational properties and electrical polarizability of the molecules and the energy transition: The Anti-Stokes Raman scattering (inelastic scatter), the Stokes scattering (inelastic scatter) and the Rayleigh scattering (elastic scatter). For the Anti-Stokes Raman scattering the energy transition takes place from the molecule to the photon, so that the energy level of the molecule is then lower, and the energy level and frequency of the photon is higher. For the Stokes Raman scattering the energy transition takes place from the photon to the molecule, so that the energy level of the molecule is then higher, and the energy level and frequency of the photon is lower. For the Rayleigh scattering the photon bounces off the molecule without any change of energy.

[0120] The change in energy level and frequency, also called Raman shift, is measurable by the Raman probe and, like a fingerprint, results in a unique spectrum depending on the molecular properties of the solution under investigation. Accordingly, Raman shift may be used in the detection or quantification of any molecule (e.g. a protein) present in the solution.

[0121] Implementation

[0122] It will be appreciated that in a continuous chromatography process, Raman spectroscopic analysis may be used for monitoring samples from or within the flow path before and / or after the column. It will further be appreciated that such Raman spectroscopic analysis may be used to monitor samples from or within the flow path before and / or after the column, at various points or stages throughout a continuous chromatographic cycle. For example, Raman spectroscopic analysis may be used to monitor during washing of the column(s), cleaning of the column(s), equilibration of the column(s), loading of the column (with e.g., the protein or plasma or fraction thereof comprising the protein), elution of the column(s) which broadly includes any step comprising elution of a composition from the column(s), including any deliberate elution step where it is intended that the protein elute from the column(s), and any other step(s) that may occur in a continuous chromatographic purification process as will be known to the person skilled in the art. By so monitoring any one of the above steps, or combination thereof, the purification process may be continually adapted in real time by using the Raman analysis to determine operational changes that may optimise the process or provide some other benefit. Such benefits or outcomes of optimisation include, but are not limited to, higher yield, higher purity, higher conversion, less wastage (e.g. less buffer required), faster purification cycles, higher turnover, and other benefits. One particular advantage may be that the monitored process allows for optimisation relating to resin efficacy and lifetime. Columns typically have to be re-packed and media exchanged after the media has reached a maximum number of cycles. However, chromatographic media typically will have batch-to-batch variation in terms of binding capacity. Moreover, binding capacity decreases overtime, and therefore column loading needs to be continuously adapted to allow an efficient process and to prevent overloading of the resin / column. A particular advantage is that the Raman spectroscopic analysis may be used to determine changes in sample composition in real time, thereby allowing the adaptation of process parameters such as flow rates, buffer volumes, temperature, pressure, etc.

[0123] As stated, Raman spectroscopic analysis may be performed on the fluid eluting from the outlet of the column. Accordingly, in some embodiments, Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium. In some embodiments, Raman spectroscopic analysis is used to monitor for the protein prior to loading on the chromatographic media. It will be understood that monitoring for protein which has passed through the chromatographic medium will typically encompass monitoring of the fluid eluting from the outlet of the column (or the fluid entering into the chromatographic media). It will therefore be understood that to monitor for protein which has passed through the chromatographic medium (or the protein entering into the column), such monitoring may occur at any point(s), portion(s), or entirety, of the chromatographic process, even at point(s) and / or portion(s) during which no protein is expected to elute from the chromatographic media (nor be present in the fluid entering the chromatographic media). Accordingly, monitoring for protein having passed through the chromatographic media (or protein entering into the chromatographic media), does not preclude, and may encompass, monitoring for other constituents of the fluid eluting from the outlet of the column (or prior to the inlet of the column), or more generally, monitoring the fluid / sample that is eluting (or entering the column) at a given point(s) or portion(s) of the process, and categorising fluid / sample into one of a number of classes (or predetermined compositions). The classes into which the eluting fluid / sample is to be assigned may be predetermined by the skilled person by reference to composition(s) (or range of composition), and / or the intended purpose or destination of the composition (e.g. waste, or eluent) according to the particular chromatographic process or setup. The classes may correspond to compositions of eluent and / or solvents that are actualised or expected in the chromatographic process. Classes may indicate or be “mapped” to particular operational changes, for example, where the fluid eluting from the column is to be directed. For example, possible classes include, but are not limited to, protein-containing eluate (e.g. IgG), elution buffer, equilibration buffer, wash buffer, protein-depleted plasma or fraction thereof (e.g. IgG-depleted CPP), low conductivity buffer, plasma, waste. Accordingly, in some embodiments, monitoring for the protein encompasses Raman spectroscopic analysis of a sample that does not comprise the protein. In some embodiments, the use of the Raman spectroscopic analysis to monitor for the protein, comprises monitoring in the absence of the protein. In some embodiments, Raman spectroscopic analysis is used to monitor for a buffer prior to loading on the chromatographic media. In some embodiments, Raman spectroscopic analysis is used to monitor for a buffer after the chromatographic media.

[0124] In some embodiments, the Raman spectroscopic analysis is used to determine that a sample is a composition corresponding to at least one of one or more predetermined compositions, and / or that the composition of the sample has changed from one to another of the one or more predetermined compositions. In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer.

[0125] In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer, and protein-depleted plasma or fraction thereof.

[0126] In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer, protein-depleted plasma or fraction thereof, low conductivity buffer, plasma, and waste. It will be appreciated that in a continuous chromatographic process, protein may be expected to be present in the eluent from a column at various stages in the process. This is clearly to be expected during a deliberate elution step (for example, where a suitable buffer is passed through the column that permits resolution, movement, or passage of the protein over and / or through the media or resin towards the outlet). Thus, in some embodiments, the Raman spectroscopic analysis monitors for elution of the protein in an elution buffer. Protein may also be expected to “breakthrough” the chromatographic medium during loading of protein, due to inefficacies in binding and / or maximum binding capacity having been reached. Accordingly, in some embodiments, the Raman spectroscopic analysis monitors for breakthrough of the protein due to overloading of the chromatographic medium. Protein may also be expected to be detected in the eluent during washing of the chromatographic medium. Thus, in some embodiments, the Raman spectroscopic analysis monitors for elution of the protein in a wash solution.

[0127] Obtaining Raman spectra

[0128] The skilled person will be familiar with standard equipment that can be used for applying light sources. In the context of the methods of the present disclosure, the equipment may include use of a Raman probe adapted for use in a (large) vessel or flow path which comprises the sample, which may be in a flow path of a continuous chromatographic process either side of a chromatographic media (for example, in a portion of the flow path preceding and / or proceeding a column comprising the chromatographic media used to isolate the protein).

[0129] In one embodiment the Raman spectroscopy instrument is arranged to analyze the test sample during mixing in a (large) tank and provide inelastic scattering or Raman shift data in real-time. In certain embodiments, several probes may be connected to a single spectrometer. In an embodiment, a first probe may thus be arranged at the first position while a second probe is arranged at the second position and, if applicable, a third probe is arranged at the third position. All such probes may be connected to the same spectrometer. The skilled person will appreciate that the use of multiple Raman probes may assist with providing a more accurate range of data relating to test samples or training samples comprising the analyte of interest.

[0130] The probe of the Raman spectroscopy instrument may be in the form of an immersion probe or constitute a part of a flow cell. The whole process flow or a side stream of the flow can be lead through such a flow cell. The Raman spectroscopic analysis may be an in-line, online, at-line or off-line analysis. In particular embodiments, the Raman spectroscopic analysis is an in-line analysis. In some embodiments, the Raman spectroscopic analysis is performed in a flow cell. In some embodiments, a flow rate through the flow cell is between about 0 and about 250 mL / min, or between about 0.1 and about 250 mL / min, or about 0.5 to 50 mL / min. In some embodiments, the Raman spectroscopic analysis is performed continuously.

[0131] In some embodiments, the Raman probe is configured to enable measurement of inelastic scattering or Raman shift during mixing or flow of a sample. The optical slit of the Raman probe may be oriented parallel to the direction of the fluid stream during mixing. Typically, the optical slit of the Raman probe is oriented so that it is not directly facing the flow of the fluid stream during mixing. For example, the optical slit may be perpendicular or at an angle relative to the fluid stream during mixing. In other words, the Raman probe may be oriented downwards alongside the wall of the vessel.

[0132] Advanced data analysis models can be developed (e.g., partial least squares regression) to ultimately detect or quantify the protein at time points within the process flow, and / or to detect the character or class of the eluent (viz. in the context of the desired chromatographic outcome), e.g. whether the eluent is “washings”, “equilibration buffer”, “eluent buffer” (with or without desired protein) at any given point in time. To predict specific protein concentrations (e.g. that of IgG) typically intrinsic fluorescence effects should be avoided in the protein-rich plasma solution. Intrinsic fluorescence is a rarely occurring characteristic of several proteins mainly caused by tryptophan residues and insignificantly caused by tyrosine side chains. This can negatively influence the sensitive Raman measurement.

[0133] In some embodiments, the Raman spectroscopy may be performed in the visible, near infrared, infrared, near ultraviolet, or ultraviolet (UV) range. In some embodiments, a signal enhancement technique known as Surface Enhanced Raman Spectroscopy (SERS), which relies on a phenomenon known as surface plasmonic resonance, may be used. In some embodiments, resonance Raman spectroscopy, tip-enhanced Raman spectroscopy, polarized Raman spectroscopy, stimulated Raman spectroscopy, transmission Raman spectroscopy, spatially offset Raman spectroscopy, difference Raman spectroscopy, Fourier Transform (FT) Raman, or hyper Raman spectroscopy may be used. In some embodiments, a Raman analyzer can be used that is configured with a laser (e.g. laser diode) or other suitable light source that operates at appropriate wavelengths (e.g., those described herein, such as between 325 nm and 1064 nm).

[0134] In some embodiments, the light source has a wavelength (in nm) of at least about 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 875, 900, 925, 950, or 1000. In some embodiments, the light source has a wavelength (in nm) of about 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 875, 900, 925, 950, or 1000. In some embodiments, the light source has a wavelength (in nm) of one or more of 532, 785, and 993, optionally 532, 785, and 993. In some embodiments, the light source has a wavelength of about 785 nm. In some embodiments, the light source is in the visible spectrum.

[0135] It will be appreciated that Raman spectroscopy comprises irradiating the sample with light from the light source for a period of time (the exposure time), and that the length of this period of time may vary. In the context of continuous spectroscopy, shorter exposure times may be preferred so that the overall time for analysis and any subsequent determination of one or more operational changes is reduced, which may be desirable in light of the system being continuous. On the other hand, shorter exposure times may produce Raman shifts with weaker intensity, and affect the quality of the analysis. It may be preferable to strike a suitable balance, which may be different depending on the anticipated composition of the eluent due to the current cycle of the process (e.g. loading, or elution).

[0136] Accordingly, in some embodiments, the Raman spectroscopic analysis comprises applying a light source to a sample with an exposure time of about or less than about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,

[0137] 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,

[0138] 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 seconds; or between about 1 and about 20 seconds, or between about 2 and about 10 seconds.

[0139] In some embodiments, the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, is performed with an exposure time between about 6 and about 10 seconds. In some embodiments, the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, is performed with an exposure time or about or less than about 60, 50, 40, 30, 20 or 10 seconds.

[0140] In some embodiments, the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, is performed with an exposure time about or less than about 60, 50, 40, 30, 20 or 10 seconds. In some embodiments, the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, is performed with an exposure time about or less than about 5, 4, 3, 2 or 1 seconds.

[0141] Processing In some embodiments, the Raman spectra are trimmed by removing peaks that are distorted. In some embodiments, peaks that are distorted are peaks that are laterally shifted or inverted. However, it should be appreciated that distorted peaks may include any peak that fails to meet certain criteria (e.g., intensity, signal -to-noise (S / N) ratio, shape, closeness to other peaks). Distorted peaks can be identified by visual inspection or by using a computer program that identifies (and removes) peaks that do not meet certain criteria. For example, peaks may be excluded because they are laterally shifted or inverted by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared with a reference peak (e.g., a non-distorted peak). Similarly, peaks may be excluded because they have a S / N ratio that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less than the S / N of a reference peak (e.g., a non-distorted peak).

[0142] In some embodiments, only a portion of a Raman spectrum is evaluated. For example, data relating to only a portion of the Raman spectrum is evaluated and the remaining data is filtered or otherwise removed prior to analysis. In some embodiments, the distorted peaks that are removed are lateral peak shifts. In some embodiments, a lateral peak shift looks like a 2-dimensional peak that has been stretched out. This peak distortion is likely the result of a component in the sample that is interacting with one of the bonds on the molecule of interest / analyte, the presence of a bond with similar character, solvent distortion, or any combination of these phenomena. In some embodiments, the laterally shifted peak or inverted peak is shifted by more than 5 cm-1in a concentration dependent fashion. In some embodiments, the lateral peak is removed if it is shifted by more than 1 cm-1, more than 2 cm-1, more than 5 cm-1, more than 10 cm-1, or more than 20 cm-1or more. In some embodiments, the lateral peak is removed if it is shifted by more than 1 cm-1, more than 2 cm-1, more than 5 cm-1, more than 10 cm-1, or more than 20 cm-1or more, in a concentration dependent fashion.

[0143] In some embodiments, the distorted peaks that are removed are inversion peaks (also called “inverted peaks” herein). An inversion peak is a peak where it appears that the lower concentration data is higher in magnitude than the high concentration data, when this relationship did not exist in the basis peaks. This type of distortion is usually due to a molecular species within the sample that has similar vibrational properties and therefore similar peaks. In some embodiments, the inverted peak is removed if there is a lack of baseline. Mathematical and / or statistical analyses, e.g. multivariate models

[0144] Any appropriate statistical model may be used in methods disclosed herein. In some embodiments, the model is a regression model that relates predicted variables (e.g., protein concentration) and observable variables (e.g., Raman spectral data). In some embodiments, the regression model is a partial least squares model. In some embodiments, the model is a bilinear factor model that projects predicted variables (e.g., protein or ethanol concentration) and observable variables (Raman spectral data) into a new space. In some embodiments, the model is a regression model that uses principal components analysis (PC A) for estimating unknown regression coefficients in the model. However, other multivariate analytical techniques may be used including, for example, support vector machines, multivariate linear regression, and others.

[0145] Typically, inelastic scattering produced spectra contain hundreds of variables and therefore some form of multivariate data analysis method is preferably used to analyze raw data from the measurements. Such multivariate data analysis methods are well known in the art and includes Partial least squares regression (PLS); PLS Discriminant Analysis (PLS-DA); Ordinary Least Squares (OLS) regression; MLR (multiple linear regression); OPLS (Orthogonal-PLS); SVM (support vector machines); GLD (general discriminant analysis); GLMC (generalized linear model); GLZ (generalized linear and non-linear model); LDA (Linear Discriminant Analysis); spectral distance; principal component analysis (PCA); principal component analysis - quadratic discriminant analysis (PCA-QDA); partial least square - discriminant analysis (PLS-DA); classification trees; cluster analysis; neural networks; and Pearson correlation.

[0146] Fluorescent background can also be managed by employing preprocessing and baseline normalization techniques such as smoothing and / or rubber band subtraction, background correction algorithms or derivative spectroscopy, to Raman spectral data, including first and second differentiation, Savitzky-Golay smoothing differentiation, SNV, multiplicative signal correction (MSC), extended multiplicative signal correction (EMSC) polynomial fitting, Fourier Transform, wavelet analysis, orthogonal signal correction (OSC), and extended inverted signal correction (EISC) among others. In some embodiments, the model is generated using partial least squares (PLS) regression of processed wavelength spectra of samples having known concentrations of the protein or total protein. In some embodiments, the method further comprises a step of applying preprocessing and baseline normalization techniques, background correction algorithms or derivative spectroscopy to the spectrum or spectra to manage the background fluorescence. In some embodiments, optionally after preprocessing and baseline normalization techniques, a data filter is used to select wavelength ranges or Raman shift regions that are of interest.

[0147] Methods for generating models / reference data sets

[0148] The skilled person will be familiar with general approaches for preparing a reference data set or developing a model of representative spectra against which the spectra from test samples can be compared for the purposes of determining protein presence or concentration.

[0149] The reference data set may be from one or more samples comprising a known concentration of the protein, wherein the concentration of the protein has been determined by a method that is appropriate given the composition of the reference and test samples. For example, in the context of turbid solutions or suspensions comprising proteins, an appropriate method for confirming protein concentration may be the Dumas method which is based on determining total nitrogen content, or other methods may be appropriate for determining protein concentration, such as the Biuret assay, BCA assay, Bradford assay, Kjeldahl method, or absorbance at 280 nm.

[0150] Representative spectra can then be obtained for the reference or training samples for which protein concentration has been determined, such that the representative spectra can be used to form the basis of a model against which test spectra can be assessed.

[0151] The skilled person will appreciate that in most cases the greater the number of representative spectra or training spectra provided, the greater the accuracy of the model. There may be a need to apply spectral pre-treatments to data (whether the test spectra or the reference or training spectra used to derive a suitable model). These pre-treatments can be applied to emphasise spectral changes. Examples of suitable spectral pretreatments include vector normalisation, first order derivative, min-max normalisation, straight line subtraction, multiplicative scatter correction, 2ndorder derivative, baseline corrections and combinations thereof. In some embodiments, the pre-treatment applied to the test spectra or the reference or training spectra used to derive a suitable model is vector normalisation or 1storder derivative. In one embodiment, the pre-treatment applied to the test spectra or the reference or training spectra used to derive a suitable model is vector normalisation in combination with 1storder derivative. Optionally, the pre-treatment further comprises Standard Normal Variate (SNV). Optionally, the pretreatment further comprises a smoothing. Optionally, the pre-treatment further comprises a standardization, such as wherein the standardization is performed by area normalization. The model may be generated using a multivariate calibration algorithm, such as Multiple Linear Regression (MLR), Principal Component Regression (PCR), or Partial Least Squares (PLS)-Regression. In some embodiments, the model is generated using Partial Least Squares (PLS)-Regression, such as that described herein. The PLS algorithm is described in (Haaland, Thomas, Anal. Chem 60 (1998) 1193; Martens, Naes, Multivariate Calibration, J. Wiley & Sons, New York (189): Chapter 3.5; Brown, Apply. Spectosc. 49, No. 12 (1995) 14A; and Bouveresse, Hartmann, Massard, Last, Prebble, Anal. Chem. 68, No. 6 (1996) 982).

