Method and system for assessing the integrity of a chromatography column

Abstract

The chromatography column analog can include a chromatography medium. The chromatography column analog can further include a void configured to create a zone of preferential flow within the chromatography medium, a block configured to create a zone of reduced flow within the chromatography medium, or a void and a block. The block can include a top surface, a bottom surface, and a thickness between the top and bottom surfaces. The width of the top or bottom surface of the block can be about 50 percent to about 90 percent of the inner diameter of the analog.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 387,717, filed December 16, 2022, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to systems and methods for assessing the integrity of chromatography columns. Some aspects of the disclosure relate to systems and methods for assessing process risks in the biological production of therapeutics related to the integrity of chromatography columns. [Background technology]

[0003] Introduction Biopharmaceuticals (e.g., antibodies, fusion proteins, adeno-associated viruses (AAV), proteins, tissues, cells, polypeptides, or other therapeutic products of biological origin) are increasingly being used to treat and prevent infectious, genetic, autoimmune, and other diseases. The production of biopharmaceuticals requires chromatography to purify, characterize, and validate the product. Disruptions in the function of chromatography systems, for example, a loss of the integrity of the chromatography column, can affect the purity of biopharmaceuticals produced using chromatography.

[0004] Because of the tight tolerances required in the production of biopharmaceuticals, even a partial loss of chromatographic column integrity can render all product that comes into contact with the damaged column unusable. The exact impact of a reduced chromatographic column integrity on the processed biopharmaceutical is unknown. The effectiveness of studying the extent and mechanism of the impact of chromatographic column integrity on biological product production is limited primarily by the cost of the large amounts of unusable product that would need to be generated to conduct such studies. Commercially available bench-scale chromatographic columns cannot reproduce the observed failure modes of production-scale chromatographic column integrity. Summary of the Invention

[0005] Aspects of the present disclosure relate to chromatographic column analogs. The chromatographic column analogs can include a chromatographic medium. The chromatographic column analogs can include voids configured to create zones of preferential flow within the chromatographic medium. The chromatographic column analogs can include blocks configured to create zones of reduced flow within the chromatographic medium.

[0006] The analog may include a tube having a top opening and a bottom opening. A gap may be between the top and bottom openings of the tube. The gap may have a length of about 1.0 cm to about 10 cm. The gap may have a width of about 0.5 cm to about 1.0 cm. The analog may include a first filter screen in contact with the top opening and a second filter screen in contact with the bottom opening. The first and second filter screens may be impermeable to the chromatography medium. The tube may include a wall between the top and bottom openings. The wall may be in contact with the chromatography medium. The tube may include stainless steel, glass, or another material impermeable to water. The block may include a top surface, a bottom surface, and a thickness between the top and bottom surfaces. The bottom surface may have a width of about 0.5 cm to about 2.5 cm. The analog may have a total volume of about 15 mL to about 4600 mL.

[0007] In another aspect, the present disclosure is directed to a chromatography column analog comprising a chromatography medium. The chromatography column analog can include a void that does not contain the chromatography medium, the void being permeable to water and / or the chromatography medium. The chromatography column analog can include a block that does not contain the chromatography medium, the block being impermeable to water.

[0008] The analog may include a lumen having a top opening and a bottom opening, with the gap being between the top opening and the bottom opening. The lumen may be parallel to the longitudinal axis of the analog. A first portion of the chromatography medium may be above the top opening, and a second portion of the chromatography medium may be below the bottom opening. The block may be below the top opening of the lumen and above the bottom opening of the lumen. The width of the block may be equal to or greater than the thickness of the block. The thickness of the block may be substantially parallel to the longitudinal axis of the analog. The width of the block may be about 50 percent to about 90 percent of the inner diameter of the analog. The block may be a first block, and the analog may include a second block.

[0009] In another aspect, the present disclosure is directed to a method for determining the relationship between column integrity and product quality. The method may include performing a first iteration of a chromatography run using a chromatography column, thereby producing a first chromatogram and a first product pool. The method may further include performing a second iteration of the chromatography run using a channeling analog, thereby producing a second chromatogram and a second product pool. The method may further include performing a third iteration of the chromatography run using a blocking analog, thereby producing a third chromatogram and a third product pool. The method may further include analyzing the product quality of the first, second, and third product pools and determining one or more peak characteristics of the first, second, and third chromatograms. The method may also include determining the relationship between product quality and one or more peak characteristics.

[0010] Analyzing product quality may include Pico Microchip-Capillary Electrophoresis (PICO MCE) purity analysis, size-exclusion ultra-performance liquid chromatography (SE-UPLC) purity analysis, imaging capillary isoelectric focusing (iCEIF), glycan analysis, host cell DNA analysis, and / or host cell protein analysis. The chromatographic operation may include introducing a mobile phase containing a salt slug. Determining one or more peak characteristics of the first, second, and third chromatograms may include determining a first peak onset, a first peak maximum, and a first peak end for a first peak in the first chromatogram, determining a second peak onset, a second peak maximum, and a second peak end for a second peak in the second chromatogram, and determining a third peak onset, a third peak maximum, and a third peak end for a third peak in the third chromatogram. The first, second, and third peaks may correspond to the elution of salt slugs.

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments. Any feature of an embodiment or example (e.g., composition, formulation, method, etc.) described herein may be combined with any other embodiment or example, and all such combinations are encompassed by the present disclosure. Furthermore, the described systems and methods are not limited to any single aspect or embodiment thereof, nor to any combination or permutation of such aspects and embodiments. For the sake of brevity, certain permutations and combinations are not separately described and / or illustrated herein. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graphical representation of an asymmetry coefficient calculation according to an aspect of the present disclosure.

[0013] [Figure 2] 1 is a chromatogram generated during a chromatography run using a hydrophobic interaction chromatography column, according to an embodiment of the present disclosure.

[0014] [Figure 3] 1 is a chromatogram generated during a chromatography run using a hydrophobic interaction chromatography column, according to an embodiment of the present disclosure.

[0015] [Figure 4] 1 is a chromatogram generated during a chromatography run using an affinity chromatography column according to an embodiment of the present disclosure.

[0016] [Figure 5] 1 is a chromatogram generated during a chromatography run using an affinity chromatography column according to an embodiment of the present disclosure.

[0017] [Figure 6]1 is a chromatogram generated during a chromatography run using an ion exchange chromatography column in accordance with an embodiment of the present disclosure.

[0018] [Figure 7] 1 is a chromatogram generated during a chromatography run using an ion exchange chromatography column, according to an embodiment of the present disclosure.

[0019] [Figure 8A] FIG. 1 is a schematic diagram of a cross section of a channeling analog, according to an embodiment of the present disclosure.

[0020] [Figure 8B] FIG. 8B is a vertical cross-sectional view of the analogue of FIG. 8A.

[0021] [Figure 9A] FIG. 1 is a schematic diagram of a cross section of a fouling analog according to an embodiment of the present disclosure.

[0022] [Figure 9B] FIG. 9B is a vertical cross-sectional view of the analogue of FIG. 9A.

[0023] [Figure 10A] 1 is a chromatogram generated from a pre-use evaluation of a chromatography column, according to an embodiment of the present disclosure.

[0024] [Figure 10B] 1 is a chromatogram generated from a pre-use evaluation of a channeling analogue, according to an embodiment of the present disclosure.

[0025] [Figure 10C] 1 is a chromatogram generated from a pre-use evaluation of fouling analogs according to an embodiment of the present disclosure.

