Method and system for evaluating chromatographic column integrity

Abstract

The chromatography column mimics may include a chromatography medium. The chromatographic column simulant may further include a void configured to create a region of preferential flow within the chromatographic medium, a block configured to create a region of reduced flow within the chromatographic medium, or a void and a block. The block may include a top surface, a bottom surface, and a thickness between the top surface and the bottom surface. The width of the top or bottom surface of the block may be from about 50% to about 90% of the inner diameter of the mimic.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 387,717, filed on Dec. 16, 2022, the entire content of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to systems and methods for assessing column integrity. Some aspects of the present disclosure relate to systems and methods for assessing the risk of processes for the bioproduction of therapeutic agents related to column integrity. Background Art

[0004] Biopharmaceutical products (such as antibodies, fusion proteins, adeno-associated virus (AAV), proteins, tissues, cells, polypeptides, or other therapeutics of biological origin) are increasingly being used for the treatment and prevention of infectious diseases, genetic diseases, autoimmune diseases, and other diseases. The production of biopharmaceutical products requires chromatography for purifying, characterizing, and validating the products. Disruption of the function of a chromatography system, such as a decrease in column integrity, can affect the purity of biopharmaceutical products produced using chromatography.

[0005] Since biopharmaceutical product manufacturing requires narrow tolerances, even partial loss of column integrity can render unusable all product that contacts the compromised column. The exact impact of decreased column integrity on processed biopharmaceutical products is not well understood. The efficacy of studying the extent and mechanisms of the impact of column integrity on biologic production is limited, mainly due to the cost of generating large amounts of unusable product for such studies. Commercially available small chromatography columns cannot replicate the failure modes of column integrity observed at manufacturing scale. Summary of the Invention

[0006] Aspects of the present disclosure relate to column analogs. A column analog may comprise a chromatographic medium. A column analog may comprise voids configured to create regions of preferential flow within the chromatographic medium. A column analog may comprise blocks configured to create regions of reduced flow within the chromatographic medium.

[0007] The analog may include a tube having a top opening and a bottom opening. A void may be located between the top opening and the bottom opening of the tube. The void may have a length of from about 1.0 cm to about 10 cm. The void may have a width of from about 0.5 cm to about 1.0 cm. This analog may include a first screen in contact with the top opening and a second screen in contact with the bottom opening. The first screen and the second screen may be impermeable to the chromatographic medium. The tube may include a wall located between the top opening and the bottom opening. The wall may be in contact with the chromatographic medium. The tube may be made of stainless steel, glass, or other materials impermeable to water. The block may include a top surface, a bottom surface, and a thickness between the top surface and the bottom surface. The bottom surface may have a width of from about 0.5 cm to about 2.5 cm. The analog may have an overall volume of from about 15 mL to about 4600 mL.

[0008] In another aspect, the present disclosure relates to a chromatographic column analog comprising a chromatographic medium. The chromatographic column analog may include a void that does not contain the chromatographic medium, wherein the void is permeable to water and / or the chromatographic medium. The chromatographic column analog may include a block that does not contain the chromatographic medium, wherein the block is impermeable to water.

[0009] The analog may include a lumen that includes a top opening and a bottom opening, wherein a void is located 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 chromatographic medium may be located above the top opening, and a second portion of the chromatographic medium may be located below the bottom opening. A block may be located below the top opening of the lumen and above the bottom opening of the lumen. The width of the block may be greater than or equal to 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 from about 50% to about 90% of the inner diameter of the analog. The block may be a first block, and the analog may include a second block.

[0010] In another aspect, the present disclosure relates to a method for determining the relationship between column integrity and product quality. The method may include performing a first iteration of a chromatographic operation using a chromatographic column to generate a first chromatogram and a first product pool. The method may further include performing a second iteration of the chromatographic operation using a channel analog to generate a second chromatogram and a second product pool. The method may include performing a third iteration of the chromatographic operation using a blockage analog to generate a third chromatogram and a third product pool. The method may also include analyzing the product quality of the first product pool, the second product pool, and the third product pool and determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram. The method may also include determining the relationship between the product quality and the one or more peak characteristics.

[0011] Analyzing the product quality may include Pico microchip-capillary electrophoresis (PICO MCE) purity analysis, size-exclusion ultraperformance liquid chromatography (SE-UPLC) purity analysis, imaged 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 a first chromatogram, a second chromatogram, and a third chromatogram may include determining a first peak start point, a first peak maximum, and a first peak end point of a first peak of the first chromatogram; determining a second peak start point, a second peak maximum, and a second peak end point of a second peak of the second chromatogram; and determining a third peak start point, a third peak maximum, and a third peak end point of a third peak of the third chromatogram. The first peak, the second peak, and the third peak may correspond to the elution of the salt slug. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings, which are included 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 (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein may be combined with any other embodiment or example, and all such combinations are included in the present disclosure. In addition, the systems and methods described are not limited to any single aspect or its embodiments, nor to any combination or arrangement of the aspects and embodiments. For the sake of brevity, certain permutations and combinations are not separately discussed and / or illustrated herein.