[0152] Methods for assessing the quality of a given model (including to then determine whether further training data are required for further developing the model) are described herein. In certain examples, criteria that may be considered when assessing the model quality of the different chemometric models or multivariate models include:

[0153] Rank: corresponds to the number of factors of the chemometric model. A lower rank usually leads to increased model stability;

[0154] Root mean square error of cross validation (RMSEC V): The RMSEC V should be minimized;

[0155] Residual prediction deviation (RPD): model performance indicator. The RPD should be maximized; and

[0156] R2: coefficient of determination, describes the relation between spectral data and the concentration data. The R2should be maximized to close to 100.

[0157] The following criteria may also be considered when assessing the predictive ability of the chemometric models or multivariate models on an independent data set:

[0158] Bias: Average difference between reference values and predicted values. Should be close to 0;

[0159] Root mean square error of prediction (RMSEP): accuracy indicator for prediction of independent test samples. The RMSEP should be minimized;

[0160] Residual prediction deviation (RPD): model performance indicator. The RPD should be maximized;

[0161] R2: coefficient of determination, describes the relation between spectral data and the concentration data. The R2should be maximized to close to 100; and

[0162] MAPE: mean absolute percentage error, measures the prediction accuracy of a forecasting model. The MAPE should be minimized.

[0163] Further examples, forjudging the model include but are not limited to, statistical parameters including the number of latent variables (PLS factors) in the model, Bias, RMSEC; RMSECV, RMSEP for independent test samples, Rank, R2, RPD value, Uncertainty, MAPE, False positive rate, False negative rate, Accuracy, Sensitivity (Recall), Specificity, Precision, Confusion matrix, and combinations thereof. The generation of the model may involve training samples that may comprise a representative set of samples that cover variables, such as different paste type, sample temperature, instrument variability, operator handling, raw materials, and plasma source. Using such varied reference samples to capture such variables in the generation of the training model will further ensure the robustness of the model when it comes to assessing a variety of samples comprising analytes of unknown concentration.

[0164] In any method described herein, the method may further comprise a step of determining the presence of, amount of, or concentration of, a protein in a sample after comparing the test spectra with reference spectra, comparing the test spectrum with a reference spectrum, or comparing the test spectra to a reference data set.

[0165] Further Examples

[0166] It will be appreciated that Raman spectroscopic analysis used in the method may comprise multiple independent analyses at different points in time, and / or of samples at different points within the purification process, e.g. samples eluting from the chromatographic medium, or samples that are being loaded onto the chromatographic medium.

[0167] It will be appreciated that the sample analysed in the Raman spectroscopic analysis, does not need to be an “isolated” sample. In other words, the term “sample” is intended to simply indicate a small part or quantity of a larger whole or bulk or the larger whole or bulk itself. The latter one is relevant in terms of an inline measurement approach. The analysis is intended to include at-line, in-line and off-line methods whereby the light source is applied to a small part of a larger bulk solution and where the light source can be applied to the small part of the bulk solution in situ, or to an aliquot of the solution that has been removed (isolated) from the larger bulk.

[0168] As used herein, the term “in-line” refers to a method of analysis whereby a probe, or sampling interface or sensor (e.g. for providing a light source) can be placed directly in a process vessel or tubing or in line with a stream of flowing material to conduct the analysis. The process may involve placing a probe in a flow system, precipitation vessel or processing unit. Such process may allow analysis without having to remove the probe or any material or samples from the bulk (i.e. the sample remains “zn situ” for the analysis). In some embodiments, multiple Raman spectra are obtained from different locations within a solution, for example an in-line processing solution. The data from such multiple spectra may be averaged if appropriate.

[0169] As used herein “on-line” refers to a method of analysis without having to remove the material or samples from the bulk. However, it may involve separating from the main process line and performing measurements on just a portion of the bulk. This may be accomplished by adding a sampling loop which directs a sample of the bulk material towards the probe or sensor, and whereby the diverted sample may be re-introduced to the process stream, flow or bulk of material, or disposed of, depending on the application.

[0170] As used herein, the term “at-line” refers to a method which includes manual sampling followed by discontinuous sample preparation, measurement and evaluation. When measuring at-line, analysis is typically completed at or near the process stream, flow or bulk of material.

[0171] As used herein, the term “off-line” refers to a method that involves the most physical difference between the process stream, flow or bulk of material and the analysis of the sample. Similarly to at-line measurement, off-line measurement involves removing an analytical sample from the larger bulk of material. Off-line analysis typically involves taking the sample or sometimes multiple samples to be analysed in a formal lab setting.

[0172] In some embodiments, each Raman spectroscopic analysis comprises; applying a light source to a sample: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: reference spectra obtained from reference samples having known concentrations of the protein or total protein; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein or total protein, to determine the presence, amount, or concentration, or a change thereof, of the protein or total protein present in the sample.

[0173] It will be appreciated that Raman spectroscopic analysis as used in the method may not require the protein or total protein to be quantified in terms of amount or concentration per se, but rather that changes in the presence, amount or concentration thereof may be inferred on the basis of spectral features or changes in spectral features, such as peak intensity, or the area under the curve.

[0174] Light sources, reference spectra and reference data sets are described elsewhere herein. In some embodiments, comparing the test spectra with the reference spectra or reference data set, comprises comparing a peak height, an area under the curve, spectral distance, or a peak profile of the test spectra, with a corresponding peak height, area under the curve, or peak profile from the reference spectra or reference data set.

[0175] In some embodiments, the concentration of protein or total protein in the reference or training samples is determined using the Dumas assay, optical density measurement, the Kjeldahl method, and / or any other method of protein determination.

[0176] It will be understood that it may be advantageous for the reference data or reference spectra to be based on reference samples having concentrations that are relevant or commensurate to those that are to be predicted according to the Raman spectroscopic analysis as used in the method disclosed herein. Accordingly, in some embodiments, the reference samples include concentrations of the protein or total protein across a range of concentrations of samples eluting from the chromatographic medium, or being loaded onto the chromatographic medium. In some embodiments, the model is generated using peak integration. In some embodiments, the model is generated using hard modelling. In some embodiments, the model is generated using multivariate analysis. In some embodiments, the model is generated using PLS multivariate analysis. In some embodiments, when the Raman spectroscopic analysis is used to monitor for breakthrough of the protein due to overloading of the chromatographic medium, the model is generated using PLS multivariate analysis. In some embodiments, when the Raman spectroscopic analysis is used to monitor for protein eluting in an elution buffer, the model is generated using peak integration. In some embodiments, when the Raman spectroscopic analysis is used to monitor for protein eluting in an elution buffer, the model is generated using PLS multivariate analysis. In some embodiments, when the Raman spectroscopic analysis is used to monitor for different solutions, which may or may not contain protein, the model is generated using spectral distance analysis.

[0177] In some embodiments, the test spectra comprise a spectral signal in the visible, near infrared, infrared, near ultraviolet, or ultraviolet (UV) range. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 5000 cm'1to 0 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 3200 cm'1to 0 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 3500 cm'1to 300 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1500 cm'1to 340 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1050 cm'1to 300 cm' f In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 2500 cm'1to 500 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1100 cm'1to 900 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 800 cm'1to 700 cm' f In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1300 cm'1to 1150 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1600 cm'1to 1300 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 3000 cm'1to 2800 cm'1. In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1620 cm'1to 350 cm'1.

[0178] In some embodiments, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one or more of the following regions: 700 cm'1and 800 cm'1; 900 cm'1and 1100 cm'1; 1150 cm'1and 1300 cm'1; 1300 cm'1and 1600 cm'1; 2800 cm'1and 3000 cm'1; 300 cm'1and 3500 cm'1; 340 cm'1and 1500 cm'1; 300 cm'1and 1050 cm'1; and / or 500 cm'1and 2600 cm'1.

[0179] It will be appreciated that the composition of the eluent may change through the chromatographic cycle, and that accordingly, it may be advantageous for the Raman spectroscopic analysis to be adjusted according to the particular phase of the chromatographic cycle and / or anticipated composition of the eluent.

[0180] In some embodiments, the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein or total protein, to determine the presence, amount, or concentration, or change thereof, of protein or total protein present in the sample.

[0181] In some embodiments, the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using peak integration of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount, or concentration, or change thereof, of protein present in the sample.

[0182] In a particular example, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one of more of the following regions:

[0183] 700 cm'1and 800 cm'1;

[0184] 900 cm'1and 1100 cm'1;

[0185] 1150 cm'1and 1300 cm'1;

[0186] 1300 cm'1and 1600 cm'1; and / or

[0187] 2800 cm'1and 3000 cm'1.

[0188] In a further example, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 900 cm'1to 1100 cm'1.

[0189] In one example, the concentration of protein is determined by comparing the area under the curve (AUC) of at least one Raman shift from the sample against an AUC of a corresponding Raman shift from the reference data set, wherein each measured Raman shift is independently selected from a Raman shift that has the highest maximum intensity relative to any other Raman shift within one of the following ranges:

[0190] 700 cm'1and 800 cm'1;

[0191] 900 cm'1and 1100 cm'1;

[0192] 1150 cm'1and 1300 cm'1;

[0193] 1300 cm'1and 1600 cm'1; and / or

[0194] 2800 cm'1and 3000 cm'1.

[0195] In one example, the concentration of protein present in the sample is determined by comparing the area under the curve (AUC) of a single Raman shift from the sample against an AUC of a corresponding Raman shift from the reference data set, wherein the single Raman shift has the highest maximum intensity relative to any other Raman shift from 900 cm'1to 1100 cm'1.

[0196] In one example, the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount, or concentration, or change thereof, of protein present in the sample.

[0197] In a particular example, the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one of more of the following regions:

[0198] 300 cm'1and 3500 cm'1;

[0199] 340 cm'1and 1500 cm'1;

[0200] 300 cm'1and 1050 cm'1; and / or

[0201] 500 cm'1and 2600 cm'1.

[0202] In one example, each Raman spectroscopic analysis comprises; applying a light source to a sample either: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: reference spectra obtained from reference samples comprising one or more predetermined compositions; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples comprising one or more predetermined compositions, to determine that the sample is a composition corresponding to at least one of the one or more predetermined compositions, and / or that the composition of the sample has changed from one to another of the one or more predetermined compositions.

[0203] In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, and wash buffer.

[0204] In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer, and protein-depleted plasma or fraction thereof. In a particular example, the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer, protein-depleted plasma or fraction thereof, low conductivity buffer, plasma, and waste.

[0205] Operational Changes

[0206] It will be appreciated that the outputs of monitoring the process by Raman spectroscopic analysis can be used to inform the control of the continuous chromatographic process. Thus, in embodiments, the monitoring by Raman spectroscopic analysis determines one or more operational change in the continuous chromatography. Operational changes that may be made on the basis of, or determined by, Raman spectroscopic analysis include the ceasing of column loading (to prevent overloading of a column, and subsequent product loss), the re-direction of column eluent containing the desired protein to a second column to prevent product-loss while maintaining efficacy of the overall process, the commencement or maintenance of a wash step (e.g. the wash step may be continued so long as residual proteins are detected, or may be stopped earlier to save buffer and time), the commencement and cessation of a (deliberate) elution step in which the isolated protein is collected (depending on the presence of absence of the desired protein in the eluent). Other suitable operational changes that may be made on the basis of a Raman spectroscopic analysis in a continuous chromatographic process will be apparent to the person skilled in the art, and all such are contemplated to be within the scope of the present disclosure. Accordingly, in some embodiments, the one or more operational change comprises collecting the protein eluting from the chromatographic medium. In some embodiments, the one or more operational change comprises ceasing loading the chromatographic medium with the protein. In some embodiments, the one or more operational change comprises loading an amount of the protein eluting from the chromatographic medium onto a further chromatographic medium. In one example thereof, both chromatographic media are the same resin. In a particular example, the further chromatographic medium is provided in a separation unit that is distinct from a separation unit providing the (first) chromatographic medium. In some embodiments, the one or more operational change comprises ceasing elution of the protein with an elution buffer. In some embodiments, the one or more operational change comprises ceasing collection of the protein. In some embodiments, the one or more operational change comprises washing the chromatographic medium.

[0207] It will be appreciated that operational changes may be made on the basis of a variety of outputs from the Raman spectroscopic analysis, including but not limited to, spectral feature (peak(s), area(s) under the curve, spectral distance), or predicted amounts or concentrations of protein or total protein on the basis of e.g. spectra features or modelling.

[0208] The person skilled in the art will therefore appreciate that the Raman spectroscopic analysis may comprise at least one of a variety of determinations or inferences about the composition of the analysed sample, in particular determinations or inferences relating to the presence, quantity and / or concentration of the protein, or even total protein. These determinations or inferences may be isolated determinations or inferences upon which operational change to the purification process may be determined and / or made, or they may be determinations or inferences which are comparable to previously made determinations or inferences. For example, the Raman spectroscopic analysis may result in real-time determination of eluent protein concentration. In another example, the Raman spectroscopic analysis may not result in absolute determination of the eluent protein concentration per se, but may determine that the eluent protein concentration has increased. In another example, the Raman spectroscopic analysis may result in determination of the presence of protein in the eluent.

[0209] Thus, in some embodiments, the Raman spectroscopic analysis comprises determination of a change in the Raman spectra of a solution eluting from the chromatographic medium, and inferring a change in an amount or concentration of the protein or total protein. In some embodiments, the Raman spectroscopic analysis comprises determination of a change in the amount or concentration of the protein. In some embodiments, the Raman spectroscopic analysis comprises determination of a change in the amount or concentration of total protein. In some embodiments, the Raman spectroscopic analysis comprises determination of a concentration of the protein. In some embodiments, the Raman spectroscopic analysis comprises determination of a concentration of total protein.

[0210] It will be appreciated that operational changes one or more operational change may be determined on the basis of a single Raman spectroscopic analysis, or determined by at least two, three, four, or five Raman spectroscopic analyses. By withholding determining an operational change on the basis of a single Raman spectroscopic analysis, greater efficiency or other benefits may be achieved in light of greater confidence and / or reduced error in the Raman analyses performed.

[0211] In some embodiments, the one or more operational change is determined by the amount or concentration of the protein or total protein eluting from the chromatographic medium, or change thereof, having been determined and / or inferred to be about or greater than about a predetermined upper threshold. It will be understood that the predetermined upper threshold is a threshold that may be predetermined by the person skilled in the art depending on the implementation, and desired level of efficiency required for the particular implementation.

[0212] In some embodiments, the predetermined upper threshold indicates that the chromatographic medium is at about or greater than about 50, 60, 70, 80, 90, 95, 99 or 100% of a dynamic binding capacity (DBC) of the chromatographic medium.

[0213] In some embodiments, the predetermined upper threshold is about or greater than about 0.1, 0.5, 1, 2, 3, 4 or 5 mg / mL.

[0214] In some embodiments, the one or more operational change is determined by the amount or concentration of the protein or total protein eluting from the chromatographic medium, or change thereof, having been determined and / or inferred to be about or less than about a predetermined lower threshold.

[0215] In some embodiments, the predetermined lower threshold indicates that the chromatographic medium is at about or less than about 20%, 10%, 5% or 1% of a dynamic binding capacity (DBC) of the chromatographic medium.

[0216] In some embodiments, the predetermined lower threshold (mg / mL) is less than about 5, 4, 3, 2, 1, 0.5, or 0.1 mg / mL.

[0217] Continuous Chromatography

[0218] The term “continuous chromatography” shall be taken to mean a chromatographic method comprising one or more column(s) packed with resin(s), which may be identical, wherein each column comprises one or more zones. A zone is a column, or a region of a column, comprising the resin where one or more chromatography steps can be performed. For example, a zone is selected from a group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, a stripping zone, or a combination thereof. In one example, a zone is selected from a group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, or a combination thereof.

[0219] Continuous chromatography comprising more than one column involves the columns being connected in an arrangement that allows the columns to be operated in series and / or in parallel. In principle, protein may be loaded on a first and / or subsequent columns while other columns (or other zones of a column) are going through an equilibration, wash, elution, and / or regeneration simultaneously. Examples of continuous chromatography will be apparent to the skilled person and / or described herein.

[0220] In some embodiments, the continuous chromatography is “continuous affinity chromatography”, which shall be taken to mean a continuous chromatographic method comprising one or more column(s) packed with affinity resin(s). Examples of columns which may be used to perform the continuous chromatography (e.g. continuous affinity chromatography) method will be apparent to the skilled person and / or described herein. For example, the continuous chromatography method may be performed using Tricorn 5 / 100 (Cytiva). In another example, the continuous chromatography method may be performed using BioSMB PD System (Sartorius). Suitable examples of continuous affinity chromatography processes and sets up include, but are not limited to, those described in W02023007445A1, the contents of which is hereby incorporated by reference herein. Further examples include the Sartorius BioSC (e.g. Resolute BioSC), Contichrom CUBE, and Contichrom TWIN LPLC (YMC America).

[0221] In some embodiments, the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, periodic countercurrent chromatography (PCC), continuous counter-current tangential chromatography (CCTC), and continuous counter-current spiral chromatography (CCSC). In one example, the continuous affinity chromatography is simulated moving bed (SMB) chromatography. In another example, the continuous affinity chromatography is periodic counter-current chromatography (PCC). In a further example, the continuous affinity chromatography is continuous counter-current tangential chromatography (CCTC). In one example, the continuous affinity chromatography is continuous counter-current spiral chromatography (CCSC).