[0026] [Figure 11] 11A-11H are chromatograms generated from pre-use evaluations of analogs according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any suitable methods and materials (e.g., similar or equivalent to those described herein) can be used in the practice or testing of this disclosure, certain exemplary methods are described herein. All publications mentioned are incorporated herein by reference.

[0028] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, and thus a process, method, article, or apparatus that includes a list of elements does not include only those elements, but may include elements not expressly listed, as well as other elements inherent to such process, method, article, or apparatus. The word "exemplary" is used in the sense of "illustrative" rather than "exemplary." The terms "for example" and "such as," and their grammatical equivalents, are understood to be accompanied by the phrase "without limitation," unless expressly stated otherwise.

[0029] As used herein, the term "about" is intended to account for variations due to experimental error. When applied to a numerical value, the term "about" may indicate a ±5% variation from the disclosed numerical value unless a different variation is specified. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, all ranges are understood to include the endpoints; for example, 1 centimeter (cm) to 5 cm includes lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0030] It should be noted that all numerical values ​​disclosed or claimed herein (including all disclosed values, limits, and ranges) can have a variation of ±5% from the disclosed numerical value unless a different variation is specified.

[0031] As used herein, the term "polypeptide" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. A protein comprises one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide can be a protein, and a protein can comprise multiple polypeptides to form a single functional biomolecule.

[0032] Post-translational modifications can modify or alter the structure of polypeptides. For example, disulfide bridges (e.g., S-S bonds between cysteine ​​residues) can be formed post-translationally in some proteins. Some disulfide bridges are essential for the proper structure, function, and interaction of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins can be subjected to other post-translational modifications, such as lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of a phosphate group to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface interactions involving polypeptides.

[0033] As used herein, the term "protein" includes biological therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Protein of interest (POI) can include any polypeptide or protein that is desired to be isolated, purified, or otherwise prepared. POI can include polypeptides produced by cells, including antibodies.

[0034] The term "antibody" as used herein includes immunoglobulins consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Typically, antibodies have a molecular weight of greater than 100 kDa, e.g., 130 kDa to 200 kDa, e.g., about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may also be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may also be abbreviated as LCDR1, LCDR2, and LCDR3).

[0035] For example, a class of immunoglobulin called immunoglobulin G (IgG) is common in human serum and contains four polypeptide chains: two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cystine disulfide bond, and the two heavy chains are linked to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass differs in their constant region and, as a result, may have different effector functions. In some embodiments described herein, a biopharmaceutical may contain a target polypeptide comprising IgG. In at least one embodiment, the target polypeptide comprises IgG4.

[0036] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0037] Biopharmaceuticals (e.g., target molecules, polypeptides, antibodies) can be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia species), or mammalian systems (e.g., CHO cells and CHO-derived cells such as CHO-K1 cells). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, such as, for example, hybridomas or quadromas. In some embodiments, the cells can be human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell may be a eukaryotic cell and may be selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cells may contain one or more viral genes, for example, may be retinal cells that express viral genes (eg, PER.C6™ cells).

[0038] The term "target molecule" may be used herein to refer to a target polypeptide (e.g., an antibody, antibody fragment, or other protein or protein fragment) or other molecule (e.g., an adeno-associated virus (AAV) or other molecule for therapeutic use) intended to be produced, isolated, purified, and / or included in a pharmaceutical product. While methods according to the present disclosure may refer to a target polypeptide, the methods may be equally applicable to other target molecules. For example, AAV can be prepared according to a suitable method (e.g., depth filtration, affinity chromatography, etc.), and a mixture containing AAV may be subjected to a method according to the present disclosure. Before or after subjecting the mixture to one or more methods of the present disclosure, the mixture containing AAV may be subjected to additional procedures (e.g., removal of "empty cassettes" or AAV that do not contain a target sequence).

[0039] In some embodiments, the target molecule is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody, or a tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0040] In some embodiments, the target molecule (e.g., an antibody) is an anti-Programmed Cell Death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication No. 2015 / 0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-Angiopoietin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), an anti-Angiopoietin-like 3 antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,018,356), an anti-Platelet-Derived Growth Factor Receptor antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,265,827), or an anti-Platelet-Derived Growth Factor Receptor antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,265,827). anti-PDGFR antibodies), anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies described in U.S. Pat. No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies described in U.S. Pat. No. 9,132,192 or anti-EGFRvIII antibodies described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-Proprotein Convertase Subtilisin Kexin-9 antibodies (e.g., anti-PCSK9 antibodies described in U.S. Pat. No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-Growth And Differentiation Antibodies Growth and Differentiation Factor-8 (GDF8) antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, described in U.S. Pat. No. 8,871,209 or U.S. Pat. No. 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibodies described in U.S. Patent Application Publication No. 2015 / 0337045A1 or U.S. Patent Application Publication No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (e.g., anti-IL4R antibodies described in U.S. Patent Application Publication No. 2014 / 0271681A1 or U.S. Pat. No. 8,735,095 or U.S. Pat. No. 8,945,559), anti-interleukin-6 receptor antibodies (e.g., U.S. Pat. No. 7,582,298, U.S. Pat. No. 8,043,515), anti-IL1R antibodies, anti-IL4R antibodies, anti-IL4R antibodies, anti-IL4R antibodies, anti-IL5R antibodies, anti-IL6R antibodies, anti-IL7R antibodies, anti-IL8R antibodies, anti-IL9R antibodies, anti-IL10R antibodies, anti-IL11R antibodies, anti-IL12R antibodies, anti-IL13R antibodies, anti-IL14R antibodies, anti-IL15R antibodies, anti-IL16R antibodies, anti-IL17R antibodies, anti-IL18R antibodies, anti-IL19R ​​antibodies, anti-IL20R antibodies, anti-IL21R antibodies, anti-IL22R antibodies, anti-IL23R antibodies, anti-IL24R antibodies, anti-IL25R antibodies, anti-IL26R antibodies, anti-IL27R antibodies, anti-IL28RNos. 617, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 670, 671, 672, 6 anti-cluster of differentiation 3 (e.g., anti-CD3 antibodies described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1, and U.S. Application No. 62 / 222,605), anti-cluster of differentiation 20 (e.g., anti-CD20 antibodies described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1, and U.S. Patent No. 7,879,984), anti-cluster of differentiation 48 (e.g., anti-CD48 antibodies described in U.S. Patent No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Pat. No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody), anti-Ebola virus antibody (e.g., Regeneron's REGN-EB3), anti-CD19 antibody, anti-CD28 antibody, anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-Erb3 antibody, anti-Zika virus antibody, anti-Lymphocyte Activation Gene 3 (e.g., anti-LAG3 antibody or anti-CD223 antibody), and anti-Actin A antibody. Each of the U.S. patents and U.S. patent publications mentioned in this paragraph is incorporated by reference in its entirety.

[0041] In some embodiments, the targeting molecule (e.g., bispecific antibody) is selected from the group consisting of anti-CD3 x anti-CD20 bispecific antibody, anti-CD3 x anti-mucin 16 bispecific antibody, and anti-CD3 x anti-prostate-specific membrane antigen bispecific antibody. In some embodiments, the targeting molecule is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesbacumab, dupilumab, trevoglumab, evinacumab, and linucumab.