[0222] In one example, the continuous chromatography is simulated moving bed (SMB) chromatography. The term “simulated moving bed chromatography” or “SMB chromatography” refers to a chromatography method first described in US patent 2,985,589. Examples of SMB chromatography setup and / or apparatus will be apparent to the skilled person and / or described herein. The concept of simulated moving bed involves the use of multiple smaller columns (rather than one large column) containing a solid absorbent (e.g. affinity resin) and performing one or more continuous chromatography steps (i.e. equilibration, binding, washing, eluting or stripping) simultaneously on different columns in a continuous loop.

[0223] An example of a SMB chromatography set up has columns arranged into four sections with one or more columns per section. Two inlet streams (feed and eluent) and two outlet streams (extract and raffinate) are directed in alternating order to and from the column ring. The inlet and outlet positions are switched at regular time intervals in the direction of the liquid flow, thus simulating counter-current movement of columns. A feed (containing adsorbable components (extract)) is loaded onto one or more columns of the SMB chromatography setup, and the extract binds to the resin within the columns. Meanwhile, less adsorbed components (raffinate) in the feed pass through the column. The raffinate may be loaded onto one or more subsequent column(s) or removed from the SMB chromatography system as waste. An eluent is loaded onto the column to collect the extract. For example, an eluate may be collected from a first column while more feed is loaded onto one or more subsequent column(s).

[0224] In one example, the continuous chromatography is periodic counter-current chromatography (PCC). Examples of PCC setup and / or apparatus will be apparent to the skilled person and / or described herein. The concept of PCC involves the use of multiple columns containing a solid absorbent (e.g. affinity resin) and performing the chromatography steps in parallel in a quasi -continuous manner. The buffers used in binding, washing, and / or elution steps flow counter-current to the affinity resin.

[0225] An example of PCC setup involves the use of two columns. In a first step, a sample is loaded onto a first column above the DBC of the resin so that unbound product (e.g. IgG) breaks through the first column and is captured by the second column. In a second step, the first column is washed, eluted, cleaned and / or re-equilibrated independently of the second column being loaded with a further sample. In a third step, an additional sample is loaded onto the second column above the DBC of the resin so that unbound product breaks through the second column and is captured by the first column. In a fourth step, the second column is washed, eluted, cleaned and / or reequilibrated independently of the first column being loaded with a further sample. The process steps are continuously cycled between the two columns.

[0226] Another example of PCC setup involves the use of multiple columns. For example, a variation of the above PCC setup can involve use of multiple columns to capture unbound product which simulates use of a large column.

[0227] In one example, the continuous chromatography is continuous counter-current tangential chromatography (CCTC). Examples of CCTC setup and / or apparatus will be apparent to the skilled person and / or described herein. The concept of CCTC involves using the affinity resin in a slurry form where the slurry is continuously directed through a number of static mixers and hollow fiber membranes which separate the fluid phase from the resin. CCTC is ordinarily performed at low pressures.

[0228] An example of a CCTC process involves binding, first wash, second wash, elution, stripping and / or equilibration steps. Another example of a CCTC process involves binding, first wash, second wash, elution and / or equilibration steps. For example, the CCTC process does not involve a stripping step. Sample (e.g. plasma or fraction thereof) and the affinity resin is passed through static mixers and hollow fiber membranes in a binding step. Impurities are removed in the flow through of the hollow fiber membranes in the washing step, while resin bound product (i.e. IgG) is retained by the membrane. The hollow fibres retain the resin and allow the product to flow through in the elution step. The resins are stripped and / or equilibrated and process repeated.

[0229] In one example, the continuous chromatography is continuous counter-current spiral chromatography (CCSC). Examples of CCSC setup and / or apparatus will be apparent to the skilled person and / or described herein. The concept of CCSC involves the use of a compact rotating coil separation column mounted onto a centrifuge rotary frame. There are two separation column designs currently available: the spiral disk assembly and the spiral tube support assembly.

[0230] An exemplary CCSC process involves a coiled separation column revolving around a central axis of the centrifuge while it synchronously rotates about its own axis (at e.g., 1,000 to 1,200 rpm). A mobile phase can be passed through the centrifuge rotor without rotary seals, and a large amount of a stationary phase is retained while the two phases are mixed along the length of the column to produce a highly efficient solute separation.

[0231] In one example, the method comprises loading the plasma or fraction thereof onto the affinity chromatography resin.

[0232] Suitable times for contacting the resin with the plasma or fraction thereof, wash buffers, equilibration buffers, elution buffers, and the like will be apparent to the person skilled in the art, and may for example, be at least 0.1 minutes, for example between about 0.1 and about 5 minutes.

[0233] In one example, the method further comprises washing the one or more subsequent column(s) with a wash buffer described herein and collecting the bound protein from the one or more subsequent column(s).

[0234] The continuous chromatography method provides for use of buffers which enable efficient protein loading and elution from the resin. Generally, plasma or fraction thereof are at a neutral pH (pH of about 7.4). Generally, the resin is equilibrated with an equilibration buffer and / or washed with a wash buffer having a buffering range which covers the neutral pH. Suitable wash buffers comprise buffering agents having a dissociation constant (pKa) between 6.8 and 8.5 at 25°C.

[0235] Suitable wash and elution buffers will be apparent to the skilled person and / or described herein. Suitable examples include but are not limited to, wash buffers comprising a buffering agent selected from a group consisting of sodium dihydrogen phosphate, sodium citrate, imidazole, Tris, glycylglycine, 3 -morpholinopropane- 1- sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[(2- Hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), bis[(2- hydroxy ethyl)amino]acetic acid (Bicine), 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), sulfurous acid, 4-(2 -Hydroxy ethyl)- 1- piperazinepropanesulfonic acid (EPPS), N-(Hydroxyethyl)piperazine-N'-2- hydroxypropanesulfonic acid (HEPPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS), Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), N- [Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), Tricine, triethanolamine (TEA) and combinations thereof. The buffering agent(s) of the wash buffer may be at a concentration of between 5mM to 200mM. The wash buffer may further comprise sodium chloride, for example, at a concentration of up to 1000 mM, for example between about 5mM and 50mM. The wash buffer may further comprise a divalent salt, for example, at a concentration of up to 1000 mM, for example between about 5mM and 50mM. Divalent salts are known in the art, and suitable examples include but are not limited to, magnesium chloride, calcium chloride, barium chloride, copper (II) chloride, nickel chloride, manganese chloride, and combinations thereof. In one example, the wash buffer comprises 20 mM sodium dihydrogen phosphate, 145 mM sodium chloride and is at a pH of 7.4. In one example, the wash buffer comprises 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride and is at a pH of 7.4.

[0236] Suitable examples of elution buffers include, but are not limited to, those comprising an agent selected from sodium acetate, acetic acid and sodium citrate. In one example, the elution buffer comprises sodium acetate, acetic acid, sodium citrate and sodium dihydrogen phosphate. In one example, the elution buffer is or comprises a sodium phosphate buffer and / or an acetate buffer. For example, the elution buffer comprises sodium acetate. For example, the elution buffer comprises acetic acid. For example, the elution buffer comprises sodium citrate. For example, the elution buffer comprises sodium dihydrogen phosphate. The agent(s) of the elution buffer may be at a concentration of between 5mM to 200mM. The elution buffer may further comprise sodium chloride, for example, at a concentration of up to 1000 mM, for example between about 5mM and 50mM. The elution buffer may further comprise a divalent salt, for example, at a concentration of up to 1000 mM, for example between about 5mM and 50mM.

[0237] In one example, the resin is equilibrated. In one example, the affinity chromatography resin is equilibrated prior to and / or post loading with an equilibration buffer. In one example, the method further comprises equilibrating the resin with the equilibration buffer having a pH of between 7 and 8.

[0238] In one example, the resin is equilibrated i) after stripping the resin or ii) without stripping the resin. For example, In one example, the resin is equilibrated after stripping the resin. In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH between 7 and 8 after stripping the resin.

[0239] In one example, the method optionally comprises stripping the resin with a stripping buffer after collecting the protein from the resin.

[0240] In one example, the resin is equilibrated without stripping the resin. For example, the method comprises equilibrating the resin with the equilibration buffer after collecting the bound protein from the resin and without stripping the resin with a stripping buffer. In one example, the method comprises equilibrating the resin with the equilibration buffer having a pH of between 7 and 8 after collecting the bound protein from the resin and without stripping the resin with a stripping buffer.

[0241] In one example, the method further comprises stripping and / or equilibrating the first column at the time the bound protein is collected from the one or more subsequent column(s). In one example, the method further comprises equilibrating the first column at the time the bound protein is collected from the one or more subsequent column(s). For example, the method does not comprise stripping the first column at the time the bound IgG is collected from the one or more subsequent column(s).

[0242] In one example, the method further comprises stripping and / or equilibrating the one or more subsequent column(s) at the time bound protein is collected from the first column. In one example, the method further comprises equilibrating the one or more subsequent column(s) at the time bound protein is collected from the first column. For example, the method does not comprise stripping the one or more subsequent column(s) at the time bound protein is collected from the first column. In one example, the method further comprises stripping and / or equilibrating the first column at the time the one or more subsequent column(s) are washed with a wash buffer described herein. In one example, the method further comprises equilibrating the first column at the time the one or more subsequent column(s) are washed with a wash buffer described herein. For example, the method does not comprise stripping the first column at the time the one or more subsequent column(s) are washed with a wash buffer described herein. In one example, the method further comprises stripping and / or equilibrating the one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein. In one example, the method further comprises equilibrating the one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein. For example, the method does not comprise stripping the one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein. One example of a buffering agent of the equilibration and / or wash buffer is sodium dihydrogen phosphate, where the phosphoric acid component of sodium dihydrogen phosphate has three dissociation constants (pKa: 2.16, 7.21 and 12.32). Phosphoric acid has a dissociation constant at about the pH of an elution and / or stripping buffer used in a continuous affinity chromatography method according to the present disclosure, where the protein is an IgG. However, phosphoric acid does not have a dissociation constant between the pH of the equilibration and / or wash buffer (higher pH) and the elution and / or stripping buffer (lower pH) used in the continuous affinity chromatography method. This enables a fast switch between wash and elution steps, and stripping and equilibration steps, giving more defined peaks and shorter chromatography phases. An advantage of using such equilibration and / or wash buffers is that smaller buffer volumes can be used, thereby increasing the efficiency of the continuous affinity chromatography method.

[0243] Other suitable buffering agents of the equilibration and / or wash buffer include, but are not limited to, imidazole (pKa: 7.0), Tris (pKa: 8.30), glycylglycine (pKa: 8.40), MOPS (pKa: 7.2), PIPES (pKa: 6.8), TES (pKa: 7.40), Bicine (pKa: 8.35), HEPES (pKa: 7.55), EPPS (pKa:8.00), HEPPSO (pKa: 7.85), MOBS (pKa: 7.60), POPSO (pKa: 7.78), TAPSO (pKa: 7.61), Tricine (pKa: 8.05), TEA (pKa: 7.76).

[0244] In one example, the method further comprises regenerating the resin.

[0245] In one example, the method further comprises sanitising the resin.

[0246] The resin in continuous chromatography may undergo multiple cycles (e.g. at least 50 cycles) of resin equilibration, IgG loading, binding, elution, stripping, sanitising, and / or regeneration per batch of plasma or fraction thereof used. Multiple batch runs (e.g. 4 to 10 batches) may be performed using the continuous chromatography resin. The total life time of the resin can be in the range of 200 to 500 cycles (if not more) before the resin is unusable. Resin regeneration is generally performed to allow multiple uses of the resin.

[0247] It will be apparent to the skilled person that the number of cycles per batch of plasma or fraction thereof will be dependent on the volume and / or weight of plasma or fraction thereof.

[0248] Chromatographic media / resin

[0249] It will be appreciated that any chromatographic media capable of isolating the desired protein from the plasma or fraction thereof may be used. Suitable examples of chromatographic media include, but are not limited to, ion exchange chromatographic media, an affinity chromatographic media, a hydrophobic interaction chromatographic media, a mixed mode chromatographic media, an adsorption chromatographic media and a partition chromatographic media.

[0250] The term “chromatographic media” or “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. For example, the chromatography medium is a chromatography resin. Exemplary chromatography resins include MabSelect® SuRe® (Cytiva), MabSelect® SuRe® LX (Cytiva), POROS® PI50 (ThermoFischer), CaptureSelect® FcXP affinity chromatography resins (Thermo Fisher), CaptureSelect® FcXL affinity resin (Thermo Fisher), CaptureSelect® IgG-CHl affinity resin (Thermo Fisher), 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). Further exemplary affinity chromatography resins include Protein A ligand affinity resin, and IgG-CHl-XL affinity resin (Thermo Fisher). For example, the chromatography medium is a chromatography membrane. Exemplary chromatography membranes include Purexa® A (Purilogics), Mustang Q (Pall Corporation).

[0251] The term “ion exchange medium” shall be taken to mean a chromatography medium comprising a negatively charged or positively charged functional group. For example, an ion exchange medium includes anion exchange chromatography medium and cation exchange chromatography medium. Anion exchange chromatography medium is a positively charged medium with an affinity for molecules (e.g. proteins) having net negative surface charges. Exemplary anion exchange chromatography medium include POROS® PI50 anion resin (ThermoFisher), POROS® XQ anion resin (ThermoFisher), POROS® HQ 50 anion resin (ThermoFisher), and POROS® D50 anion resin (ThermoFisher). Cation exchange chromatography medium is a negatively charged medium with an affinity for molecules (e.g. proteins) having net positive surface charges. Exemplary cation exchange chromatography medium include POROS® XS strong cation exchange resin (ThermoFisher) and POROS® HS strong cation exchange resin (ThermoFisher).

[0252] The term “hydrophobic interaction chromatography medium” shall be taken to mean a chromatography medium comprising a hydrophobic ligand attached to a matrix such as, e.g., those described herein. Hydrophobic interaction chromatography (HIC) comprising a hydrophobic interaction chromatography medium separates molecules (e.g. proteins) based on the molecule’s hydrophobicity (i.e. aversion to water). Exemplary hydrophobic interaction chromatography medium include POROS® Ethyl Hydrophobic Interaction Chromatography (HIC) resin (ThermoFisher) and POROS® Benzyl Ultra Hydrophobic Interaction Chromatography (HIC) resin (ThermoFisher).

[0253] The term “mixed mode chromatography medium” shall be taken to mean a chromatography medium which allows for the separation of molecules (e.g. proteins) based on more than one form of interaction between the molecules and the medium. For example, mixed mode chromatography medium may comprise affinity chromatography ligands and ion exchange functional groups attached to a matrix. Exemplary mixed mode chromatography medium include CMM HyperCel (Satorius), MEP HyperCel (Satorius), HEA HyperCel (Sartorius), PPA HyperCel (Sartorius), and HA Ultragel® (Satorius).

[0254] The term “adsorption chromatography medium” shall be understood to mean a chromatography medium which allows the separation of a component in a mixture (e.g., proteins) by adsorption from a mobile phase into the stationary solid surface. Exemplary stationary phases included hydroxyapatite such as ceramic hydroxyapatite (CHT type I and type II, Bio-Rad Laboratories), HA Ultragel hydroxyapatite (Pall Corp.), and ceramic fluoroapatite. (CFT Type I and Type II, Bio-Rad Laboratories).

[0255] The term “partition chromatography medium” shall be understood to mean a chromatography medium which allows for the separation of mixtures based on partition of a solute between two solvents one of which is immobilized by the substance in the separation unit (e.g., column).

[0256] In some embodiments, the chromatographic media is affinity chromatographic media or resin. Accordingly, in some embodiments, the method is for isolating or purifying a protein (such as IgG or albumin) from the plasma or fraction thereof using an affinity chromatography resin. By way of example, where the protein is IgG, the affinity resin may comprise a ligand capable of specifically binding to a CH3 domain of human IgG. Accordingly, in some embodiments, the continuous chromatography is continuous affinity chromatography.

[0257] The term “affinity chromatography resin” or “affinity chromatographic media” shall be taken to mean a resin comprising an affinity chromatography ligand (e.g. camelid-derived single domain [VHH] antibody fragment) attached to a matrix such as, e.g., those described herein. Exemplary affinity chromatography resins used in a method described herein include POROS® Capture Select® FcXP affinity resin (Thermo Fisher) and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography resins include variants thereof that specifically bind to the CH3 domain of human IgG. Exemplary affinity chromatography resins are also described in US10259886. Suitable affinity chromatography resins will be apparent to the skilled person and / or described herein. In one example, the resin comprises a ligand of camelid-derived single domain [VHH] antibody fragments. The skilled person will be aware that ligands based on camelid-derived single domain [VHH] antibody fragments are capable of specifically binding to all subclasses of IgG (IgGl, IgG2, IgG3, IgG4). Exemplary resins are the Capture Select® FcXP affinity chromatography resins (Thermo Fisher), CaptureSelect® FcXL affinity resin (Thermo Fisher), Capture Select® IgG-CHl affinity resin (Thermo Fisher), 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).

[0258] In one example, the affinity chromatography resin comprises a camelid-derived single domain [VHH] antibody fragment and a cross-linked poly(styrene- divinylbenzene) matrix. For example, the affinity chromatography resin is POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher). The cross-linked poly(styrene- divinylbenzene) matrix allows the resin to withstand pressures of up to 100 bar.

[0259] In one example, the affinity chromatography resin comprises a camelid-derived single domain [VHH] antibody fragment and an agarose-based matrix. For example, the affinity chromatography resin is CaptureSelect FcXP agarose affinity resin (Thermo Fisher).

[0260] In some embodiments, the chromatographic medium is packed into a first separation unit and one or more subsequent separation units (s). In some embodiments, the separation units(s) are columns.

[0261] In one example, the chromatographic media is packed into one or more columns wherein each column comprises one or more zones. For example, the chromatographic media is packed into a series of two or more columns. For example, the chromatographic media is packed into a series of two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve columns. In one example, the chromatographic media is packed into a series of two columns. In one example, the chromatographic media is packed into a series of three columns. In one example, the chromatographic media is packed into a series of four columns.