[0042] In some embodiments, the target molecule is a recombinant protein (e.g., an Fc fusion protein) comprising an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor-Fc fusion protein, which comprises one or more extracellular domains of a receptor linked to an Fc portion. In some embodiments, the Fc portion comprises a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor-Fc fusion protein comprises two or more different receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g., rilonacept, which comprises the IL-1RAcP ligand-binding region fused to the extracellular domain of IL-1R1 fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, both of which are incorporated by reference in their entirety). In other embodiments, the Fc fusion protein is an ScFv-Fc fusion protein, which comprises one or more antigen-binding domains, such as the variable heavy and light fragments of an antibody, coupled to an Fc portion.

[0043] The term "chromatography," as used herein, refers to a process of separating components of a mobile phase (e.g., a mixture or solution containing multiple components) by passing the mobile phase through a medium such that the components pass through the medium at different velocities, and includes, but is not limited to, column chromatography, planar chromatography, thin-layer chromatography, displacement chromatography, gas chromatography, affinity chromatography (e.g., Protein A or Protein L), ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, hydrophobic interaction chromatography (HIC), fast protein liquid chromatography, high-performance liquid chromatography, countercurrent chromatography, cyclic countercurrent chromatography, chiral chromatography, or mixed-mode chromatography. While embodiments herein may be disclosed with respect to exemplary types of chromatography processes or apparatus, e.g., column chromatography, the embodiments disclosed herein may be applicable to any type of chromatography.

[0044] The systems and methods of the present disclosure include methods and systems for studying the impact of chromatography column integrity on the production of biopharmaceuticals. Chromatography columns compatible with the methods and systems herein include any columns suitable for separating and / or purifying components of a mobile phase.

[0045] A chromatography column can include a chromatography medium. For example, a chromatography column can include an amino acid medium, a ligand-specific medium, an immunoaffinity medium, an ion affinity medium, a hydrophobic interaction medium, and / or a charged medium. The medium can be a resin, beads, particles bound in a packed-bed column configuration, a membrane, or any form capable of accommodating a mixture or other liquid containing a biopharmaceutical. The medium can include a support structure such as, for example, agarose beads (e.g., Sepharose), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, or other compressible synthetic structures.

[0046] The chromatographic medium can include one or more ligands configured to interact with one or more components of the mobile phase and a support structure supporting the one or more ligands. For example, the chromatographic medium can include a ligand comprising a quaternary amine, a protein A-derived group, a protein L-derived group, a phenyl group, a sulfopropyl group, a triazabicyclodecene (TBD) group, a trimethylammonium ethyl (TMAE) group, a dimethylaminoethyl (DMAE) group, a sulfoethyl group, or a combination thereof. The support structure can include cross-linked agarose, highly-linked agarose, silica, aluminum oxide, methacrylate, glass, polyvinyl ether, or a combination thereof.

[0047] Chromatography columns used in the production of biopharmaceuticals can be configured so that the media has a depth (e.g., bed height) of about 15 centimeters (cm) to about 30 cm. In some embodiments, the chromatography column can be configured so that the internal diameter of the chromatography column is about 15 cm to about 200 cm. In some embodiments, the chromatography column has a total volume (e.g., total capacity for holding a mixture, mobile phase, or other substance) of about 25 liters (L) to about 277 L.

[0048] In addition to one or more columns, a chromatography system may include a detector. The detector may be any type of detector suitable for detecting one or more properties at the outlet of the column. Such properties may include, for example, column outlet conductivity, pH, optical density, and / or ultraviolet (UV) or visible light absorbance. In some embodiments, the detector may include a conductivity detector, a UV detector, a fluorescence detector, a refractive index detector, a pH detector, and / or a manometer. For example, the detector may measure the absorbance of UV light (e.g., at a wavelength of 280 nm), and the measured absorbance may be correlated to the protein concentration of the mobile phase exiting the column.

[0049] A chromatography operation may typically include one or more steps, including, for example, one or more pre-equilibration, equilibration, packing, washing, elution, stripping, and / or regeneration steps. Chromatography operations may be tracked and / or recorded with data collected from a detector at the outlet of the chromatography column. For chromatography operations involved in the manufacture of biopharmaceuticals, the quality, consistency, and completeness of the chromatography operation must be monitored to ensure that the manufactured biopharmaceutical meets internal quality assurance metrics and applicable regulatory agency standards.

[0050] Generally, column integrity can be determined by the characteristics of how a mobile phase flows through the stationary phase (e.g., chromatographic media) of the column. The signal detected from the detector can be plotted against the elapsed time and / or flow volume of the chromatographic run. These plots, called chromatograms, can be used to monitor the progress of the chromatographic run and determine whether it is proceeding within acceptable operating parameters. For example, the presence of abnormal characteristics in a chromatogram can indicate a decrease in column integrity.

[0051] Column integrity represents the ability of a chromatography column to perform at maximum efficiency. Variations in the axial dispersion of the chromatography media within a chromatography column can affect the ability of the chromatography column to perform at maximum efficiency. Additionally or alternatively, variations in the radial dispersion of the chromatography media within a chromatography column can affect the ability of the chromatography column to perform at maximum efficiency. Depending on the type of chromatography column (e.g., the type of chromatography media within the column), a decrease in column integrity can cause a lack of binding of mobile phase components to the column, a lack of separation between mobile phase components, and / or the introduction of impurities into the mobile phase. A chromatography column has decreased column integrity when it is not operating at maximum efficiency.

[0052] The degree to which the column integrity of a chromatography column has deteriorated can be quantified by determining the height equivalent of a theoretical plate (HETP) or the number of theoretical plates of the chromatography column. Compared to a column operating at maximum efficiency, a chromatography column with deteriorated column integrity may have an increased HETP and / or a decreased number of theoretical plates. Another method for quantifying column integrity involves transition analysis, in which chromatographic peaks are analyzed against historical or predicted chromatographic peaks. Variations in chromatographic peaks identified in transition analysis may indicate that the chromatography column used to generate the chromatogram has deteriorated column integrity.

[0053] Degradation of column integrity can result from disruptions of the chromatography media within the column, and these disruptions can adversely affect the ability of materials to flow through the column. Disruptions that can cause degradation of column integrity can be the result of repeated use of a chromatography column. Disruptions in the chromatography media can cause materials to flow too quickly through the column without contacting a sufficient amount of the chromatography media to effectively separate the components of the introduced material. Additionally or alternatively, the disruption can block the flow of materials through a region of the column, which can also result in a reduction in the efficiency of the column.

[0054] One type of fracture in a chromatographic medium, channeling, refers to the presence of voids within the chromatographic medium that can propel the mobile phase through the chromatographic medium faster than the average flow rate of the chromatographic operation. Chromatographic media that is too dense can fracture, leading to channeling and the formation of voids within the chromatographic medium. Channeling can cause a lack of separation of the components of the mobile phase because preferential flow of the mobile phase through the voids in the chromatographic medium reduces the interaction between the components of the mobile phase and the chromatographic medium.

[0055] Peak broadening and peak fronting in a chromatogram may indicate channeling in the chromatographic column used to generate the chromatogram. Additionally or alternatively, bifurcated peaks, false peaks, shifted peak elution timing, and / or the absence of expected peaks may indicate channeling.

[0056] Another type of disruption in chromatography media, fouling, refers to the blocking of pores or flow paths in the chromatography media. For example, the support structure of the chromatography media may be embedded in a filter screen within a chromatography column. The embedded media may prevent mobile phase from flowing through a portion of the chromatography column. Poor flow caused by blockages can lead to the formation of biofilms or other growths that further impede the movement of mobile phase through the chromatography column. Fouling can cause a first portion of the mobile phase to flow slower than a second portion of the mobile phase. The resulting delay in elution of the mobile phase through the column can result in poor separation of the mobile phase components. Delayed elution can also cause peak broadening and peak tailing. Thus, detection of peak broadening, peak tailing, and / or poor peak resolution can indicate fouling.

[0057] Deterioration of column integrity can be detected by the presence of asymmetric chromatographic peaks. For example, a chromatogram plotted based on a chromatographic run utilizing a column with impaired column integrity may contain fronted and / or tailed peaks. Peak fronting refers to an asymmetric peak in which the first half of the chromatographic peak is broader than the second half of the chromatographic peak. Peak tailing refers to an asymmetric peak in which the second half of the chromatographic peak is broader than the first half of the chromatographic peak.

[0058] The asymmetry of a peak may be quantified by one or more peak symmetry metrics, such as, for example, the asymmetry factor. The asymmetry factor (a) of a peak may be calculated according to Equation 1, where b h% is the width of the second half of the peak at h% of the peak height, and f h% is the width of the first half of the peak at h% of the peak height.

number

[0059] An example of asymmetry calculation at 5% of the peak height is shown in Figure 1. The peak shown in Figure 1 is symmetrical, therefore the width of the first half of the peak at 5% of the peak height, f 5%h is the width of the second half of the peak at 5% of the peak height, b 5%h A symmetric peak has an asymmetry factor (a) of 1.0. A fronted peak has an asymmetry factor less than 1.0, and a tailed peak has an asymmetry factor greater than 1.0.

[0060] Figure 2 shows an exemplary chromatogram of a chromatography run using a hydrophobic interaction chromatography column. The chromatography run shown in Figure 2 is a flow-through process in which eluate collection occurs while a mobile phase (e.g., a mixture containing a biopharmaceutical) and wash buffer are introduced into the column. The collected eluate contains the desired biopharmaceutical, while undesired molecules (e.g., host cell proteins) remain bound to the column. Subsequent washing and stripping steps remove the undesired molecules bound to the column, so the column can be regenerated and reused. To generate the chromatogram shown in Figure 2, the ultraviolet absorbance (black line), conductivity (gray line), and pH (dashed line) of the solution exiting the column are measured by one or more detectors and plotted against the volume passing through the column.

[0061] Events related to the chromatographic run are marked along the x-axis. At T0, a pre-equilibration step is initiated, followed by an equilibration step beginning at T1. A mixture containing the biopharmaceutical is introduced at T2 and eluate collection begins. After the mixture is introduced onto the column, a wash buffer is introduced at T3. Eluate collection ends at T4. One or more stripping buffers are introduced onto the column at T5, T6, and T7.

[0062] The chromatogram shown in Figure 3 is an example of a chromatogram generated during a chromatography run involving a hydrophobic interaction chromatography column with compromised column integrity. Similar to the chromatogram shown in Figure 2, a pre-equilibration step begins at T0, followed by an equilibration step beginning at T1. A mixture containing a biopharmaceutical is introduced at T2, and eluate collection begins. After the mixture is introduced into the column, a wash buffer is introduced at T3. Eluate collection ends at T4. One or more stripping buffers are introduced into the column at T5, T6, and T7. Still referring to Figure 3, the black line represents UV absorbance, the gray line represents conductivity, and the dashed line represents pH.

[0063] Chromatogram features can be monitored or analyzed to determine whether a chromatography column is operating within intended parameters or whether the column's integrity is deteriorating. For example, referring to FIG. 2, after a mixture containing a biopharmaceutical is introduced into the column, the absorbance increases almost immediately. Compared to the chromatogram shown in FIG. 2, the absorbance peak in the chromatogram shown in FIG. 3 has a more gradually sloping increase near T2. In addition, the absorbance peak in the chromatogram of FIG. 3 is less symmetrical than the absorbance peak in the chromatogram of FIG. 2. The sloping increase in the absorbance peak and the asymmetric peak distribution indicate deteriorating column integrity.

[0064] Figure 4 shows an exemplary chromatogram of a chromatography run using an affinity chromatography column. The black line represents UV absorbance, the gray line represents conductivity, and the dashed line represents pH. Events in the affinity chromatography run are marked on the x-axis. At T0, a mixture containing a biopharmaceutical is introduced into the column, which may be referred to as the loading step. At T1 and T2, one or more wash buffers are introduced into the column. At T3, an elution buffer is introduced into the column and eluate collection begins. One or more wash buffers and / or strip buffers may be introduced into the column after the elution buffer. At T4, eluate collection is stopped, and at T5, an equilibration buffer is introduced into the column.

[0065] The chromatogram shown in Figure 5 is an example of a chromatogram generated during a chromatography run involving an affinity column with reduced column integrity. Similar to the chromatogram shown in Figure 4, the black line represents UV absorbance, the gray line represents conductivity, and the dashed line represents pH. Events in the affinity chromatography run are marked on the x-axis. At T0, a mixture containing the biopharmaceutical is introduced into the column, which may be referred to as the loading step. At T1 and T2, one or more wash buffers are introduced into the column. At T3, an elution buffer is introduced into the column and eluate collection begins. One or more wash buffers and / or strip buffers may be introduced into the column after the elution buffer. At T4, eluate collection is stopped, and at T5, an equilibration buffer is introduced into the column.

[0066] The absorbance peak in Figure 5 corresponding to elution (i.e., the peak between T3 and T4) is broader than the absorbance peak in Figure 4 corresponding to elution (i.e., the peak between T3 and T4). In addition, the absorbance peak in Figure 5 corresponding to elution is more asymmetric than the absorbance peak corresponding to elution in Figure 4. The absorbance peak corresponding to elution shown in Figure 6 is tailing (i.e., the second half is broader than the first half).

[0067] Figure 6 shows an exemplary chromatogram of a chromatography run using a cation exchange chromatography column. The black line represents UV absorbance, the gray line represents conductivity, and the dashed line represents pH. Events in the ion exchange chromatography run are marked on the x-axis. At T0, a mixture containing a biopharmaceutical is introduced into the column, which may be referred to as the loading step. At T1, a wash buffer is introduced into the column, and at T2, an elution buffer is introduced into the column. At T3, eluate collection begins, and at T4, eluate collection ends. At T5 and T6, one or more equilibration buffers may be introduced into the column.

[0068] The chromatogram shown in Figure 7 is an example of a chromatogram generated during a chromatography run involving a cation exchange column with compromised column integrity. Similar to the chromatogram shown in Figure 6, the black line represents UV absorbance, the gray line represents conductivity, and the dashed line represents pH. Events in the ion exchange chromatography run are marked on the x-axis. At T0, a mixture containing the biopharmaceutical is introduced into the column, which may be referred to as the loading step. At T1, a wash buffer is introduced into the column, and at T2, an elution buffer is introduced into the column. At T3, eluate collection begins, and at T4, eluate collection ends. At T5 and T6, one or more equilibration buffers may be introduced into the column.

[0069] The absorbance peak corresponding to the elution shown in Figure 7 (i.e., the peak between T2 and T4) is broader compared to the absorbance peak corresponding to the elution shown in Figure 6 (i.e., the peak between T2 and T4). In addition, the absorbance peak shown in Figure 7 exhibits load breakthrough at approximately T1. In other words, during the loading step, proteins comprising the biopharmaceutical flowed through the column instead of binding to the column as intended. Load breakthrough is the result of a loss of column integrity.