[0262] For example, a zone is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, a stripping zone, and a combination thereof. In another example, a zone is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, and a combination thereof. In one example, the zone is an equilibration zone. In another example, the zone is a binding zone. In a further example, the zone is a wash zone. In one example, the zone is an elution zone. In another example, the zone is a stripping zone. In one example, there is no stripping zone. In a further example, the zone is a wash / elution zone. In one example, the zone is an equilibration / binding zone. In another example, the zone is a binding / wash zone.

[0263] Methods for packing chromatographic media or resin will be known to the person skilled in the art. In one example, the chromatographic media is packed into a first column and one or more subsequent column(s). In one example, the chromatographic media has a total bed height between about 0.5 cm and about 70 cm, or between about 0.5 and about 30 cm. In one example, the column has a diameter of between 5 cm and 200 cm, for example a diameter of 5 cm, 20 cm, 50 cm, 100 cm or 200 cm.

[0264] In one example, the first column is loaded with protein at a concentration at or above the dynamic binding capacity (DBC) of the resin. Determining the DBC of a chromatographic media will be apparent to a skilled person and / or described herein. For example, the DBC of a chromatographic media may be determined by loading protein on the column and monitoring the concentration at which unbound protein flows through the column e.g. by Raman spectroscopic analysis or offline analytical tests based on immunoturbidimetry. For example, the DBC of the resin is 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg, or 50 mg, or 60 mg, or 70 mg protein per mL of resin. In one example, the DBC of the resin is at least 5 mg protein per mL of resin. In one example, the DBC of the resin is at least 10 mg protein per mL of resin. In one example, the DBC of the resin is at least 20 mg protein per mL of resin. In one example, the DBC of the resin is 40 mg protein per mL of resin.

[0265] In one example, the first column is loaded with protein at a concentration of more than 5mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg, or 50 mg, or 60 mg, or 70 mg IgG per mL of resin. In one example, the first column is loaded with protein at a concentration up to the DBC of the resin. For example, the first column is loaded with protein at a concentration of up to 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg protein per mL of resin. In one example, the first column is loaded with protein at a concentration of up to 40 mg IgG per mL of resin. In one example, the one or more subsequent column(s) are loaded with protein at a concentration up to the DBC of the resin. In one example, the one or more subsequent column(s) are loaded with protein at a concentration of up to 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg IgG per mL of resin. In one example, the one or more subsequent column(s) are loaded with protein at a concentration of up to 40 mg IgG per mL of resin. Plasma and Proteins

[0266] The methods of the present disclosure relate to isolating a protein from plasma or a fraction thereof. The protein may be endogenous to the plasma or fraction thereof, or an additive, i.e. an exogenous component added during the process, and is not naturally found in plasma. Suitable examples of proteins that may be isolated from plasma or a fraction thereof include, but are not limited to, plasma protein, peptide hormone, growth factor, cytokine, polyclonal immunoglobulin.

[0267] In one example, the plasma protein is selected from the group consisting of an immunoglobulin G (IgG), a RhD immunoglobulin protein product, an apolipoprotein Al, an albumin, a protease, a protease inhibitor, plasminogen, a fibrinogen, a von Willebrand factor, a clotting factor or activated form thereof, a cofactor or activated form thereof, a contact system factor, a prekallikrein activator (PKA), a prothrombin, thrombin, prothrombin complex factor or activated form thereof, a protein C, an anti-thrombin III, alpha acid glycoprotein, a transport protein, Factor H, a component of the complement pathway, inhibitors of any component of the complement pathway, a highly glycosylated protein Protein S, histidine-rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC-globulin, erythropoietin, interferon, tumor factors, tPA, yCSF, an oligomeric form or degradation product of any of the foregoing and combinations thereof.

[0268] In one example, the plasma protein is an IgG.

[0269] In one example, the plasma protein is a RhD immunoglobulin protein product

[0270] In one example, the plasma protein is an apolipoprotein Al.

[0271] In one example, the plasma protein is an albumin. For example, a-globulins and / or P-globulins.

[0272] In one example, the plasma protein is a protease. For example, a serine protease or a plasmin, or a kallikrein or a FX / FXa.

[0273] In one example, the plasma protein is a protease inhibitor. For example, a serine protease inhibitor, a Cl esterase inhibitor, an alpha- 1- antitrypsin, an anti-thrombin, a P- antithrombin, an a-antithrombin, an a-2-macroglobulin, a tissue factor pathway inhibitor (TFPI), a heparin cofactor II, a protein C inhibitor (PAI-3), an a-1 esterase inhibitor protein or antiangionetic proteins. In one example, the plasma protein is an alpha- 1- antitrypsin. In one example, the plasma protein is a Cl esterase inhibitor. In one example, the plasma protein is an anti-thrombin. For example, P-antithrombin or a-antithrombin or an anti-thrombin III. In one example, the plasma protein is a P-antithrombin. In one example, the plasma protein is an a-antithrombin. In one example, the plasma protein is an a-2-macroglobulin. In one example, the plasma protein is a tissue factor pathway inhibitor (TFPI). In one example, the plasma protein is a heparin cofactor II. In one example, the plasma protein is a protein C inhibitor (PAI-3). In one example, the plasma protein is an a-1 esterase inhibitor protein. In one example, the plasma protein is an anti angiogenic protein. For example, latent-anti-thrombin. In one example, the plasma protein is plasminogen.

[0274] In one example, the plasma protein is a fibrinogen.

[0275] In one example, the plasma protein is a von Willebrand factor.

[0276] In one example, the plasma protein is a prekallikrein activator (PKA).

[0277] In one example, the plasma protein is a prothrombin complex factor or activated forms thereof. For example, the plasma protein is a factor Xl / XIa, a factor II / IIa, a factor Vll / VIIa, a factor IX / IXa, or a X / Xa. In one example, the plasma protein is a factor Xl / XIa. In one example, the plasma protein is a factor II / IIa. In one example, the plasma protein is a factorVII / VIIa. In one example, the plasma protein is a factorIX / IXa. In one example, the plasma protein is a factor X / Xa.

[0278] In one example, the plasma protein is a prothrombin.

[0279] In one example, the plasma protein is a thrombin.

[0280] In one example, the plasma protein is a protein C.

[0281] In one example, the plasma protein is alpha acid glycoprotein.

[0282] In one example, the plasma protein is a transport protein. For example, the plasma protein is haptoglobin. In one example, the plasma protein is hemopexin. In one example, the plasma protein is transferrin. In one example, the plasma protein is a ceruloplasmin. In one example, the plasma protein is a hemoglobulin.

[0283] In one example, the plasma protein is Factor H.

[0284] In one example, the plasma protein is a clotting factor or activated form thereof. For example, the clotting factor is factor X / Xa, factor Vll / VIIa, factor Vlll / VIIIa, a factor IX / IXa, factor Xll / XIIa, factor Xlll / XIIIa and / or factor Xl / XIa. In one example, the coagulation factor is factor X / Xa. In another example, the coagulation factor is factor Vll / VIIa. In a further example, the coagulation factor is factor Vlll / VIIIa. In one example, the coagulation factor is factor IX / IXa. In another example, the coagulation factor is factor Xl / XIa. In a further example, the coagulation factor is factor Xll / XIIa. In one example, the coagulation factor is factor Xlll / XIIa.

[0285] In one example, the plasma protein is a cofactor or activated forms thereof. For example, the plasma protein is factor V / Va, factor Vlll / VIIIa or factor XIH / XHIa. In one example, the plasma protein is factor V / Va. In one example, the plasma protein is FVIII / VIIIa. In one example, the plasma protein is factor Xlll / XIIIa. In one example, the plasma protein is a contact system factor. For example, the plasma protein is a factor Xl / XIa, factor Xll / XIIa or plasma kallikrein. In one example, the plasma protein is a factor Xl / XIa. In one example, the plasma protein is a factor Xll / XIIa. In one example, the plasma protein is a plasma kallikrein.

[0286] In one example, the plasma protein is a component of the complement system.

[0287] In one example, the plasma protein is a Protein S.

[0288] In one example, the plasma protein is a highly glycosylated protein. For example, a- 1 -acid glycoprotein, anti chymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein or a C-reactive protein. In one example, the plasma protein is an antichymotrypsin. In one example, the plasma protein is an a- 1 -acid glycoprotein. In one example, the plasma protein is an inter-a-trypsin inhibitor. In one example, the plasma protein is an a-2-HS glycoprotein. In one example, the plasma protein is a C-reactive protein.

[0289] In one example, the plasma protein is a histidine-rich glycoprotein.

[0290] In one example, the plasma protein is a mannan binding lectin.

[0291] In one example, the plasma protein is a C4-binding protein.

[0292] In one example, the plasma protein is a fibronectin.

[0293] In one example, the plasma protein is a GC-globulin.

[0294] In one example, the plasma protein is an erythropoietin.

[0295] In one example, the plasma protein is an interferon.

[0296] In one example, the plasma protein is a tumor factors.

[0297] In one example, the plasma protein is a tPA.

[0298] In one example, the plasma protein is a yCSF.

[0299] In one example, the plasma protein is an oligomeric form or degradation product of any of the foregoing plasma proteins.

[0300] It will be apparent to the skilled person from the disclosure herein that reference to a clotting factor (e.g., Factor X or FX) includes reference to the zymogen (or inactive form) as well as the activated form thereof (e.g., Factor Xa or FXa) and vice versa.

[0301] In one example, the plasma protein is a natural or recombinant plasma protein. In one example, the plasma protein is a naturally occurring plasma protein. In another example, the plasma protein is a recombinantly produced plasma protein.

[0302] In one example, the plasma protein is derived from serum, plasma, a fermentation broth, a cell culture harvest or a protein suspension.

[0303] In one example, the sample is serum, plasma, a plasma fraction, or a purified or partially purified plasma or plasma fraction thereof, a fermentation broth or a purified or partially purified fermentation broth thereof, a cell culture harvest or a purified or partially purified cell culture harvest thereof or a protein suspension. In one example, the plasma or plasma fraction is selected the group consisting of a human blood plasma sample, an IgG intermediate product, a cryo-rich plasma, a cryopoor plasma, a Supernatant I (SN I), a Cohn Fraction II (Fr II), a Cohn Fraction II+III (Fr II+III), a Cohn Fraction (I+)II+III (Fr(I+)II+III), a Kistler / Nitschmann Precipitate A (KN A), a Kistler / Nitschmann Precipitate B (KN B), a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an IgG depleted intermediate product and combinations thereof. In one example, the plasma or plasma fraction is a human blood plasma sample. In another example, the plasma fraction is an IgG intermediate product. In one example, the plasma fraction is cryo-rich plasma. In another example, the plasma fraction is cryopoor plasma. In a further example, the plasma fraction is Supernatant I (SN I). In one example, the plasma fraction is Cohn Fraction II (Fr II). In another example, the plasma faction is Cohn Fraction II+III (Fr II+III). In a further example, the plasma fraction is Cohn Fraction (I+)II+III (Fr(I+)II+III). In one example, the plasma fraction is Kistler / Nitschmann Precipitate A (KN A). In another example, the plasma fraction is Kistler / Nitschmann Precipitate B (KN B). In a further example, the plasma fraction is Kistler / Nitschmann Precipitate of Supernatant B (KN B+l). In one example, the plasma fraction is an IgG depleted intermediate product.

[0304] In one example, the plasma fraction is a suspended paste. For example, the suspended paste is selected from a group consisting of Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction (I+)II+III ((FrI+)II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), and combinations thereof. In one example, the suspended paste is a Cohn Fraction II (Fr II) paste. In one example, the suspended paste is a Cohn Fraction II+III (Fr II+III) paste. In another example, the suspended paste is a Cohn Fraction (I+)II+III ((FrI+)II+III) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate A (KN A) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate B (KN B) paste. In a further example, the suspended paste is a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l) paste.

[0305] In one example, the plasma fraction is selected from the group consisting of a mammalian plasma fraction, a human plasma fraction, an equine plasma fraction, and a bovine plasma fraction. In one example, the plasma fraction is a mammalian plasma fraction. In one example, the plasma fraction is a human plasma fraction. In one example, the plasma fraction is an equine plasma fraction. In one example the plasma fraction is a bovine plasma fraction. In one example the plasma fraction is a bovine plasma fraction comprising human polyclonal antibodies. In one example, the plasma sample or fraction thereof is a human blood plasma sample from one or more subjects.

[0306] In one example, the plasma is hyperimmune plasma. For example, hyperimmune anti-D, tetanus and / or hepatitis B plasma. The plasma, whether pooled from more than one or several hundred individuals, or whether obtained from a single individual, may be hyperimmune plasma. For example, the plasma may be obtained from the blood of individual(s) who have / has mounted an immune response to an infection, and have recovered (and are therefore otherwise healthy individuals).

[0307] In one example, the plasma or plasma fraction has been purified or partially purified.

[0308] In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to one or more steps selected from the group consisting of clarification, ethanol precipitation, octanoic acid fractionation, ammonium sulphate precipitation, filtration and combinations thereof.

[0309] In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to clarification. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ethanol precipitation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to octanoic acid fractionation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ammonium sulphate precipitation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to filtration.

[0310] In one example, the plasma or fraction thereof is clarified. Methods of clarification of the plasma or fraction thereof will be apparent to the skilled person and / or described herein. For example, the plasma or fraction thereof is clarified using a separator, centrifuge and / or continuous centrifuge. For example, the plasma or fraction thereof is clarified by passing the plasma or fraction thereof through a filter. For example, a depth or membrane filter can be used or a cake filtration using filter aid can be used. For example, the plasma or fraction thereof is passed through a combination of filters. For example, the combination may be a 1.2 and 0.45 / 0.22 pm membrane filter combination (e.g. Sartorius Sartopure® PP3 and Sartorius Sartobran® P). For example, the plasma or fraction thereof is clarified by passing the plasma or fraction thereof through a depth filter (e.g. BECO® depth filter). In one example, the plasma or fraction thereof is clarified by passing the plasma or fraction thereof through a filter press (e.g. BECO® integra plate or compact plate) comprising one or more depth filter(s). In one example, the filter press further comprises one or more filter aid(s) (e.g. cellulose-based filter aids such as Diacel® 150). In one example, the plasma or fraction thereof is clarified by passing the plasma or fraction thereof through a lipid-specific filter (e.g. Zeta Plus ™ DEL Series filter). For example, the plasma fraction is clarified Supernatant I (SN I). For example, the plasma fraction is clarified Cohn Fraction II (Fr II). For example, the plasma faction is clarified Cohn Fraction II+III (Fr II+III). For example, plasma fraction is clarified Cohn Fraction (I+)II+III ((FrI+)II+III). For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate A (KN A). For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate B (KN B). For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate of Supernatant B (KN B+l). In one example, the plasma is clarified cryo-rich plasma. In one example, the plasma fraction is clarified cryo-poor plasma.

[0311] In one example, the plasma or fraction thereof is at a temperature in the range of 1 °C to 35 °C before the continuous affinity chromatography step. In one example, the plasma or fraction thereof is at the temperature for up to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 48 hrs.

[0312] In one example, the plasma or fraction thereof is stored at a temperature of 1 °C and then warmed up to 18 to 22 °C before both clarifying filtration and the subsequent continuous chromatography step is performed. In one example, the plasma or fraction thereof is at a first temperature in the range of 30°C to 38°C and then at a second temperature in the range of 2°C to 28°C before the continuous affinity chromatography step and / or prior to loading the plasma or fraction thereof onto the chromatographic media. For example, the plasma or fraction thereof is warmed to a first temperature in the range of 30°C to 35°C or 30°C to 38°C and then cooled to a second temperature in the range of 18°C to 25°C or 2°C to 28°C before the continuous chromatography step.

[0313] In one example, the plasma or fraction thereof is at the first and / or second temperature for up to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 48 hrs.

[0314] In one example, the method is performed at large scale. For example, the method is performed on an industrial or a commercial scale. Methods of performing on an industrial or a commercial scale will be apparent to a skilled person and / or described herein. For example, the method performed on an industrial scale comprises large scale purification of protein from the plasma or fraction thereof.

[0315] In one example, large scale purification of protein is performed using at least 500kg of the plasma or fraction thereof. For example, large scale purification of protein is performed using between 500kg to 1000kg, or 1000kg to 2500kg, or 2500kg to 5000kg, or 5000kg to 7500kg, or 7500kg, or 10000kg, or 10000kg to 12500kg, or 12500kg to 15000kg of the plasma or fraction thereof. In one example, large scale purification of protein is performed using at least 1000kg, or 2500kg, or 5000kg, or 7500kg, or 10000kg, or 12500kg, or 15000kg of the plasma of fraction thereof.

[0316] Analysis of protein composition

[0317] Methods of determining yield, purity and identify, class or subclass of the protein following purification will be apparent to the skilled person and / or described herein.

[0318] In one example, purity is determined by SDS-PAGE and MALDI-TOF-MS peptide fingerprint analysis. Briefly, purified protein (e.g. IgG), a protein-enriched preparation or protein-containing pharmaceutical composition described herein is loaded onto a suitable SDS-PAGE gel (e.g. 8-16% TRIS-glycine), along with a protein size marker and a positive control for the protein under reduced and non-reduced conditions. Proteins are separated based on size and protein bands of interest are isolated, processed and analysed by MALDI-TOF-MS.

[0319] In another example, where the protein is an IgG, impurities in the IgG-enriched preparation or IgG-containing pharmaceutical composition described herein are measured in an Enzyme-Linked Immunosorbent Assay (ELISA) using impurity (e.g. IgA) specific antibodies. For example, the ELISA is performed using commercially available methods, for example with a Cedex Bio HT Analyzer. In one example, purity, yield and / or subclass distribution of IgG is determined by nephelometry. In one example, purity of IgG is determined by nephelometry. In one example, yield of IgG is determined by nephelometry. In one example, subclass distribution of IgG is determined by nephelometry. For example, the light scattering patterns of purified IgG, an IgG-enriched preparation or IgG-containing pharmaceutical composition described herein is measured by nephelometry and compared to light scattering profiles of compositions with known IgG subclass distributions. In one example, IgG concentration and / or yield of IgG and / or purity of IgG is determined by immunoturbidimetry.

[0320] In one example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof. In another example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof following the continuous chromatography method. For example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof following the continuous chromatography method without further purification steps. For example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof following the continuous chromatography method with further purification steps. For example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof following an ion exchange chromatography step. In one example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof following an anion exchange chromatography step. In one example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from the plasma or fraction thereof, wherein the protein (e.g. IgG, albumin) is derived from at least 500 kg of plasma or fractions thereof. For example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the protein (e.g. IgG, albumin) is recovered from large scale purification of the plasma or fraction thereof.

[0321] In one example, the protein (e.g. IgG, albumin) has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In another example, the protein (e.g. IgG, albumin) has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% following the continuous chromatography method. In one example, the protein (e.g. IgG, albumin) has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% following the continuous chromatography method without further purification steps. In one example, the protein (e.g. IgG, albumin) has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% following the continuous chromatography method with further purification steps. In one example, the protein (e.g. IgG, albumin) having a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% is derived from at least 500kg of plasma or fraction thereof. For example, the protein (e.g. IgG, albumin) having a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% is recovered from large scale purification of the plasma or fraction thereof. In one example, the protein (e.g. IgG, albumin) has a purity of 90%. In one example, the protein (e.g. IgG, albumin) has a purity of 95%. In one example, the protein (e.g. IgG, albumin) has a purity of 96%. In one example, the protein (e.g. IgG, albumin) has a purity of 97%. In one example, the protein (e.g. IgG, albumin) has a purity of at least 98%. In another example, the protein (e.g. IgG, albumin) has a purity of at least 98% following the continuous chromatography method. In one example, the protein (e.g. IgG, albumin) has a purity of at least 98% following the continuous chromatography method without further purification steps. In one example, the protein (e.g. IgG, albumin) has a purity of at least 98% following the continuous chromatography method with further purification steps. In one example, the protein (e.g. IgG, albumin) having a purity of at least 98% is derived from at least 500kg of plasma or fraction thereof. For example, the protein (e.g. IgG, albumin) having a purity of at least 98% is recovered from large scale purification of the plasma or fraction thereof.

[0322] Stability of plasma and fractions thereof

[0323] The stability of the plasma or fraction thereof for loading onto a resin described herein can be determined by assessing the pro-coagulant activity, proteolytic activity and particle size of the plasma or fraction thereof. Methods for assessing pro-coagulant activity, proteolytic activity and particle size will be apparent to a skilled person and / or described herein. Briefly, the plasma or fraction thereof is freeze / thawed in one or more cycles, stored at between 1°C and 37°C (e.g. 1°C, 2°C, 10°C, 18°C, 21°C, 28°C, 32°C, 35°C or 37°C) for 12 or 24 or up to 48 hrs and analysed using one or more of the methods described below. In one example, the plasma or fraction thereof is thawed in one or more cycles at a temperature of 32°C, stored for 12 or 24 hours, or up to 48 hours and analysed using one or more of the methods described below. In another example, the plasma or fraction thereof is thawed in one or more cycles at a temperature of 32°C, stored for 12 or 24 hours, or up to 48 hours and analysed using one or more of the methods described below and then cooled and stored at a temperature of 21 °C. In one example, the plasma or fraction thereof is thawed at a temperature of 32°C and at a temperature of 21°C before the continuous affinity chromatography.

[0324] In one example, the pro-coagulant activity in the plasma or fraction thereof can be determined using an in vitro coagulation assay, e.g., activated partial thromboplastin time (NaPTT) assay. The NaPTT assay measures the rate at which one or more coagulation factors are activated in plasma, or a fraction thereof.

[0325] In one example, proteolytic activity in the plasma or fraction thereof can be assessed by measuring the activity of thrombin, serine proteases, kallikrein, plasmin and FXa e.g. using commercially available kits, such as thrombin activity assay kit (S-2238), serine protease assay kit (S-2288), kallikrein activity assay kit (S-2302), plasmin activity assay kit (S-2251) and FXa activity kit (S-2765).

[0326] In one example, the size of any particles in the plasma or fraction thereof is assessed by microflow imaging (MFI) and polydispersity index is calculated. Calculation of the poly dispersity index will be apparent to the skilled person.

[0327] Additional Purification Steps

[0328] Additional purification steps may be performed before or after the continuous chromatography step. In one example, additional purification steps may be performed before the continuous chromatography step. In one example, additional purification steps may be performed after the continuous chromatography step.

[0329] In one example, the method further comprises one or more steps selected from a group consisting of ethanol precipitation, octanoic acid fractionation, ion exchange chromatography, viral inactivation, viral filtration and ultrafiltration / diafiltration. Additional purification steps will be apparent to the skilled person and / or described herein.

[0330] In one example, the method further comprises ethanol precipitation. For example, cold ethanol may be used to isolate and enrich IgG by removing albumin and a- and P- globulins from the plasma or fractions thereof. For example, as described in WO201 1 / 149472.

[0331] In one example, the method further comprises immunoaffinity chromatography. For example, the method further comprises isoagglutinin affinity chromatography using Eshmuno anti-A and anti-B resin. For example, isoagglutinin affinity chromatography may be used to remove isoagglutinins A and B.

[0332] In one example, the method further comprises octanoic acid fractionation. Octanoic acid may be used to remove of plasma lipids and plasma proteins (other than IgG). For example, as described in WO2011 / 131787.

[0333] In one example, the method further comprises ion exchange chromatography. In one example, the ion exchange chromatography is anion exchange chromatography. For example, anion exchange chromatography may be used to remove IgA, remaining IgM and other plasma components (other than IgG).

[0334] The anion exchanger can be a resin-based anion exchanger, an anion exchange membrane adsorber, or any other format of anion exchanger with a positively charged substrate for capturing negatively charged particles. In one example, the anion exchanger is an anion exchange membrane adsorber. In another example, the anion exchanger is a resin-based anion exchanger. In a further example, the anion exchanger is a monolithic anion exchanger.

[0335] In one example, the method further comprises anion exchange chromatography using a resin-based anion exchanger. For example, the anion exchange chromatography resin is a strong anion exchanger. In one example, the strong anion exchange resin comprises a matrix consisting of a poly(styrene-divinylbenzene) matrix. In one example, the strong anion exchanger comprises a quaternized polyethyleneimine functional group. Suitable resin-based anion exchanges will be apparent to the skilled person and include, for example, POROS™ HQ 50. In one example, the anion exchange chromatography step is performed in flow through mode. In another example, the anion exchange chromatography step is performed in bind-and-elute mode. In another example, the method further comprises an anion exchange chromatography step using a strong anion exchange resin operated in flow through mode.

[0336] In one example, the anion exchange chromatography step comprises a buffer selected from the group consisting of sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium dihydrogen phosphate, Bis-Tris, phosphate, L-histidine and combinations thereof. In one example, the anion exchange chromatography step comprises a buffer comprising MES buffer. In another example, the anion exchange chromatography step comprises phosphate buffer.

[0337] In one example, the method further comprises viral inactivation. For example, viral inactivation may be effected by adjusting the solution to low pH. Low pH may be a pH of between 2 to 4. In one example, low pH viral inactivation is performed in the presence of caprylate. In another example, viral inactivation may be effected by contacting the plasma or fraction thereof, or an IgG-enriched preparation or IgG- containing pharmaceutical composition with w-Octyl-P-D-Glucopyranoside (OG), thereby forming an OG-IgG mixture. In a further example, low pH viral inactivation is performed in the presence of A,A-dimethylmyristylamine A-oxide (TDAO).

[0338] In a further example, viral inactivation may be effected by exposing the plasma or fraction thereof, or an IgG-enriched preparation or IgG-containing pharmaceutical composition to a solvent-detergent inactivation step. Suitable solvent-detergent treatments would be apparent to the skilled person and include, for example environmentally friendly detergents. Exemplary environmentally friendly detergents suitable for use in the present disclosure and in particular for use in inactivating lipid enveloped viruses include N, A-dimethylmyristylamine A-oxide (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced), and a non-ionic surfactant prepared from glucose and alcohol (e.g., Simulsol™ formulations). In one example, the detergent is A( A-dimethylmyristylamine A -ox ide (TDAO). In one example, the detergent is polysorbate 80. In another example, the detergent is polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced). In a further example, the detergent is a non-ionic surfactant prepared from glucose and alcohol.

[0339] In one example, the method further comprises viral filtration. For example, viral filtration membranes of pore sizes from 15-20 nm may be used to remove microbes and viruses from a solution or eluate or pharmaceutical composition. Exemplary nanofilters include Planova S20N (Asahi), Virosart HC (Sartorius) and Planova 20N (Asahi). In one example, the method further comprises ultrafiltration / diafiltration. An exemplary ultrafiltration / diafiltration membrane is Pellicon 2 Cassettes (Millipore) or Poly ethersulfone or Hydrosart cassettes (Sartorius).

[0340] Pharmaceutical Compositions

[0341] Purified proteins provided by the methods of the present disclosure that are active ingredients (e.g. IgG, albumin), are useful for formulating into a pharmaceutical composition for parenteral administration, such as intravenous administration or subcutaneous administration, for therapeutic and prophylactic treatment.

[0342] The compositions for administration will commonly comprise a solution of the purified protein of the disclosure dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable carriers as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

[0343] The concentration of the purified protein of the present disclosure in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives. For example, the pharmaceutical composition comprises proline as a stabilising agent.

[0344] Suitable pharmaceutical compositions in accordance with the disclosure will generally include an amount of the purified protein of the present disclosure admixed with an acceptable pharmaceutical carrier, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980.

[0345] For example, the protein concentration of the pharmaceutical composition is 1 to 5% w / v, 5 to 15% w / v, or 8 to 12% w / v. For example, the protein concentration of the pharmaceutical composition is 1%, 2%, 3%, 4%, 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15% w / v. For intravenous use, 1% w / v (i.e. 10g protein / L may be used. For intravenous use, 10% w / v (i.e. 100g protein / L) may be used. For subcutaneous administration, a higher concentration may be used. For example, 15 to 35% w / v, or 20 to 30% w / v. In one example, the protein concentration of the pharmaceutical composition is 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26% w / v.

[0346] Method of use

[0347] The present disclosure provides a method of treating, preventing and / or delaying progression of a condition in a subject, comprising administering a protein or a pharmaceutical formulation thereof to the subject, wherein the protein or pharmaceutical formulation thereof are as described according to any aspect, embodiment or example provided herein.

[0348] The present disclosure also provides for use of a protein or a pharmaceutical formulation according to any aspect, embodiment or example provided herein, in the manufacture of a medicament for treating, preventing and / or delaying progression of a condition in a subject.

[0349] In one example, the condition is an immunodeficiency, autoimmune disease or acute infection. For example, the condition is allogenic bone marrow transplant, chronic lymphocytic leukaemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), kidney transplant with a high antibody recipient or with an ABO incompatible donor, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves' ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, Lupus erythematosus, multifocal motor neuropathy, multiple sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus B19 infection, pemphigus, posttransfusion purpura, renal transplant rejection, spontaneous Abortion Miscarriage, stiff person syndrome, opsoclonus Myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X- linked agammaglobulinemia, hypogammaglobulinemia, primary immune deficiency, RRMS, Alzheimer's disease, and Parkinson's disease.

[0350] In one example, the condition is selected from a group consisting of primary immunodeficiency disease (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP). In one example the condition is primary immunodeficiency disease (PI). In one example, the condition is chronic inflammatory demyelinating polyneuropathy (CIDP). In one example, the condition is chronic immune thrombocytopenic purpura (ITP). In one example of any method described herein, the subject is a mammal, for example a primate such as a human.

[0351] EXAMPLE EMBODIMENTS

[0352] The present disclosure can also be described by reference to one or more of the following example embodiments. It will be appreciated that the specific embodiments presented below are not intended to be limiting to the scope. It will be appreciated that persons skilled in the art may incorporate one or more of the elements, features or embodiments in the listing below (indeed, any such aspect or embodiment described herein) into combinations not specifically set forth herein. All such embodiments are considered to be within the scope of the disclosure.

[0353] 1. A method of isolating a protein from plasma or a fraction thereof using continuous chromatography, the method comprising:

[0354] (a) loading an amount of plasma, or fraction thereof, comprising the protein onto a chromatographic medium capable of separating the protein from at least a portion of the amount of plasma or fraction thereof;

[0355] (b) eluting the protein from the chromatographic medium; and

[0356] (c) collecting the protein; wherein, Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium and / or which is in the amount of plasma or fraction thereof being loaded onto the chromatographic medium.

[0357] 2. The method of example embodiment 1, wherein the Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium.

[0358] 3. The method of example embodiment 1 or example embodiment 2, wherein the Raman spectroscopic analysis monitors for breakthrough of the protein due to overloading of the chromatographic medium.

[0359] 4. The method of any one of example embodiments 1 to 3, wherein the Raman spectroscopic analysis monitors for elution of the protein in an elution buffer. 5. The method of any one of example embodiments 1 to 4, wherein the Raman spectroscopic analysis monitors for elution of the protein in a wash solution.

[0360] 6. The method of any one of example embodiments 1 to 5, wherein the Raman spectroscopic analysis comprises determination of a change in the Raman spectra of a solution eluting from the chromatographic medium, and inferring a change in an amount or concentration of the protein or total protein.

[0361] 7. The method of any one of example embodiments 1 to 6, wherein the Raman spectroscopic analysis comprises determination of a change in the amount or concentration of the protein.

[0362] 8. The method of any one of example embodiments 1 to 7, wherein the Raman spectroscopic analysis comprises determination of a change in the amount or concentration of total protein.

[0363] 9. The method of any one of example embodiments 1 to 8, wherein the Raman spectroscopic analysis comprises determination of a concentration of the protein.

[0364] 10. The method of any one of example embodiments 1 to 9, wherein the Raman spectroscopic analysis comprises determination of a concentration of total protein.

[0365] 11. The method of any one of example embodiments 1 to 10, wherein the use of the Raman spectroscopic analysis to monitor for the protein, comprises monitoring in the absence of the protein.

[0366] 12. The method of any one of example embodiments 1 to 11, wherein the Raman spectroscopic analysis is used to determine that a sample is a composition corresponding to at least one of one or more predetermined compositions, and / or that the composition of the sample has changed from one to another of the one or more predetermined compositions.

[0367] 13. The method of any one of example embodiments 1 to 12, wherein the monitoring by Raman spectroscopic analysis determines one or more operational change in the continuous chromatography. 14. The method of example embodiment 13, wherein the one or more operational change is determined by the amount or concentration of the protein or total protein eluting from the chromatographic medium, or change thereof, having been determined and / or inferred to be about or greater than about a predetermined upper threshold.

[0368] 15. The method of example embodiment 14, wherein the predetermined upper threshold indicates that the chromatographic medium is at about or greater than about 50, 60, 70, 80, 90, 95, 99 or 100% of a dynamic binding capacity (DBC) of the chromatographic medium.

[0369] 16. The method of example embodiment 14 or example embodiment 15, wherein the predetermined upper threshold is about or greater than about 0.1, 0.5, 1, 2, 3, 4 or 5 mg / mL.

[0370] 17. The method of any one of example embodiments 13 to 16, wherein the one or more operational change comprises collecting the protein eluting from the chromatographic medium.

[0371] 18. The method of any one of example embodiments 13 to 17, wherein the one or more operational change comprises ceasing loading the chromatographic medium with the protein.

[0372] 19. The method of any one of example embodiments 13 to 18, wherein the one or more operational change comprises loading an amount of the protein eluting from the chromatographic medium onto a further chromatographic medium. 0. The method of example embodiment 19, wherein both chromatographic media are the same resin. 1. The method of example embodiment 19 or example embodiment 20, wherein the further chromatographic medium is provided in a separation unit that is distinct from a separation unit providing the (first) chromatographic medium. 22. The method of example embodiments 13 to 21, wherein the one or more operational change is determined by the amount or concentration of the protein or total protein eluting from the chromatographic medium, or change thereof, having been determined and / or inferred to be about or less than about a predetermined lower threshold.

[0373] 23. The method of example embodiment 22, wherein the predetermined lower threshold indicates that the chromatographic medium is at about or less than about 20%, 10%, 5% or 1% of a dynamic binding capacity (DBC) of the chromatographic medium.

[0374] 24. The method of example embodiment 22 or example embodiment 23, wherein the predetermined lower threshold (mg / mL) is less than about 5, 4, 3, 2, 1, 0.5, or 0.1 mg / mL.

[0375] 25. The method of any one of example embodiments 22 to 24, wherein the one or more operational change comprises ceasing elution of the protein with an elution buffer.

[0376] 26. The method of any one of example embodiments 22 to 25, wherein the one or more operational change comprises ceasing collection of the protein.

[0377] 27. The method of example embodiment 22 to 26, wherein the one or more operational change comprises washing the chromatographic medium.

[0378] 28. The method of any one of example embodiments 13 to 27, wherein the one or more operational change is determined by at least three, four, or five Raman spectroscopic analyses.

[0379] 29. The method of any one of example embodiments 1 to 28, wherein the Raman spectroscopic analysis is an in-line, online, at-line or off-line analysis.