[0070] As noted above, a decrease in column integrity can be determined by monitoring the signal from a detector (e.g., plotting conductivity, absorbance, and / or pH as a function of volume passed through the column). Certain characteristics of chromatographic peaks can indicate a decrease in column integrity. For example, broad elution peaks, fronting peaks, tailing peaks, and load breakthrough can all indicate a decrease in column integrity.

[0071] During the manufacture of biopharmaceuticals, signals generated during a chromatographic operation may be monitored by a detector to assess the column integrity of a column used in the chromatographic operation. For example, a chromatogram plotted based on the signal received from the detector may be monitored for the presence of indicators of a decrease in column integrity. If a loss of column integrity is detected, the column may be deemed unsuitable for use. Product that comes into contact with a column deemed unsuitable for use may be deemed unusable. Additionally, production must be stopped while the unsuitable column is regenerated, repaired, or replaced. Unusable product and stopped production time increase the cost and time required to manufacture a biopharmaceutical.

[0072] Because the impact of reduced chromatography column integrity is poorly understood, larger quantities of biopharmaceuticals may be deemed unusable by quality control and regulatory standards when in fact they are only affected by reduced chromatography column integrity. With a better understanding of the relationship between loss of chromatography column integrity and the quality of the manufactured biopharmaceutical, more narrowly tailored chromatography process control can be implemented. Narrowly tailored chromatography process control results in less wasted product and fewer production interruptions compared to traditional chromatography process control.

[0073] Factors limiting understanding the relationship between loss of chromatography column integrity and the quality of manufactured biopharmaceuticals include the cost of investigating the relationship and the lack of precision to evaluate failed columns. For example, there are currently no models or commercially available analogs for columns with reduced column integrity. Traditional studies of the effects of column integrity require columns that have naturally lost column integrity over the course of multiple chromatography processes. Therefore, columns used in such studies are poorly controlled, and the pattern of breakdown within the chromatography media is rarely uniform. Additionally, the quantity of biopharmaceuticals and the operation of manufacturing-scale equipment to investigate the effects and mechanisms of column integrity loss can be cost prohibitive.

[0074] The interaction between a biopharmaceutical and a chromatographic medium can depend on the identity, structure, and properties of the biopharmaceutical. For example, the distribution and size of hydrophobic regions within a biopharmaceutical can affect the interaction between the chromatographic medium and the product. Additionally or alternatively, the feed stream composition of the mobile phase can depend on the structure and / or properties of the biopharmaceutical within the mobile phase. As a result, the relationship between the integrity of the chromatography column and the quality of the produced biopharmaceutical can be unique to each product and chromatography run.

[0075] Therefore, there is a need for a system and method for modeling chromatography systems having columns with reduced column integrity. Specifically, there is a need for analogs of chromatography columns with reduced column integrity, and a system for investigating the effect of column integrity on processed biopharmaceuticals.

[0076] The present disclosure includes analogs of chromatography columns with reduced column integrity. Additionally, the present disclosure includes methods for analyzing the impact of column integrity on the quality of processed biopharmaceuticals and methods for developing chromatography process controls. For example, a system can include an analog configured to cause a mobile phase passed through the analog to behave similarly to a mobile phase passed through a chromatography column with reduced column integrity.

[0077] In some embodiments, the analog may include a chromatographic medium, such as an amino acid medium, a ligand-specific medium, an immunoaffinity medium, an ion affinity medium, a hydrophobic interaction medium, and / or a charged medium. The analog may also include one or more regions that simulate, model, and / or mimic a region of a chromatographic column that involves disruption of the chromatographic medium. For example, the analog may include a region that simulates, models, and / or mimics channeling. Additionally or alternatively, the analog may include a region that simulates, models, and / or mimics fouling.

[0078] The one or more regions of the analogue that simulate, model, and / or mimic a region of a chromatography column involving disruption of the chromatography media may include one or more of a disk, block, void, lumen, tube, or other structure that may disrupt the chromatography media.

[0079] The analogs can be configured to fit into standard chromatography systems. For example, a chromatography system including a pump, inlets, a detector, and one or more chromatography columns can have removable, interchangeable, and / or replaceable columns. The analogs of the present disclosure can be configured to be used in place of a chromatography column in a chromatography system.

[0080] 8A and 8B, the channeling analog 110 can include a chromatographic medium 115 and a void 225. The void 225 is a region of the analog 110 that does not include the chromatographic medium 115. In some embodiments, the void 225 can be formed as a tube inserted into the analog 110, the lumen of a channel, or other space formed within a three-dimensional structure. For example, a tube comprising glass, stainless steel, polystyrene, plastic, or other suitable rigid material that is impermeable to the mobile phase and chemically inert may be inserted into the chromatographic medium 115. The void 225 can include a space within a tube or other three-dimensional structure within the chromatographic medium 115 of the analog 110. The void 225 can include one or more screens that are permeable to the mobile phase. For example, the top opening of the void 225 can include a first screen, and the bottom opening of the void 225 can include a second screen.

[0081] Analog 110 can be configured so that the chromatography medium 115 has a bed height 107 (e.g., depth) of about 15 cm to about 30 cm. Analog 110 can have an inner diameter 103 of about 1 cm to about 14 cm. In some embodiments, analog 110 can have a total volume (e.g., total capacity for holding a mixture, mobile phase, or other substance) of about 15 milliliters (mL) to about 4600 mL.

[0082] The void 225 may have an elongated shape (e.g., a cylindrical shape). The longitudinal axis of the void 225 may be parallel to the longitudinal axis of the analog 110. For example, the longitudinal axis of the void 225 may be parallel to the sidewall of the analog 110. The void 225 may have a width 223 (e.g., a diameter) of about 0.5 cm to about 1.0 cm. The void 225 may have a height 227 of about 1.0 cm to about 10 cm.

[0083] The width 223 of the gap 225 can be between about 10 percent and about 40 percent of the inner diameter 103 of the analog 110. The height 227 of the gap 225 can be between about 10 percent and about 30 percent of the bed height 107 of the analog 110.

[0084] The gap 225 can be positioned from about 1 cm to about 29 cm from the bottom edge of the chromatography medium 115. In some embodiments, the gap 225 can be positioned from about 1 cm to about 29 cm from the top of the edge of the chromatography medium 115. The gap can be positioned from about 1 cm to about 6 cm from the sidewall of the analog 110.

[0085] 8A and 8B includes one void 225, but this is just one example. The channeling analog 110 may include two, three, four, or more voids 225 depending on the degree of column integrity degradation being simulated. For example, to simulate or model a chromatography column with severe loss of column integrity, the analog 110 may include two or more voids 225. Additionally or alternatively, the size of one or more voids 225 may be adjusted to achieve a desired level of disruption within the chromatography medium 115. Larger and more voids 225 simulate a greater degradation of column integrity.

[0086] 9A and 9B, the fouling analog 120 can include a chromatographic medium 115 and one or more blocks 235, 235'.

[0087] Analog 120 can be configured so that chromatography medium 115 has a bed height 107 (e.g., depth) of about 15 cm to about 30 cm. Analog 120 can have an inner diameter 103 of about 1 cm to about 14 cm. In some embodiments, analog 120 can have a total volume (e.g., total capacity for holding a mixture, mobile phase, or other substance) of about 15 mL to about 4600 mL.