[0380] 30. The method of example embodiment 29, wherein the Raman spectroscopic analysis is an in-line analysis. The method of any one of example embodiments 1 to 30, wherein the Raman spectroscopic analysis is performed in a flow cell. The method of example embodiment 31 , wherein a flow rate through the flow cell is between about 0 and about 250 mL / min, or between about 0.1 and about 250 mL / min, or between about 0.5 and about 50 mL / min. The method of any one of example embodiments 1 to 32, wherein the monitoring by Raman spectroscopic analysis is performed continuously. The method of any one of example embodiments 1 to 33, wherein the Raman spectroscopic analysis comprises the use of Surface Enhanced Raman Spectroscopy (SERS), resonance Raman spectroscopy, tip-enhanced Raman spectroscopy, polarized Raman spectroscopy, stimulated Raman spectroscopy, transmission Raman spectroscopy, spatially offset Raman spectroscopy, difference Raman spectroscopy, Fourier Transform (FT) Raman, or hyper Raman spectroscopy. The method of any one of example embodiments 1 to 34, wherein the Raman spectroscopic analysis comprises the use of a Raman analyzer configured with a laser or other suitable light source that operates at defined wavelengths, optionally in the range between 325 nm and 1064 nm. The method of example embodiment 35, wherein the light source has a wavelength (in nm) of at least about 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 875, 900, 925, 950, or 1000. The method of example embodiment 35, wherein the light source has a wavelength (in nm) of about 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 875, 900, 925, 950, or 1000. The method of example embodiment 35, wherein the light source has a wavelength (in nm) of one or more of 532, 785, and 993, optionally 532, 785, and 993. 39. The method of example embodiment 35, wherein the light source has a wavelength of about 785 nm.

[0381] 40. The method of example embodiment 35, wherein the light source is in the visible spectrum.

[0382] 41. The method of any one of example embodiments 1 to 40, wherein the Raman spectroscopic analysis comprises applying a light source to a sample with an exposure time of about or less than about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 seconds; or between about 1 and about 20 seconds, or between about 2 and about 10 seconds.

[0383] 42. The method of any one of example embodiments 1 to 41, wherein the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, is performed with an exposure time between about 6 and about 10 seconds.

[0384] 43. The method of any one of example embodiments 1 to 42, wherein the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, is performed with an exposure time less than about 5, 4, 3, 2 or 1 second(s).

[0385] 44. The method of any one of example embodiments 1 to 43, wherein each Raman spectroscopic analysis comprises; applying a light source to a sample: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: reference spectra obtained from reference samples having known concentrations of the protein or total protein; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein or total protein, to determine the presence, amount or concentration, or a change thereof, of the protein or total protein present in the sample. The method of any one of example embodiments 1 to 43, wherein each Raman spectroscopic analysis comprises; applying a light source to a sample: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: reference spectra obtained from reference samples having known concentrations of the protein; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount or concentration, or a change thereof, of the protein present in the sample. The method of any one of example embodiments 1 to 43, wherein each Raman spectroscopic analysis comprises; applying a light source to a sample: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: reference spectra obtained from reference samples comprising one or more predetermined compositions; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples comprising one or more predetermined compositions, to determine that the sample is a composition corresponding to at least one of the one or more predetermined compositions, and / or that the composition of the sample has changed from one to another of the one or more predetermined compositions. The method of example embodiment 46, wherein the one or more predetermined compositions are selected from a group comprising: protein-containing eluate, elution buffer, equilibration buffer, wash buffer, protein-depleted plasma or fraction thereof, low conductivity buffer, plasma, and waste. The method of any one of example embodiments 44 to 47, wherein comparing the test spectra with the reference spectra or reference data set, comprises comparing a peak height, an area under the curve, spectral distance, or a peak profile of the test spectra, with a corresponding peak height, area under the curve, spectral distance, or peak profile from the reference spectra or reference data set. The method of any one of example embodiments 44 to 48, wherein the concentration of protein or total protein in reference or training samples is determined using the Dumas assay, optical density measurement, the Kjeldahl method, and / or any other method of protein determination. The method of any one of example embodiments 44 to 49, wherein the reference samples include concentrations of the protein or total protein across a range of concentrations of samples eluting from the chromatographic medium, or being loaded onto the chromatographic medium. The method of example embodiment any one of example embodiments 44 to 50, wherein the model is generated using peak integration. The method of any one of example embodiments 44 to 50, wherein the model is generated using hard modelling. The method of any one of example embodiments 44 to 50, wherein the model is generated using a multivariate analysis selected from the group consisting of: Partial least squares regression (PLS); PLS Discriminant Analysis (PLS-DA); Ordinary Least Squares (OLS) regression; MLR (multiple linear regression); OPLS (Orthogonal-PLS); SVM (support vector machines); GLD (general discriminant analysis); GLMC (generalized linear model); GLZ (generalized linear and non-linear model); LDA (Linear Discriminant Analysis); spectral distance, principal component analysis (PCA), principal component analysis - quadratic discriminant analysis (PCA-QDA), partial least square - discriminant analysis (PLS-DA), classification trees; cluster analysis; neural networks; and Pearson correlation. The method of any one of example embodiments 44 to 50 or 53, wherein the model is a model generated using partial least squares (PLS) regression of processed wavelength spectra of samples having known concentrations of the protein or total protein. The method of any one of example embodiments 44 to 54, wherein the model generated is judged using the following statistical parameters:

[0386] Number of latent variables (PLS factors) in the model,

[0387] Bias,

[0388] RMSEC,

[0389] RMSECV,

[0390] RMSEP for independent test samples,

[0391] Rank,

[0392] R2,

[0393] RPD value,

[0394] Uncertainty,

[0395] MAPE,

[0396] False positive rate,

[0397] False negative rate,

[0398] Accuracy,

[0399] Sensitivity (Recall),

[0400] Specificity,

[0401] Precision, and / or

[0402] Confusion matrix. The method of any one of example embodiments 44 to 55, wherein the method comprises applying at least one spectral pre-treatment to the wavelength spectra. 57. The method of example embodiment 56, wherein the spectral pre-treatment is 1storder derivative, 2ndorder derivative, or vector normalization, or a combination thereof.

[0403] 58. The method of any one of example embodiments 44 to 57, further comprising a step of applying preprocessing and baseline normalization techniques, background correction algorithms or derivative spectroscopy to the spectrum or spectra to manage the background fluorescence.

[0404] 59. The method of any one of example embodiments 44 to 58, further comprising using a data filter to select wavelength ranges or Raman shift regions that are of interest.

[0405] 60. The method of any one of example embodiments 44 to 59, wherein the test spectra comprise a spectral signal in the visible, near infrared, infrared, near ultraviolet, or ultraviolet (UV) range.

[0406] 61. The method of any one of example embodiments 44 to 60, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 5000 cm'1to 0 cm'1.

[0407] 62. The method of any one of example embodiments 44 to 61 , wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 3200 cm'1to 0 cm'1.

[0408] 63. The method of any one of example embodiments 44 to 62, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1100 cm'1to 900 cm'1.

[0409] 64. The method of any one of example embodiments 44 to 63 , wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 1620 cm'1to 350 cm'1.

[0410] 65. The method of any one of example embodiments 44 to 64, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one or more of the following regions: 700 cm'1and 800 cm'1; 900 cm'1and 1100 cm'1; 1150 cm'1and 1300 cm'1; 1300 cm'1and 1600 cm'1; 2800 cm'1and 3000 cm'1; 300 cm'1and 3500 cm'1; 340 cm'1and 1500 cm'1; 300 cm'1and 1050 cm'1; and / or 500 cm'1and 2600 cm'1.

[0411] 66. The method of any one of example embodiments 1 to 65, wherein the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein or total protein, to determine the presence, amount, or concentration, or change thereof, of protein or total protein present in the sample.

[0412] 67. The method of any one of example embodiments 1 to 65, wherein the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using peak integration of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount, or concentration, or change thereof, of protein present in the sample.

[0413] 68. The method of example embodiment 67, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one of more of the following regions:

[0414] 700 cm'1and 800 cm'1; 900 cm'1and 1100 cm'1;

[0415] 1150 cm'1and 1300 cm'1;

[0416] 1300 cm'1and 1600 cm'1; and / or 2800 cm'1and 3000 cm'1. The method of example embodiment 67 or example embodiment 68, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift from 900 cm'1to 1100 cm'1. The method of any one of example embodiments 67 to 69, wherein the concentration of protein is determined by comparing the area under the curve (AUC) of at least one Raman shift from the sample against an AUC of a corresponding Raman shift from the reference data set, wherein each measured Raman shift is independently selected from a Raman shift that has the highest maximum intensity relative to any other Raman shift within one of the following ranges:

[0417] 700 cm'1and 800 cm'1;

[0418] 900 cm'1and 1100 cm'1;

[0419] 1150 cm'1and 1300 cm'1;

[0420] 1300 cm'1and 1600 cm'1; and / or

[0421] 2800 cm'1and 3000 cm'1. The method of any one of example embodiments 67 to 70, wherein the concentration of protein present in the sample is determined by comparing the area under the curve (AUC) of a single Raman shift from the sample against an AUC of a corresponding Raman shift from the reference data set, wherein the single Raman shift has the highest maximum intensity relative to any other Raman shift from 900 cm'1to 1100 cm'1. The method of any one of example embodiments, wherein the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount, or concentration, or change thereof, of protein present in the sample.

[0422] 73. The method of example embodiment 72, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one of more of the following regions:

[0423] 300 cm'1and 3500 cm'1;

[0424] 340 cm'1and 1500 cm'1;

[0425] 300 cm'1and 1050 cm'1; and / or

[0426] 500 cm'1and 2600 cm'1.

[0427] 74. The method of any one of example embodiments 1 to 73, wherein the chromatographic medium is selected from the group consisting of an ion exchange chromatographic medium, an affinity chromatographic medium, a hydrophobic interaction chromatographic medium, a mixed mode chromatographic medium, an adsorption chromatographic medium and a partition chromatographic medium.

[0428] 75. The method of any one of example embodiments 1 to 73, wherein the chromatographic medium is an affinity chromatographic medium.

[0429] 76. The method of any one of example embodiments 1 to 75, wherein the continuous chromatography is continuous affinity chromatography.

[0430] 77. The method of example embodiment 76, wherein the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, periodic counter-current chromatography (PCC), continuous counter-current tangential chromatography (CCTC), and continuous counter-current spiral chromatography (CCSC).

[0431] 78. The method of any one of example embodiments 1 to 77, wherein the chromatographic medium is packed into a first separation unit and one or more subsequent separation units (s). 79. The method of example embodiment 78, wherein the separation units(s) are columns.

[0432] 80. The method of example embodiment 78 or example embodiment 79, wherein the separation unit(s) are fluidly connected and separated by fluid conduits comprising inlet and outlet valves.

[0433] 81. The method of any one of example embodiments 78 to 80, wherein the first separation unit is loaded with the protein at a concentration above the DBC of the chromatographic medium.

[0434] 82. The method of any one of example embodiments 78 to 81, wherein the one or more subsequent separation units(s) are loaded with the protein at a concentration up to the DBC of the chromatographic medium.

[0435] 83. The method of any one of example embodiments 78 to 82, wherein the DBC of the chromatographic medium is at least 5 mg of the protein per mL of chromatographic medium.

[0436] 84. The method of any one of example embodiments 78 to 83, wherein the one or more subsequent column(s) are loaded with the protein at a concentration of up to 40 mg of the protein per mL of chromatographic medium.

[0437] 85. The method of any one of example embodiments 78 to 84, wherein the chromatographic medium has a total bed height of at between 0.5 cm and 30 cm.

[0438] 86. The method of any one of example embodiments 1 to 85, wherein the plasma or a fraction thereof is obtained from processing of blood-derived plasma obtained from human blood.

[0439] 87. The method of any one of example embodiments 1 to 86, wherein the plasma or a fraction thereof is obtained or derived from the processing of blood-derived plasma that comprises fresh plasma, cryo-poor plasma, or cryo-rich plasma. 88. The method of any one of example embodiments 1 to 87, wherein the plasma or a fraction thereof is obtained from a number of donations and / or subjects, and pooled.

[0440] 89. The method of any one of example embodiments 1 to 88, wherein the plasma or a fraction thereof is obtained or derived from hyperimmune plasma.

[0441] 90. The method of any one of example embodiments 1 to 89, wherein the plasma or a fraction thereof is a resuspension of a precipitate or paste obtained from blood- derived plasma.

[0442] 91. The method of any one of example embodiments 1 to 90, wherein the plasma or a fraction thereof is a filtrate obtained from blood-derived plasma.

[0443] 92. The method of any one of example embodiments 1 to 91, wherein the plasma or a fraction thereof is selected from the group consisting of cryo-rich plasma, cryopoor plasma, Supernatant I (SN I), Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction (I+)II+III ((FrI+)II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), and combinations thereof.

[0444] 93. The method of any one of example embodiments 1 to 92, wherein the protein is immunoglobulin (IgG).

[0445] 94. The method of any one of example embodiments 1 to 92, wherein the protein is albumin.

[0446] 95. The method of any one of example embodiments 1 to 94, wherein at least 75% of the protein is recovered from the plasma or fraction thereof.

[0447] 96. The method of any one of example embodiments 1 to 95, wherein the collected protein has a purity of at least 90%.

[0448] 97. The method of any one of example embodiments 1 to 96, wherein the method further comprises regenerating the resin. 98. The method of any one of example embodiments 1 to 97, wherein the method further comprises sanitizing the resin.

[0449] 99. The method of any one of example embodiments 1 to 98, wherein the method further comprises one or more steps selected from a group consisting of ethanol precipitation, octanoic acid fractionation, ion exchange chromatography, viral inactivation, viral filtration and ultrafiltration / diafiltration.

[0450] 100. The method of any one of example embodiments 1 to 99, wherein the method further comprises an anion exchange chromatography step using a strong anion exchange resin operated in flow through mode.

[0451] 101. The method of any one of example embodiments 1 to 100, wherein the method further comprises formulating the protein into a pharmaceutical composition.

[0452] 102. The method of any one of example embodiments 1 to 101, wherein the Raman spectroscopic analysis is an in-line analysis.

[0453] EXAMPLES

[0454] In order that the disclosure may be more clearly understood, particular embodiments of the invention are described in further detail below by reference to the following nonlimiting experimental materials, methodologies and examples.

[0455] Example 1: Detection of IgG elution during elution step of a continuous immunoaffinity chromatography process

[0456] The aim of this study was to develop an inline Raman spectroscopy method as a tool to monitor IgG elution from affinity resin in real-time during continuous immunoaffinity chromatography.

[0457] This study comprises three different experimental phases. Firstly, training data was gained for model development. Secondly, validation of the most reliable model, and finally, implementation of the developed inline Raman spectroscopy method during continuous immunoaffinity chromatography runs performed with BioSMB system. Method - Model Development

[0458] Eluate samples containing purified IgG present in eluate buffer were adjusted to different IgG concentrations to simulate real time BioSMB operation. Raman spectra were measured with two different probe types (Flow Cell and Raman Bio-Optics) using a RAMAN Rxn2 Analyser from Endress + Hauser.

[0459] The Bio-Optic probe was installed into a metal beaker. Aluminium foil was used to prevent light shining into the beaker as light would disturb the measurement of BioOptic probe. The Flow Cell does not need to be darkened as it comes with a build in dark chamber. A pump was continuously circulating the suspension to supply a constant flow within the Flow Cell. The solution was mixed in the beaker using a magnetic stirrer and plate.

[0460] Firstly, 100 % elution buffer was added to a metal beaker. The focus function of the device was used to suggest a proper exposure time for the intermediate. After sampling 20 mL, the calculated amount of IgG-eluate (45 mL) was added to form an 80 % elution buffer / 20 % eluate mixture. After measuring three spectra of each mixture using each probe, sampling was performed. Amounts were added according to Table 1. After the final mixture of 20 % elution buffer / 80 % eluate was analysed, the test system was cleaned using WFI, 70% Isopropanol, WFI and air. Afterwards it was filled with 100 % IgG-eluate. The intermediate was sampled, and spectra were measured. The samples were additionally tested offline using Cedex Bio HT to analyse IgG levels based on an immunoturbidimetric assay.

[0461] Table 1. Calculation of Sample Mixtures

[0462] Method - Validation

[0463] For the validation of the developed model based on Raman spectroscopy, conditions comparable to those of a continuous chromatography run were simulated and testing was performed accordingly. To simulate continuously changing IgG concentrations in the outlet, eluate containing purified IgG present in eluate buffer was added continuously to eluate buffer via pump. The eluate / eluate buffer mixture was pumped through the Flow Cell with a flow rate of 39 mL / min using a second pump (a rate comparable to that of the BioSMB). Spectra was obtained continuously, and samples were taken directly using a three-way-valve after the flow cell (for offline testing using Cedex Bio HT).

[0464] Method - Feasibility

[0465] After development and validation of the model, it was tested directly in the BioSMB environment. The Flow Cell with the three-way-valve was added into the outlet flow used for the elution step. Sampling was performed using the three-way-valve and analytical at-line measurements were assigned respective to spectra.

[0466] An affinity chromatography resin POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher) was used. In this example, the affinity chromatography resin contained a ligand capable of binding to a CH3 domain of human IgG, specifically camelid-derived single domain [VHH] antibody fragment, and a matrix of cross-linked poly(styrene- divinylbenzene). The affinity chromatography resin was packed in YMC Eco plus columns (diameter 1.5 cm) and chromatography performed on a BioSMB PD System (Sartorius) at room temperature (approx. 20°C). More information about the continuous chromatography process is described in Table 2 below, and Figure 13.

[0467] Table 2. BioSMB operation - Equipment, Material and Buffer

[0468] Cryo-poor plasma (CPP) (prepared by CSL Behring) was filtered through a 1.2 pm (Sartorius Sartopure® PP3 Capsule 1.2 pm; approx. 0.0026 m2 / L CPP) and 0.45+0.2 pm (Sartorius Sartobran® P Midicap 0.45 + 0.2 pm; approx. 0.005 m2 / L CPP) capsule filters at pressure between 0.5-1.0 bar. Filtered CPP at room temperature was used as the feed material. Evaluation of Measurement Settings

[0469] For Raman spectroscopy measurement, the parameters exposure time and frequency need to be defined. Exposure time is defined as length of time the sample is exposed to the laser. It should be adjusted in a way that 3 to 99% of detector saturation are reached. Frequency describes how many spectra are recorded during a single measurement. If frequency is above one, then all collected spectra of this measurement are joined to one spectrum which will be reported by the system. Devices may be provided by the vendor with a build-in focus tool. Instead of measuring a sample several times with different exposure time settings, one can use the focus function and a range for the exposure time will be suggested by the system.

[0470] Table 3 summarises the results of the flow cell conditions applied. With a measurement exposure time of 9.5 s at a frequency of 1, a detector saturation of 40 % was reached. For an exposure time of 16.7 s and a frequency of 1 a saturation of 71 % was reached. The exposure time is preferably as short as possible since the measurement is planned to be implemented in line with changing matrix of the solution. Therefore, an exposure time of 10 s with a frequency of 1 was preferred. The same procedure was performed to evaluate the settings for the Bio Optics. Based on the data and spectra quality, an exposure time of 30 s with frequency of 1 was preferred. When comparing the Flow Cell and Bio Optics, Bio Optics needed three times longer (30 s vs. 10 s) to measure a spectrum with the same intensity when compared to Flow Cell device.

[0471] Table 3. Exposure time and Saturation (Flow Cell)

[0472] Table 4. Exposure time and Saturation (Bio Optics)

[0473] The Flow Cell was used for applications at different flow rates. As there is a possibility of variance coming from this parameter the same solution was measured at different flow rates from 0.5 to 50.0 mL / min, however no significant variation on the spectra was observed for flow rates within this range.

[0474] Spectra Analysis

[0475] Spectra were analysed using PEAXACT Software (Version 5.8). The software has different tools to preprocess the spectra. As the spectra is the basis for model building the aim of preprocessing is to enhance the intensity and difference between spectra with the background of modelling, to focus on Raman shift regions which are specific for measurement of the analyte(s), to exclude regions that are not directly affected by the analyte(s) to not negatively influence the prediction of the analyte(s), to increases specificity and robustness of the model, and to reduce negative effects caused by peaks affected by other substances present in the sample.

[0476] The first preprocessing step was the rubber band subtraction approach to eliminate the background signal caused by sample fluorescence. Rubber band determines support points by finding the convex hull of each spectrum. The baselines are then piecewise linear or (smoothing) splines through the support points.

[0477] An aim of preprocessing is also to cut out areas of the spectrum which are not relevant. The peak at the left site in the Raman shift range from 3400 - 3600 cm'1is not relevant as no correlation between Raman intensity and IgG concentration was observed in this specific range. Therefore, the Raman shift range relevant to assess Raman intensity in dependency of the IgG concentration measured was set from 0 - 3000 cm'1to exclude the areas that are irrelevant for this study.

[0478] As the last prepossessing step, baseline nodes were used to pull down the peaks to baseline. The nodes were placed at 200, 500, 748, 770, 866, 1023, 1297.47, 1434, 1487.22, 1560 and 1781 cm'1. The spectra after application of the pretreatment are shown in Figures 1A-1C.

[0479] Model Building

[0480] A peak integration model was investigated. Specific peaks need to be elaborated from the spectrum to use the peak integration approach as model type. In this case, to remove the impact of the different probe types on model building, a separate model was developed for each probe (Flow Cell / Bio Optics). The peaks were selected on the basis they fulfilled two requirements: (1) that peak area increases linearly in relation to the concentration measured; and (2) if the concentration is zero, the peak area also needs to be zero.

[0481] Table 5. Model Properties - Flow Cell

[0482] For each selected peak, one model was built. In addition, one model using all five peaks and one model using peak no. 1 to 4 were built to compare performance of different models with each other. For each model, the true result (measured with Cedex Bio HT) was plotted against the results which are predicted by the model.

[0483] Calibration properties and error marks are provided in Table 6. Although suitability of all of the models investigated was demonstrated, the model based on peak 2 was judged to provide the best results. It has the highest R2and lowest RMSEC, RMSEP. For RMSECV, the model of peak 1 to 5 is minimally lower.

[0484] Table 6 - Calibration Properties - Flow Cell

[0485] The same procedure was performed for the Bio Optics probe, with the peaks defined in Table 7, and calibration properties and error marks in Table 8. For the Bio Optics probe the preferred model was based on peaks 1 to 4. The other models developed were shown to be suitable for application, in light of good performance during calibration and testing.

[0486] Table 7 - Model Properties - Bio Optics Table 8 - Calibration Properties - BioOptics

[0487] Model Validation

[0488] The model is aimed to be implemented inline during chromatographic purification. Accordingly, validation was undertaken with the model built on the Flow Cell data.

[0489] The model based on peak 2 showed preferable performance when comparing the error metrics between all models, and was therefore used for validation. Model validation is important to assess if the model is also generally applicable or if it is only providing reliable predictions for the training data set.

[0490] For the validation of the developed model based on Raman spectroscopy, conditions comparable to those of a continuous chromatography run were simulated and testing was performed accordingly. To simulate continuously changing IgG concentrations in the outlet, eluate containing purified IgG present in eluate buffer was added continuously to eluate buffer via pump. The eluate / eluate buffer mixture was pumped through the Flow Cell with a flow rate of 39 mL / min using a second pump. Spectra were conducted continuously, and samples were taken directly using a three- way -valve after the Flow Cell.

[0491] Figure 2 A shows the predicted IgG content in relation to the true content which was measured at-line as described before. The plot contains the training samples used for calibration (grey) and the validation samples (black). Specially in the range from 0 to 4 mg / mL IgG, predicted and true result are laying very close to the identity line. The sample from 8 to 10 mg / mL IgG show more variation. Nevertheless, the highest difference is still low with 1 mg / mL.

[0492] A similar result was obtained for run 2 (Figure 2B). The difference was slightly higher with 1.15 mg / mL when compared to run 1, however, a robust prediction is also given for this run, indicating the ability for generalization of the model for new data. For the third run validation values are also located near the identity line (Figure 2C). The maximum difference is 1.74 mg / mL, and therefore represents the highest value of all three runs. It is believed that differences can also be caused due to the sampling method. The sampling is performed manually. Opening and closing the valve needs to be done on point if the sample should represent the flowthrough of the Flow Cell during the measurement. Therefore, already slight delays in the sampling process even just for some seconds can cause differences between at-line IgG results and the predicted result aside from variations coming from the model itself. Such issues should be resolved by inline use.

[0493] All three runs were found to have relatively low Bias (Table 9). RMSEP (root mean squared error of prediction) shows a good comparability to the RMSEP from the calibration. This data also indicates the ability of generalization of the model, and validation of the model was found to be successful.

[0494] Table 9. Bias and RMSEP of validation runs

[0495] Feasibility Study

[0496] The feasibility during a routine BioSMB run was shown as the final test of the developed model. As a pretest, the Flow-Cell was integrated into the elution outlet to see if it would cause any pressure increases for the BioSMB system. This was not observed to be the case.

[0497] As the next step, the model was activated in the software of Raman Rxn2 Analyzer, and the prediction of IgG was monitored in real time. As last step of the feasibility study, at-line samples were taken using a three-way -valve in parallel to Raman spectra measurement. The IgG result from each sample measured at-line was assigned to its corresponding spectrum. The model was validated against this new data.

[0498] Figure 3 displays the predicted result in correlation to the true result obtained via at-line measurement. The first thing that stands out is the high IgG content of approx. 28 mg / mL as the calibration of the model just covered an IgG range from 0 to 9 mg / mL. Nevertheless, the mechanistic model developed based on the peak integration approach was able to predict results relatively close to the true content. Exposure Time

[0499] A new target was set to build a model with a shorter measurement time which is capable of providing more prediction points of the eluate peak during elution. To fulfill this new target, the model development was repeated two times each with two different exposure times: 2 s and 5 s.

[0500] For both exposure times, a linear correlation was demonstrated, however the Raman intensity with a the 2s exposure time spectra was found to be very low with just 1500. For the 10s spectra, it was around 8000.

[0501] Table 10 compares the two newly built models with the original one showing that the model based on 5 s data led to even better results than the 10 s one. The 2 s model showed higher results for RMSEP as most important criteria. Moreover, RMSECV and RMSEC were higher. R2was lower but still acceptable. The 2s and 5s models were taken for further validation.

[0502] Table 10 - Calibration properties - exposure time study

[0503] The 5 s model was utilised in the BioSMB set up. The 5 s model was validated by comparing at-line measurements with predicted results. Figure 4A show the plotted validation data. The difference between true and predicted results increased to a maximum of 5 mg / mL IgG. For the validation approach under controlled conditions, the maximum difference was around 1 mg / mL. However, it needs to be considered that the IgG range is wider (0 to 40 mg / mL IgG) whereas the range for the controlled validation just reached from 0 to 9 mg / mL IgG. Therefore, a difference of 5 mg / mL is still acceptable as it only accurses for higher IgG values. Also, the before mentioned manual sampling process can lead to differences as it is not automated and brings more variance caused by handling. Overall, the validation was considered successful.

[0504] Therefore, a continuous chromatography run using BioSMB with a Raman spectra measurement with 2 s exposure time was performed as a feasibility run. During the process, sampling and at-line IgG testing was performed using the Cedex Bio HT system. The prediction of IgG based on the 2 s spectra demonstrated an even better modelling of the elution peaks during the continuous process compared to the 5 s measurement (Figure 4B). With the shorter measurement time, at least four points per peak can be predicted.

[0505] Example 2: Detection of IgG breakthrough during column loading step of a continuous immunoaffinity chromatography process

[0506] The aim of this study was to develop an inline Raman spectroscopy method as a tool to monitor IgG breakthrough in real-time during the column loading step of a continuous immunoaffinity chromatography process. CPP was used as feed solution.

[0507] For this study, four different experimental approaches were followed. Firstly, spectra were generated for the development and calibration of the model. The second approach was used for the validation of the most reliable model and the third one was used to simulate an IgG breakthrough. The last design focused on spiking of IgG into CPP.

[0508] Model Development

[0509] The flowthrough of the loading step can be considered as CPP without IgG so called IgG-depleted plasma. CPP samples and IgG-depleted-CPP samples were mixed in different variations to create samples with different IgG values in a range from 0 to 6 mg / mL of IgG. Raman spectra were measured with two different probe types (Flow Cell and Bio-Optic) using a RAMAN Rxn2 Analyser.

[0510] The Bio-Optic probe was installed into a metal beaker. Aluminium foil was used for light exclusion to prevent light shining into the beaker, as the light would disturb the measurement using the Bio-Optic probe. The Flow Cell does not need to be darken as it is equipped with a built-in dark chamber. A pump was continuously circulating the suspension to supply a constant flow within the Flow Cell. The solution was mixed in the beaker using a magnetic stirrer and plate. Firstly, 100% IgG-depleted plasma was added to a metal beaker. The focus function of the device was used to suggest a proper exposure time of the sample. After sampling of 20 mL, the calculated amount of CPP was added to form a 99% IgG-depleted plasma / 1% CPP mixture. After measuring three spectra of this mixture with each probe, sampling was performed. Amounts were added according to Table 11. After the final mixture of 20% IgG depleted plasma and 80% CPP was analysed, the setup was cleaned using WFI, 70% isopropanol, WFI and air. Afterwards it was filled with 100% CPP. The intermediate was sampled, and spectra were measured. The samples were analyzed at-line using the Cedex Bio HT system to quantify IgG and total protein.

[0511] Table 11. Preparation of CPP mixtures

[0512] Validation

[0513] To validate the model, process conditions were simulated that were comparable to those of a continuous immunoaffinity chromatography (CIAC) run. The highest flow rate of the CIAC process was used (39 mL / min). To simulate constantly changing IgG concentrations measured in the outlet of the BioSMB system, CPP was added continuously to IgG-depleted CPP via pump. The mixture was pumped through the Flow Cell with a flow of 39 mL / min using a second pump. Spectra were collected continuously, and samples were taken using a three-way-valve directly after the Flow Cell to conduct additional at-line testing.

[0514] Feasibility

[0515] The feasibility experiments simulated a breakthrough of IgG by overloading the immunoaffinity column. The continuous chromatography method performed with a BioSMB system (the same resin, buffer, column dimensions, plasma, temperature, preprocessing, etc. as described above for Example 1) was performed with an Akta system. For this approach, only one of the immunoaffinity columns was used with the Akta system. Hence, the method displays one cycle of the continuous chromatography process performed with just one immunoaffinity column. In total, two different methods were tested, which were called “4 CV method” and “12 CV method” (Table 12). The “4 CV method” means that 4 CVs of CPP were loaded onto the column, which is the same amount of CPP that is used for the CIAC process using the BioSMB system as described in Example 1. But the CIAC process uses the loopback approach, meaning that the flow- through fraction is directly loaded onto a second column after 2 CVs. This loopback procedure was not applied for the Akta run. The second method increased the loading volume to 12 CV (approx. 150 mL) of CPP with the aim to cause an IgG breakthrough. The overloading experiments were monitored in real time with the Raman system. Additionally, at-line samples were taken and tested for IgG and total protein using Cedex Bio HT (immunoturbidimetric assay).

[0516] Table 12. - Modified methods of CIAC used for Akta overloading experiments

[0517] IgG spiking into CPP

[0518] IgG spiking experiments aimed at increasing the IgG levels of CPP samples. Therefore, lyophilised IgG was reconstituted in WFI with a final concentration of 25 mg / mL IgG in the dissolved solution. In total, three different spike volumes were added according to Table 13. The spectra were measured as described under “Model development”. Table 13. - Pipetting schemes for spiking experiments

[0519] Evaluation of Measurement Settings

[0520] Table 14 summarizes the optimal setting used for the Flow Cell that was identified based on the results of this study. For CPP, 5.4 to 9.5 s were suggested by the focus function applied. With an exposure time of 5.4 s and a frequency of 1, a detector saturation of 40% was reached. For the setting exposure time of 9.5 s with a frequency of one, a saturation of 71% was reached. The exposure time is preferably as short as possible since the measurement is planned to be implemented in line with changing matrix of the solution. Therefore, an exposure time of 6 s with a frequency of one was selected for further validation.

[0521] Table 14. - Focus function used for the measurement of CPP (by using the Flow Cell)

[0522] The same procedure was performed to evaluate the settings for the Bio Optics. The results are listed below. On the basis of the experimental results (Table 15), an exposure time of 20 s was selected for further validation.

[0523] Table 15. - Focus function used for CPP (Bio Optics)

[0524] The Flow Cell will be used for different applications at different flow rates. As there is a possibility of variance coming from this parameter, meaning that different flow rates could theoretically have an effect on the Raman spectrum, the CPP was measured at different flow rates ranging from 0.5 to 50.0 mL / min, however no significant variation on the spectra was observed for flow rates within this range. Model Building

[0525] The aim of building a Raman model was to predict IgG levels in the outlet of the column during the loading step of the continuous immunoaffinity chromatography (CIAC) process by using inline Raman spectroscopy measurement. To investigate the change in spectra in relation to IgG present in IgG-depleted cryo-poor plasma or cryo-poor plasma, different mixtures of cryo-poor plasma (CPP) and IgG depleted CPP (flowthrough) were prepared, and Raman spectra were conducted according to the experimental design described above. A range from 0 to 6 mg / mL of IgG was covered.

[0526] PEAXACT software was used to build a partial least square (statistical approach; PLS) model. Table 16 provides an overview of the preprocessing strategy and data filter applied for the model. The aim of preprocessing is to convert the spectra in a way that robust modelling of a changing IgG content is possible. Changes in spectra are preferably only caused by variation of IgG concentration. PLS as statical model always evaluates a correlation in the data generated. Thus, it is preferable to exclude as much as background noise as possible, or other influences not related to a change in IgG concentration, as these signals would otherwise negatively affect the model.

[0527] Table 16 - Preprocessing approach used in this study for PLS model

[0528] The model was calibrated using the dataset previously obtained using mixtures of CPP and IgG-depleted CPP in variations from 0 % to 100% CPP. In total, two runs were performed. Table 17 displays a summary of the calibration parameters and metrics for the model. Rank is a parameter that can be selected during calibration and displays the complexity of the model where Rank 1 has the lowest complexity and rank 10 the highest. The preferred rank is as small as possible but also has a small RMSE and large R2. “Diff. min and max” displays the difference between true value and predicted value. R2is preferably higher than 0.95. RMSEC is the root mean square error of calibration. This value is preferably minimized as it displays the error of the model for the prediction of the training data used for calibration (C). If a maximum mean error of 10% was defined for our method, this would have been resulted in a target of the RMSEP of < 0.6, if setting 6 mg / mL IgG represents 100%. It is noted that the RMSE is the mean value of all errors, and that some errors can be higher than 0.6. Accordingly, evaluation of RMSEP is preferably combined with “Diff min. and max.” which displays the absolute value that is representative for differences between true and predicted results over the whole dataset. Based on Table 17, the model showed a good performance for the calibration and testing against the test dataset. Hence, the model was further investigated in validation runs.

[0529] Table 17 - Calibration metrics of the PLS model

[0530] Model Validation

[0531] Model validation aims to show the performance of a model based on complete unknown data (unknown for the model). The true value is obtained from at-line analytics.

[0532] Two validation runs were performed. The model was validated against the unknown data to evaluate their ability to generalize, meaning to assess their performance on unknown data (data which was not part of the calibration process). Table 18 summarizes the performance during the two validation runs for the model. On the basis of the results, the model was considered for further validation.

[0533] Table 18 - Validation metrics for the PLS model

[0534] Feasibility Study

[0535] Model validation identified the PLS model as suitable for predicting IgG concentrations present in the samples investigated. The ability of the Raman model to detect an IgG- breakthrough after the immunoaffinity column was further evaluated. Raman spectroscopy was conducted using the Flow Cell and the established PLS model to predict the IgG values based on the measured spectra. In total, four runs were performed using the 4 CV and 12 CV method, respectively. 4 CV

[0536] At-line analytics with the Cedex Bio HT system to analyze IgG and total protein (TP) revealed no breakthrough of IgG in the flowthrough fractions sampled in case the column is loaded with 4 CV of CPP (Figure 5). Two runs were performed. The at-line results were correlated to the corresponding Raman spectra. This dataset was then used to evaluate the performance of the model and its ability to predict an IgG concentration of 0 mg / mL in the IgG-depleted flowthrough. Overall, the model was found to be suitable for the prediction for 0 mg / mL with RMSEPs of only 0.389906 or 0.432046 mg / mL IgG (Figure 6A and Figure 6B).