[0088] Blocks 235, 235' are structures inserted within analog 120 that do not contain chromatography medium 115 and that obstruct and / or prevent the passage of mobile phase through the space occupied by blocks 235, 235'. Blocks 235 may include stainless steel, glass, polystyrene, plastic, or another material with suitable properties. For example, block 235 may include a material that is impermeable to the chromatography medium. Additionally or alternatively, a suitable material for block 235 may be impermeable to the mobile phase and strong enough not to deform during packing of the chromatography medium within analog 120.

[0089] 9A and 9B, block 235 may have a disk shape including a thickness 237 between a circular top surface and a circular bottom surface. In some embodiments, the top and bottom surfaces of block 235 may have a triangular, oval, rectangular, or other shape. While blocks 235, 235' shown in FIGS. 9A and 9B have a width 233 that is greater than thickness 237, this is by way of example. In other embodiments, block 235 may have a thickness 237 that is greater than width 233.

[0090] The block 235 may have a thickness 237 of about 0.2 millimeters (mm) to about 0.6 mm. The block 235 may have a width 233 of about 0.5 cm to about 2.5 cm. The width 233 of the block 235 may be about 50 percent to about 90 percent of the inner diameter 103 of the analog 120. The height 237 of the block 235 may be about 0.001 percent to about 0.1 percent of the bed height 107 of the analog 120.

[0091] The block 235 closest to the bottom edge of the chromatography medium 115 can be positioned at least about 0.5 cm to about 1.0 cm from the bottom edge of the chromatography medium 115. In some embodiments, the block 235 closest to the top edge of the chromatography medium 115 can be positioned at least about 0.5 cm to about 1.0 cm from the top of the edge of the chromatography medium 115. The block can be positioned about 0.5 cm to about 5 cm from the nearest sidewall of the analog 120.

[0092] 9A and 9B includes two blocks, but this is by way of example. The fouling analog 120 may include one, three, four, five, six, seven, eight, nine, ten, or more blocks 235, depending on the degree of column integrity degradation being simulated. Additionally or alternatively, the size of one or more blocks 235 may be adjusted to achieve a desired level of disruption within the chromatography media 115. Larger, more numerous blocks 235 simulate greater column integrity degradation.

[0093] 9A and 9B, in embodiments in which analog 120 includes multiple blocks 235, 235', the blocks may be positioned parallel to one another. In some embodiments, multiple blocks 235, 235' may be positioned within analog 120 such that at least two blocks 235, 235' are not parallel to one another. Each block 235, 235' of the multiple blocks 235, 235' may have the same shape and size. In some embodiments, at least one block 235, 235' of the multiple blocks 235, 235' has a different shape and / or size from at least one other block 235, 235'. Each block 235, 235' may be coaxial with one another (i.e., the center point of each block 235, 235' is along a single axis of analog 120). In some embodiments, at least one block 235, 235' of the plurality of blocks 235' is at a different position relative to the central axis of the analogue 120 than at least one other block 235, 235' of the plurality of blocks 235, 235'.

[0094] As described above, analogs (e.g., channeling analog 110 and fouling analog 120) allow for investigation of the impact of column integrity of a chromatography column on the purity, yield, and quality of biopharmaceuticals processed using the chromatography column. The analogs can be configured to simulate a desired level of column integrity, for example, by varying the size, shape, and / or number of voids 225 and / or blocks 235. The analogs can allow mobile phase to flow through the analog consistently and repeatedly, similar to how it would flow through a chromatography column with reduced column integrity. The consistent and repeatable nature of mobile phase flow through the analog allows for investigation of the mechanisms and impact of column integrity on biopharmaceutical processing. [Example]

[0095] Before a chromatography column is used in the manufacture of a biopharmaceutical, a pre-use evaluation can be performed on the column to determine whether the mobile phase is flowing through the column as intended. The pre-use evaluation can include generating a baseline chromatogram using the column and generating a chromatogram based on monitoring the conductivity of the eluate containing the salt slug exiting the column. Comparing the baseline chromatogram to the monitored conductivity from the eluate containing the salt slug can enable a determination of whether the mobile phase is flowing through the column as intended. The characteristics of the peak corresponding to the salt slug can be compared to historical or predicted data using an equivalent chromatography column.

[0096] For example, peak onset (e.g., the point at which the signal first reaches at least 5% of the maximum peak height), peak maximum, and / or peak end (e.g., the point at which the signal reaches 5% of the peak height after the maximum peak height) can be compared between peaks generated from a pre-use evaluation protocol and historical peak data (e.g., peaks generated from an unused column or a column with confirmed performance efficiency). If one or more of the peak onset, peak maximum, or peak end of a peak generated from a pre-use evaluation protocol differs from historical or predicted peak data (e.g., differs by at least about 0.1 column volumes), the column used to generate the deviant peak can be considered unsuitable for use.

[0097] During biopharmaceutical manufacturing, chromatography columns exhibiting channeling and / or blocking caused by disruption of the chromatography media can be detected by pre-use evaluation. For example, peak features in a chromatogram generated using a chromatography column exhibiting channeling may appear earlier (e.g., peak fronting) compared to historical peak data. Peak features in a chromatogram generated using a chromatography column exhibiting blocking may appear later (e.g., peak tailing) compared to historical peak data. To demonstrate that the analogs of the present disclosure simulate mobile phase flow through chromatography columns exhibiting channeling and / or blocking, pre-use evaluations were performed on analogs having multiple sizes and types of disruption in the chromatography media.

[0098] One example of a pre-use evaluation protocol, Pre-Use Evaluation Protocol A, involves generating a baseline chromatogram by passing 2.5 column volumes (CV) of a buffer containing 0.1 M sodium chloride through a chromatography column at a flow rate of 125 centimeters per hour (cm / hr) and monitoring the conductivity of the eluate exiting the column. Pre-Use Evaluation Protocol A involves generating a chromatogram by monitoring the conductivity of the eluate while: (a) introducing 0.01 CV of a buffer containing 1.0 M sodium chloride into the chromatography column; and (b) washing the column with 0.1 M sodium chloride at a flow rate of 125 cm / hr until the 1.0 M sodium chloride pulse elutes and the conductivity returns to the baseline level (e.g., the conductivity of the 0.1 M sodium chloride solution). According to Pre-Use Evaluation Protocol A, the peak characteristics corresponding to 0.01 CV of the buffer containing 1.0 M sodium chloride can be used to determine the relative efficiency of the chromatography column. For example, the characteristics of the peak corresponding to 0.01 CV in a buffer containing 1.0 M sodium chloride can be used to determine whether a chromatography column contains disruptions (e.g., channeling or blocking) within the chromatography media.

[0099] Comparative Example

[0100] Figure 10A shows a chromatogram generated from Protocol A, a pre-use evaluation of a chromatography column in which no fractures were introduced into the chromatography media. The column had a bed height of approximately 20 cm, an internal diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL. The dashed line represents the measured conductivity from the baseline chromatogram, and the solid line represents the measured conductivity from the eluate containing the salt slug. The asymmetry of the eluted peak at 5% of the peak height was 1.17, as calculated according to Equation 1.

[0101] Example 1

[0102] FIG. 10B shows a chromatogram generated from Protocol A, a pre-use evaluation of a channeling analog according to an embodiment of the present disclosure. The channeling analog included a stainless steel tube positioned parallel to the longitudinal axis of the column, with a bed height of approximately 20 cm, an internal diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL. The tube included a filter screen at the top opening and a filter screen at the bottom opening to prevent chromatographic media from seeping into the tube. The space within the tube formed an air gap with a height of approximately 2 cm and an internal diameter of approximately 0.7 cm. Referring to FIG. 10B, the dashed line represents the measured conductivity from the baseline chromatogram, and the solid line represents the measured conductivity from the eluate containing the salt slug. The asymmetry of the eluted peak at 5% of the peak height was 0.85, as calculated according to Equation 1. An asymmetry of 0.85 is less than that of the comparative example, confirming that the channeling analog results in peak fronting.