[0537] 12CV

[0538] The CPP loading volume was increased from 4 CV to 12 CV to cause a breakthrough of IgG. The generated data was used to evaluate the performance of Raman spectroscopy. Additionally, samples were taken and analyzed for IgG and total protein using Cedex Bio HT. The 12 CV method was performed twice. The at-line analytics for 12 CV Run 1 showed that an IgG breakthrough occurred after approx. 150 s (Figure 7), which additionally resulted in an increase of total protein (TP).

[0539] For the evaluation of the Raman spectra, the model was considered in light of its performance in the 4 CV runs. The validation plot (Figure 8A) showed that the model gave precise predictions in the lower IgG concentration range. Moreover, the increase of IgG is modelled with less difference between predicted vs. true values. Notwithstanding, higher IgG values may not be predicted correctly. In a second run, the model was again confirmed as giving precise predictions in the lower IgG concentration range, below 2 mg / mL (Figure 8B). Overall, the study results demonstrate that an IgG breakthrough during BioSMB operation can be detected using Raman spectroscopy in combination with a PLS model.

[0540] Evaluation of 12CV Run 2

[0541] The model was optimised by altering the calibration dataset. A copy of the model was created which was calibrated using spectra data of the 12 CV Run 2 and corresponding at-line test data. The calibration plot is shown in Figure 9A. Rank 3 was used for this PLS model resulting in an RMSEC of 0.115376 and a RMSECV of 0.951913.

[0542] The model was subsequently validated against the 12 CV run 1 data to evaluate the performance of the model for data outside of the calibration dataset, meaning to assess its ability to generalize. Validation showed that the model is now more precise for higher IgG values, but samples in the absence of IgG are predicted with higher variation and go up to 2 mg / mL of predicted IgG although these sample do not contain any IgG (Figure 9B).

[0543] The other validation plot (Figure 9C) shows the predicted IgG values in relation to the sample index confirming a better performance of the model by using a different calibration dataset, especially for the prediction of higher IgG levels. For sample index 15 to 40, nearly no difference between predicted vs. true value is shown. In conclusion, with the aim to implement this model to monitor higher IgG levels during the CIAC loading step, an additional rule is implemented as well to improve accuracy of model prediction: Three consecutive predicted values have to demonstrate an increase of IgG of more than 2 mg / mL to consider the observation as being true.

[0544] IgG dissolved in WFI was spiked into CPP to investigate the influence on the resulting Raman spectra. The samples were measured twice and at-line measurement of IgG and total protein using Cedex Bio HT was conducted for each run. Additionally, mixtures with CPP and IgG-depleted CPP aka flowthrough (FT) were created and measured. The at-line testing demonstrated that total protein was decreasing due to the dilution of the CPP with the added spike material whereas IgG concentration increased as the added spike solution was highly concentrated with 25 mg / mL of IgG. The results are listed in Table 19.

[0545] Table 19. - At-line testing performed with Cedex Bio HT system to analyze mixtures of CPP and FT, and CPP spiked with IgG

[0546] The raw spectra were analysed with PEAXACT software using a global range setting of 0 to 3000 cm'1and rubber band subtraction as baseline correction with baseline nodes at 1021.45 cm'1, 1427.39 cm'1and 1493.51 cm'1as preprocessing approach. Additionally, the spectra were normalized based on the peak area of the probe peak from 380 cm'1to 480 cm'1. Figure 10A shows the spectra from the mixtures of CPP and FT together with the spectra of CPP spiked with IgG, clearly demonstrating that peak area increases in linear correlation to IgG concentration of the mixtures of CPP and FT, but for CPP spiked with IgG, a linear correlation between IgG and peak area was not observed anymore for IgG.

[0547] If the spectra are coloured according to the total protein value of the solution a linear correlation can be seen again (Figure 10B). Based on this observation it may be assumed that the peak area is changing in relation to the total protein values present in a sample.

[0548] Consequently, the model was calibrated based on the total protein at-line data and corresponding spectra of 12 CV Run 2. The calibration plot is shown in Figure 11 A. Therefore, the model was validated against the 12 CV Run 1 data with the corresponding total protein values measured at-line. This investigation demonstrated a precise prediction of the increase of total protein during the breakthrough phase of IgG (Figure 1 IB). A smaller selection of samples from the 12 CV Run 1 data which are relevant for monitoring IgG breakthrough was taken and used to validate the model to have a better understanding of the performance of the model during the loading phase and IgG breakthrough. Figure 11C depicts the respective validation plot. Here, it was demonstrated that the model makes precise predictions of the complete range of total protein values from 40 to 46 mg / mL.

[0549] Example 3: Detection of IgG elution during elution step of a continuous immunoaffinity chromatography process with a PLS model

[0550] This study aimed to evaluate the application of a PLS model to monitor via inline Raman spectroscopy the elution of IgG from FcXP affinity resin. This study utilised the data set of Raman spectra created during the in-line BioSMB feasibility study of the peak integration model described in Example 1.

[0551] Model Buildins

[0552] Four PLS models (Table 20) were implemented and calibrated against the dataset. The models differ in their pre-processing procedures covering different techniques in terms of baseline correction, smoothing, derivative and standardization. In addition, data filters were used to select Raman shift regions of interest for model building. All models were calibrated with a k-fold cross validation approach with C=3 and a maximum rank of 10.

[0553] Table 20. - Preprocessing of PLS models for protein elution monitoring

[0554] The “predicted vs true” plots for Models No 1 to 4 are depicted in Figures 12A- D respectively.

[0555] The PLS model No. 1 uses a wide global range setting from a Raman shift of 3100 to 300 cm'1. Rubber band subtraction in combination with baseline nodes was used as baseline correction approach. Smoothing with a filter length of 145 was applied to reduce the noise of the spectra coming from short measurement times with low detector saturation. Rank 5 was chosen for this model to provide the best compromise between complexity and low RMSE and high R2. PLS model No. 2 considered the fingerprint region from 1500 to 340 cm'1only.

[0556] Standardization was performed based on the peak specific for the probe. For this model, a rank of 4 provided the best results as the RMSE was reduced and R2was increased up to a high level. Starting from Rank 5, RMSEC V and RMSEP start to increase again, thereby indicating an overfitting of the model due to higher complexity. For PLS model No. 3 the Raman shift of the peak at 1003cm'1was investigated only. The relatively low rank of 2 was the optimal compromise between low rank, low RMSE and high R2. Hence, if using the Raman shift of the peak at 1003 cm'1, a PLS model with a low complexity already delivers promising predictions for IgG concentration. For PLS model No. 4, first order derivative was applied in preprocessing of this PLS model. Rank 3 was chosen as it provided low RMSE and high R2. Increasing the complexity of the model led to higher RMSEP und RMSECV indicating an overfitting to the training data.

[0557] This study showed that the PLS models can be successfully used for the prediction of IgG during the elution from an FcXP affinity resin with a BioSMB system. The models were developed by utilizing different preprocessing strategies. These PLS models were established and calibrated against a dataset of 57 Raman spectra and tested against 25 spectra. Cross validation according to the k-fold approach with grouping of 3 was performed for calibration in each case. Table 21 gives an overview of the used datasets for training and testing together with a comparison of the performance metrics R2, RMSEC, RMSECV and RMSEP as most important criteria to display the root mean square error of prediction for the test dataset. This approach demonstrates the performance of the models on a dataset which was not used for calibration to evaluate how precise the prediction will be for future data.

[0558] PLS model No. 3 and No. 4 showed preferable results based on the calculated RMSE and the selected rank. Model No. 4 had a slightly lower RMSEP of 2.33 mg / mL but also used a rank of 3 whereas Model No. 3 only needs a rank of 2 to reach a similar RMSEP of 2.35 mg / mL. Another advantage of Model No. 3 is its lower complexity and fewer preprocessing steps compared to Model No. 4 using first order derivative. It may be preferable to exclude as many variables as possible to perform the prediction only on those regions of the spectrum which are directly influenced by the presence of the target variable (IgG concentration). This strategy may make the model more robust against fluctuations which are not related to changes in the IgG concentration but also influence the Raman spectrum.

[0559] Table 21 - Performance of PLS model

[0560] Example 4: Monitoring BioSMB operation by classifying samples that are present before and after passing the immunoaffinity column

[0561] The aim of this study was to develop an inline Raman spectroscopy method based on classification models as a tool to monitor the continuous immunoaffinity chromatography process in real time. The classification models developed are used to categorizes samples and identify the belonging class of samples present in the inlets and outlets of the BioSMB system before and after passing the immunoaffinity column.

[0562] Development of measurement settings

[0563] All samples present during the continuous immunoaffinity chromatography step were investigated to determine optimal measurement settings. The focus function of the Raman Rxn2 analyzer was used to support the definition of appropriate measurement ranges: an exposure time of 6 s and a frequency of 1 was defined for generating spectra of the training and test dataset.

[0564] Table 22 - Focus suggestions for the samples investigated

[0565] Creation of a dataset

[0566] All measurements were conducted with the Rxn2 analyser from Endress + Hauser in combination with an inline flow cell from Endress + Hauser. Three batches of each sample were considered and measured using a pump which was circulating each sample through the flow cell at a flow rate of 40 mL / min. Ten Raman spectra were collected from each sample resulting in a total number of 210 spectra. Batch 1 and 3 were used to train the model (140 spectra) and batch 2 was used for testing the developed classification model using data outside of the calibration set (70 spectra).

[0567] Model Building

[0568] Models were build using the principal component analysis (PCA) approach to evaluate spectral preprocessing parameters which provided best separation of spectra of the samples investigated in case they were plotted according to their principal component 1 and 2. Based on this approach, classification models were established afterwards, as described in the following table.

[0569] Table 23. - Classification models established

[0570] Model Training and testing

[0571] All models were trained with the dataset described before. Model no. 2 to 4 showed a misclassification rate of 0 for the train dataset, the cross validation of the training data, and the test dataset. Model no. 1 misclassified one Raman spectra of the test dataset (true class: elution buffer, identified class: low conductivity buffer). Therefore, the misclassification rate of the test dataset is 0.014.

[0572] These results show that all four models were able to classify all intermediates with a high accuracy, including the test dataset which was not part of calibration. Even batch to batch variations did not affect the performance of the model. The classification models are able to detect changes between, for example, different sample buffers but can also detect the presence of protein by classifying the solution as IgG-depleted CPP, CPP or eluate instead of a buffer. Furthermore, the models are capable to classify between CPP and IgG-depleted CPP providing an additional approach for the detection of a breakthrough of IgG if the model is detecting CPP at the column outlet instead of IgG- depleted CPP. Also, the elution step can be monitored using the classification models as the developed models are able to differentiate between eluate and elution buffer. Table 24. - Performance of the classification models developed

[0573] 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

CLAIMS1. A method of isolating a protein from plasma, or a fraction thereof, using continuous chromatography, the method comprising:(a) loading an amount of plasma, or fraction thereof, comprising the protein onto a chromatographic medium capable of separating the protein from at least a portion of the amount of plasma or fraction thereof;(b) eluting the protein from the chromatographic medium;(c) collecting the protein; wherein, Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium and / or which is in the amount of plasma, or fraction thereof, being loaded onto the chromatographic medium.

2. The method of claim 1, wherein the Raman spectroscopic analysis is used to monitor for the protein which has passed through the chromatographic medium.

3. The method of claim 1 or claim 2, wherein the Raman spectroscopic analysis monitors for breakthrough of the protein due to overloading of the chromatographic medium.

4. The method of any one of claims 1 to 3, wherein the Raman spectroscopic analysis monitors for elution of the protein in an elution buffer.

5. The method of any one of claims 1 to 4, wherein the Raman spectroscopic analysis comprises determination of a concentration of the protein and / or a concentration of total protein.

6. The method of any one of claims 1 to 5, wherein the monitoring by Raman spectroscopic analysis determines one or more operational change in the continuous chromatography, optionally wherein the one or more operational change are selected from: collecting the protein eluting from the chromatographic medium; ceasing loading the chromatographic medium with the protein;loading an amount of the protein eluting from the chromatographic medium onto a further chromatographic medium, preferably wherein both chromatographic media are the same resin, further preferably wherein the further chromatographic medium is provided in a separation unit that is distinct from a separation unit providing the (first) chromatographic medium; ceasing elution of the protein with an elution buffer; ceasing collection of the protein; and / or washing the chromatographic medium.

7. The method of any one of claims 1 to 6, wherein the Raman spectroscopic analysis is an in-line, online, at-line or off-line analysis, preferably an in-line analysis, further preferably wherein the Raman spectroscopic analysis is performed continuously.

8. The method of any one of claims 1 to 7, wherein the Raman spectroscopic analysis is performed in a flow cell, preferably wherein a flow rate through the flow cell is between about 0 and about 250 mL / min, or between about 0.1 and about 250 mL / min, or between about 0.5 and about 50 mL / min.

9. The method of any one of claims 1 to 8, wherein the Raman spectroscopic analysis comprises the use of a Raman analyzer configured with a laser or other suitable light source that operates at defined wavelengths, optionally wherein the light source has a wavelength of about 785 nm, or a wavelength in the range between 325 nm and 1064 nm.

10. The method of any one of claims 1 to 9, wherein the Raman spectroscopic analysis comprises applying a light source to a sample with an exposure time between about 1 and about 20 seconds.

11. The method of any one of claims 1 to 10, wherein the Raman spectroscopic analysis, when monitoring for breakthrough of the protein due to overloading of the chromatographic medium, is performed with an exposure time between about 6 and about 10 seconds.

12. The method of any one of claims 1 to 11, wherein the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, is performed with an exposure time less than about 5, 4, 3, 2 or 1 second(s).

13. The method of any one of example embodiments 1 to 12, wherein each Raman spectroscopic analysis comprises; applying a light source to a sample: eluting from the chromatographic medium; or being loaded onto the chromatographic medium, measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, wherein each Raman spectroscopic analysis further comprises: comparing the test spectra with reference spectra obtained from reference samples having known concentrations of the protein or total protein; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein or total protein, to determine the presence, amount or concentration, or a change thereof, of the protein or total protein present in the sample; or comparing the test spectra with: reference spectra obtained from reference samples having known concentrations of the protein; or a reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount or concentration, or a change thereof, of the protein present in the sample; or comparing the test spectra with: reference spectra obtained from reference samples comprising one or more predetermined compositions; ora reference data set in the form of a model generated using peak integration, hard modelling, or multivariate analysis of processed reference spectra of reference samples comprising one or more predetermined compositions, to determine that the sample is a composition corresponding to at least one of the one or more predetermined compositions, and / or that the composition of the sample has changed from one to another of the one or more predetermined compositions, optionally wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one or more of the following regions: 700 cm'1and 800 cm'1; 900 cm'1and 1100 cm'1; 1150 cm'1and 1300 cm'1; 1300 cm'1and 1600 cm'1; 2800 cm'1and 3000 cm'1; 300 cm'1and 3500 cm'1; 340 cm'1and 1500 cm'1; 300 cm'1and 1050 cm'1; and / or 500 cm'1and 2600 cm'1.

14. The method of any one of claims 1 to 13, wherein the Raman spectroscopic analysis, when monitoring for the protein eluting in an elution buffer, comprises: applying a light source to a sample eluting from the chromatographic medium; measuring inelastic scattering or Raman shift from the sample, thereby generating test spectra, comparing the test spectra with: a reference data set in the form of a model generated using peak integration of processed reference spectra of reference samples having known concentrations of the protein, to determine the presence, amount, or concentration, or change thereof, of protein present in the sample.

15. The method of claim 14, wherein the test spectra comprise and / or consist of measurements of inelastic scattering or Raman shift within one of more of the following regions:700 cm'1and 800 cm'1;900 cm'1and 1100 cm'1;1150 cm'1and 1300 cm'1;1300 cm'1and 1600 cm'1; and / or2800 cm'1and 3000 cm'1.

16. The method of any one of claims 1 to 15, wherein the continuous chromatography is continuous affinity chromatography, optionally wherein the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, periodic counter-current chromatography (PCC), continuous counter-current tangential chromatography (CCTC), and continuous counter-current spiral chromatography (CCSC).

17. The method of any one of claims 1 to 16, wherein the plasma, or a fraction thereof, is obtained from processing of blood-derived plasma obtained from human blood, optionally wherein the plasma, or a fraction thereof, is obtained or derived from the processing of blood-derived plasma that comprises fresh plasma, cryopoor plasma, or cryo-rich plasma; and / or optionally wherein the plasma, or a fraction thereof, is obtained or derived from hyperimmune plasma; and / or optionally wherein the plasma, or a fraction thereof, is a resuspension of a precipitate or paste obtained from blood-derived plasma; and / or optionally wherein the plasma, or a fraction thereof, is a filtrate obtained from blood-derived plasma.

18. The method of any one of claims 1 to 17, wherein the plasma, or a fraction thereof, is selected from the group consisting of a human blood plasma sample, an IgG intermediate product, a cryo-rich plasma, a cryo-poor plasma, a Supernatant I (SN I), a Cohn Fraction II (Fr II), a Cohn Fraction II+III (Fr II+III), a Cohn Fraction (I+)II+III (Fr(I+)II+III), a Kistler / Nitschmann Precipitate A (KN A), a Kistler / Nitschmann Precipitate B (KN B), a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an IgG depleted intermediate product and combinations thereof.

19. The method of any one of claims 1 to 18, wherein the protein is immunoglobulin (IgG).

20. The method of any one of claims 1 to 18, wherein the protein is albumin.

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