[0103] Example 2

[0104] FIG. 10C shows a chromatogram generated from fouling simulant pre-use evaluation Protocol A according to an embodiment of the present disclosure. The fouling simulant had a bed height of approximately 20 cm, an internal diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL and contained three polystyrene blocks. Each polystyrene block had an approximately circular shape, a thickness of approximately 0.2 mm, and a diameter of approximately 1.75 cm. The dashed line represents the measured conductivity from the baseline chromatogram, and the solid line represents the measured conductivity from the eluate containing the salt slug. The asymmetry factor of the eluted peak at 5% of the peak height was 1.53, as calculated according to Equation 1. The asymmetry factor of 1.53 is greater than that of the comparative example, confirming that the fouling simulant results in peak tailing.

[0105] Although the pre-use evaluation chromatograms shown in Figures 10A-10C include lines for the baseline and the eluate containing the salt slug, the pre-use evaluation chromatograms may alternatively be presented by plotting the difference in baseline conductivity subtracted from the measured conductivity of the eluate containing the salt slug.

[0106] Additional channeling and fouling analogs were prepared according to embodiments of the present disclosure and are described in Examples 3-10. All analogs described in Examples 3-10 contain affinity chromatography media and / or ion exchange media. The analogs described in Examples 3-10 have a bed height of approximately 20 cm, an internal diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL.

[0107] Example 3

[0108] A channeling analog was prepared containing a void having a height of approximately 2.0 cm and a width of approximately 0.7 cm. The void was formed by inserting a tube containing stainless steel into the chromatography media. The tube contained a filter screen at the top opening and a filter screen at the bottom opening to prevent the chromatography media from entering the void formed in the tube.

[0109] Example 4

[0110] A channeling analog was prepared containing a gap having a height of approximately 3.0 cm and a width of approximately 0.7 cm. The gap was formed by inserting a tube containing stainless steel into the chromatography media. The tube contained a filter screen at the top opening and a filter screen at the bottom opening to prevent the chromatography media from entering the gap formed in the tube.

[0111] Example 5

[0112] A channeling analog was prepared containing a gap having a height of approximately 3.5 cm and a width of approximately 0.7 cm. The gap was formed by inserting a tube containing stainless steel into the chromatography media. The tube contained a filter screen at the top opening and a filter screen at the bottom opening to prevent the chromatography media from entering the gap formed in the tube.

[0113] Example 6

[0114] A channeling analog was prepared containing a void having a height of approximately 4.0 cm and a width of approximately 0.7 cm. The void was formed by inserting a tube containing stainless steel into the chromatography media. The tube contained a filter screen at the top opening and a filter screen at the bottom opening to prevent the chromatography media from entering the void formed in the tube.

[0115] Example 7

[0116] A fouling simulant was prepared containing three blocks, each approximately circular in shape, approximately 0.2 mm thick, and approximately 1.75 cm in diameter. The blocks consisted of polystyrene disks inserted into the chromatographic media.

[0117] Example 8

[0118] A fouling simulant was prepared containing four blocks, each approximately circular in shape, approximately 0.2 mm thick, and approximately 1.88 cm in diameter. The blocks consisted of polystyrene discs inserted into the chromatographic media.

[0119] Example 9

[0120] A fouling simulant was prepared containing six blocks, each having an approximately circular shape, a thickness of about 0.2 mm, and a diameter of about 2.00 cm. The blocks consisted of polystyrene disks inserted into the chromatographic media.

[0121] Example 10

[0122] A fouling simulant was prepared containing eight blocks, each approximately circular in shape, approximately 0.2 mm thick, and approximately 2.13 cm in diameter. The blocks consisted of polystyrene disks inserted into the chromatographic media. Figures 11A-11H show chromatograms generated from Pre-Use Evaluation Protocol A for the analogs of Examples 3-10 according to an embodiment of the present disclosure. For each plot in Figures 11A-11H, the dashed line represents the measured conductivity from the baseline chromatogram, and the solid line represents the measured conductivity from the eluate containing the salt slug. For each chromatogram, the onset, maximum, and end points of each peak were calculated in relation to the column volume through which the analog was passed. The peak onset is the first point at which the conductivity reaches 5% of its maximum value. The peak end is the last point at which the conductivity is 5% of its maximum value. The peak maximum is the point at which the conductivity reaches its maximum value. The asymmetry factor at 5% peak height was also calculated for each chromatogram according to Equation 1. The peak onset, peak maximum, peak end, and asymmetry factor for Examples 3-10 are summarized in Table 1. [Table 1]

[0123] Channeling analogs had fronted elution peaks with asymmetry factors less than 1. Fouling analogs had tailed elution peaks with asymmetry factors greater than 1.

[0124] Analogs for chromatography columns with loss of column integrity can be constructed and implemented as described herein. The analogs described herein can be used to develop or improve the efficiency of chromatography runs. For example, chromatography runs can be performed with analogs that include various integrity loss conditions. The quality of biopharmaceuticals processed using the analogs can be analyzed to determine how column integrity affects the quality of the resulting biopharmaceutical.

[0125] Product quality testing may include pico microchip-capillary electrophoresis (PICO MCE) purity analysis, size-exclusion ultra-performance liquid chromatography (SE-UPLC) purity analysis, imaging capillary isoelectric focusing (iCEIF), glycan analysis, host cell DNA analysis, and / or host cell protein analysis. PICO MCE purity analysis may involve analysis of test samples by non-reducing and reducing microchip capillary electrophoresis using a GXII instrument to estimate the purity and impurity levels (with an emphasis on determining fragmentation levels) of product pool samples. SE-UPLC purity analysis involves size-exclusion chromatography and ultra-performance liquid chromatography to separate protein species based on molecular weight. iCIEF involves imaging capillary isoelectric focusing to determine the relative abundance of charge variants of biopharmaceuticals. Glycan analysis involves determining the fucosylated glycan content of the product using reversed-phase high-performance liquid chromatography. Host cell DNA analysis involves the detection of DNA from host cells (e.g., Chinese hamster ovary cells) in product samples using real-time quantitative polymerase chain reaction (PCR) analysis. Host cell protein analysis involves the quantification of the presence of host cell proteins (HCPs) in products using enzyme-linked immunosorbent assays (ELISAs).

[0126] Based on biopharmaceutical quality testing, chromatography operations can be developed or improved. For example, a model or relationship determined based on data from product quality testing can determine a threshold level of column integrity that may adversely affect biopharmaceutical quality. Pre-use assessment protocols can be developed using the determined threshold to efficiently screen column integrity during biopharmaceutical manufacturing.

[0127] The present disclosure is further illustrated by the following non-limiting sections.

[0128] Item 1. A chromatography column analogue, a chromatography medium; and a void configured to create zones of preferential flow within the chromatography medium; or Blocks configured to create zones of reduced flow within the chromatography media. and one or more of:

[0129] Item 2. The analog of item 1, wherein the analog includes the gap and a tube having a top opening and a bottom opening, and the gap is between the top opening and the bottom opening of the tube.

[0130] Item 3. The analogue according to Item 2, wherein the gap has a length of about 1.0 cm to about 10 cm and a width of about 0.5 cm to about 1.0 cm.

[0131] Item 4. The analog of item 2, further comprising a first filter screen in contact with the top opening and a second filter screen in contact with the bottom opening, wherein the first and second filter screens are impermeable to the chromatography media.

[0132] Item 5. The analog of item 2, wherein the tube comprises a wall between the top opening and the bottom opening, the wall being in contact with the chromatography medium.

[0133] Item 6. The analog of item 2, wherein the tube comprises stainless steel, glass, or another material that is impermeable to water.

[0134] Item 7. The analog of item 1, wherein the analog includes the block, and the block includes a top surface, a bottom surface, and a thickness between the top surface and the bottom surface.

[0135] Item 8. The product according to Item 7, wherein the bottom surface has a width of about 0.5 cm to about 2.5 cm.

[0136] Item 9. The analogue according to Item 1, wherein the analogue has a total volume of about 15 mL to about 4600 mL.

[0137] Item 10. A chromatography column analogue, a chromatography medium; and a void that does not contain a chromatographic medium and is permeable to water; or A block that does not contain a chromatographic medium and is impermeable to water. and one or more of:

[0138] Item 11. The analog of item 10, wherein the analog comprises the void and a lumen having an upper opening and a lower opening, and the void is between the upper opening and the lower opening of the lumen.

[0139] Item 12. The analog of item 11, wherein the lumen is substantially parallel to the longitudinal axis of the analog.

[0140] Item 13. The analog of item 11, wherein a first portion of the chromatography medium is above the top opening and a second portion of the chromatography medium is below the bottom opening.

[0141] Item 14. The analog of Item 11, wherein the analog includes the block, the block being below the top opening of the lumen and above the bottom opening of the lumen.

[0142] Item 15. The analog according to Item 10, wherein the analog comprises the block, the width of the block is equal to or greater than the thickness of the block, and the thickness of the block is substantially parallel to the longitudinal axis of the analog.

[0143] Item 16. The analog according to Item 15, wherein the width of the block is about 50 percent to about 90 percent of the inner diameter of the analog.

[0144] Item 17. The analog of item 15, wherein the block is a first block and the analog further comprises a second block.

[0145] Item 18. A method for developing a pre-use evaluation protocol, comprising: performing a first iteration of a chromatography run using the chromatography column, thereby generating a first chromatogram and a first product pool; performing a second iteration of the chromatography run using a channeling analog, thereby generating a second chromatogram and a second product pool; performing a third iteration of the chromatography run using a blocking analog, thereby generating a third chromatogram and a third product pool; analyzing product quality of the first product pool, the second product pool, and the third product pool; determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram; and determining a relationship between product quality and said one or more peak characteristics.

[0146] Item 19. The method of item 18, wherein analyzing the product quality comprises Pico microchip-capillary electrophoresis (PICO MCE) purity analysis, size-exclusion ultra-performance liquid chromatography (SE-UPLC) purity analysis, imaging capillary isoelectric focusing (iCEIF), glycan analysis, host cell DNA analysis, and / or host cell protein analysis.

[0147] Item 20. The chromatography operation includes introducing a mobile phase containing a salt slug; Determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram comprises: determining a first peak start, a first peak maximum, and a first peak end of a first peak in the first chromatogram; determining a second peak start, a second peak maximum, and a second peak end for a second peak in the second chromatogram; determining a third peak start, a third peak maximum, and a third peak end for a third peak in the third chromatogram; Item 19. The method of item 18, wherein the first peak, the second peak, and the third peak correspond to elution of the salt slug.

[0148] Those skilled in the art will appreciate that the conception on which this disclosure is based may be readily utilized as a basis for designing other methods and systems for carrying out several purposes of the present disclosure, and therefore the scope of the appended claims should not be deemed to be limited by the foregoing description.

Claims

1. A chromatography column analogue comprising: a chromatography medium; and a void configured to create zones of preferential flow within the chromatography medium; or Blocks configured to create zones of reduced flow within the chromatography media. and one or more of:

2. The analog of claim 1 , wherein the analog comprises the void and a tube having a top opening and a bottom opening, the void being between the top opening and the bottom opening of the tube.

3. The analog of claim 2, wherein the gap has a length of about 1.0 cm to about 10 cm, and the gap has a width of about 0.5 cm to about 1.0 cm.

4. 3. The analog of claim 2, further comprising a first filter screen in contact with the top opening and a second filter screen in contact with the bottom opening, wherein the first and second filter screens are impermeable to the chromatography medium.

5. 3. The analog of claim 2, wherein the tube includes a wall between the top opening and the bottom opening, the wall being in contact with the chromatography medium.

6. 3. The analog of claim 2, wherein the tube comprises stainless steel, glass, or another material that is impermeable to water.

7. The analog of claim 1 , wherein the analog comprises the block, the block comprising a top surface, a bottom surface, and a thickness between the top surface and the bottom surface.

8. The analog of claim 7, wherein the bottom surface has a width of about 0.5 cm to about 2.5 cm.

9. 10. The analog of claim 1, wherein the analog has a total volume of about 15 mL to about 4600 mL.

10. A chromatography column analogue comprising: a chromatography medium; and a void that does not contain a chromatographic medium and is permeable to water; or A block that does not contain a chromatographic medium and is impermeable to water. and one or more of:

11. The analog of claim 10, wherein the analog comprises the gap and a lumen having a top opening and a bottom opening, the gap being between the top opening and the bottom opening of the lumen.

12. The analog of claim 11 , wherein the lumen is substantially parallel to the longitudinal axis of the analog.

13. 12. The analog of claim 11, wherein a first portion of the chromatography medium is above the top opening and a second portion of the chromatography medium is below the bottom opening.

14. The analog of claim 11 , wherein the analog includes the block, the block being below the top opening of the lumen and above the bottom opening of the lumen.

15. The analog of claim 10, wherein the analog comprises the block, the width of the block being equal to or greater than the thickness of the block, and the thickness of the block being substantially parallel to the longitudinal axis of the analog.

16. The analog of claim 15, wherein the width of the block is about 50 percent to about 90 percent of the inner diameter of the analog.

17. 16. The analog of claim 15, wherein the block is a first block and the analog further comprises a second block.

18. 1. A method for developing a pre-use assessment protocol, comprising: performing a first iteration of a chromatography run using the chromatography column, thereby generating a first chromatogram and a first product pool; performing a second iteration of the chromatography run using a channeling analog, thereby generating a second chromatogram and a second product pool; performing a third iteration of the chromatography run using a blocking analog, thereby generating a third chromatogram and a third product pool; analyzing product quality of the first product pool, the second product pool, and the third product pool; determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram; and determining a relationship between product quality and said one or more peak characteristics.

19. 20. The method of claim 18, wherein analyzing the product quality comprises Pico microchip-capillary electrophoresis (PICO MCE) purity analysis, size-exclusion ultra-performance liquid chromatography (SE-UPLC) purity analysis, imaging capillary isoelectric focusing (iCEIF), glycan analysis, host cell DNA analysis, and / or host cell protein analysis.

20. the chromatography operation includes introducing a mobile phase comprising a salt slug; Determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram comprises: determining a first peak start, a first peak maximum, and a first peak end of a first peak in the first chromatogram; determining a second peak start, a second peak maximum, and a second peak end for a second peak in the second chromatogram; determining a third peak start, a third peak maximum, and a third peak end for a third peak in the third chromatogram; 20. The method of claim 18, wherein the first peak, the second peak, and the third peak correspond to leaching of the salt slug.

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