[0013] Figure 1 A graphical representation for the calculation of the asymmetry factor according to various aspects of the present disclosure;

[0014] Figure 2 A chromatogram generated during chromatographic operation using a hydrophobic interaction chromatography column according to various aspects of the present disclosure;

[0015] Figure 3 A chromatogram generated during chromatographic operation using a hydrophobic interaction chromatography column according to various aspects of the present disclosure;

[0016] Figure 4 A chromatogram generated during chromatographic operation using an affinity chromatography column according to various aspects of the present disclosure;

[0017] Figure 5A chromatogram generated during chromatographic operation using an affinity chromatography column according to aspects of the present disclosure;

[0018] Figure 6 A chromatogram generated during chromatographic operation using an ion exchange chromatography column according to aspects of the present disclosure;

[0019] Figure 7 A chromatogram generated during chromatographic operation using an ion exchange chromatography column according to aspects of the present disclosure;

[0020] Figure 8A A schematic diagram of a cross-section of a channel mimic according to aspects of the present disclosure;

[0021] Figure 8B is Figure 8A a vertical cross-section of a mimic of

[0022] Figure 9A A schematic diagram of a cross-section of a fouling mimic according to aspects of the present disclosure;

[0023] Figure 9B is Figure 9A a vertical cross-section of a mimic of

[0024] Figure 10A A chromatogram generated from a pre-use evaluation of a chromatography column according to aspects of the present disclosure;

[0025] Figure 10B A chromatogram generated from a pre-use evaluation of a channel mimic according to aspects of the present disclosure;

[0026] Figure 10C A chromatogram generated from a pre-use evaluation of a fouling mimic according to aspects of the present disclosure;

[0027] Figures 11A to 11H A chromatogram generated from a pre-use evaluation of a mimic according to aspects of the present disclosure. DETAILED DESCRIPTION

[0028] 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 to or equivalent to those described herein) can be used in the practice or testing of the present disclosure, specific example methods are described herein. All publications mentioned are incorporated herein by reference.

[0029] As used herein, the terms "comprising," "including," or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or elements inherent to such process, method, article, or apparatus. The term "exemplary" is used in the sense of "example" rather than "ideal." For the terms "for example" and "such as" and their grammatically equivalent words, unless otherwise expressly stated, they should be understood to follow the phrase "and are not limited to."

[0030] As used herein, the term "about" is intended to account for variations caused by experimental error. When applied to a numerical value, the term "about" may mean a variation of + / - 5% from the disclosed numerical value, unless a different variation is specified. As used herein, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Additionally, all ranges should be understood to include the endpoints; for example, 1 centimeter (cm) to 5 cm will include lengths of 1 cm, of 5 cm, and all distances between 1 cm and 5 cm.

[0031] It should be noted that, unless a different variation is specified, all numerical values (including all disclosed values, limitations, and ranges) disclosed or claimed herein may have a variation of + / - 5% from the disclosed numerical value.

[0032] As used herein, the term "polypeptide" refers to any amino acid polymer having more than about 20 amino acids covalently linked by amide bonds. A protein comprises one or more chains of amino acid polymers (e.g., polypeptides). Thus, a polypeptide can be a protein, and a protein can comprise multiple polypeptides to form a single functional biomolecule.

[0033] Post-translational modifications can modify or alter the structure of a polypeptide. For example, disulfide bonds (e.g., S-S bonds between cysteine residues) can form post-translationally in some proteins. Some disulfide bonds are essential for the correct structure, function, and interactions of polypeptides, immunoglobulins, proteins, cofactors, matrices, etc. In addition to disulfide bond formation, proteins can also undergo 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 sugars to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine intermolecular interactions involving the polypeptide.

[0034] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins for 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. A protein of interest (POI) can include any polypeptide or protein that is to be isolated, purified, or otherwise prepared. A POI can include polypeptides produced by cells, including antibodies.

[0035] As used herein, the term "antibody" includes immunoglobulins that are composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Typically, an antibody has a molecular weight of more than 100 kDa, such as between 130 kDa and 200 kDa, such as about 140 kDa, 145 kDa, 150 kDa, 155 kDa or 160 kDa. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conserved regions, called framework regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2 and LCDR3).

[0036] One class of immunoglobulins, called immunoglobulin G (IgG), for example, is common in human serum and contains four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to a heavy chain via a cystine disulfide bond, and the two heavy chains are bonded to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD and IgE. For IgG, there are four subclasses: IgG1, IgG2, IgG3 and IgG4. The constant regions of each subclass are different and may thus have different effector functions. In some embodiments described herein, a biopharmaceutical product may comprise a target polypeptide, including IgG. In at least one embodiment, the target polypeptide comprises IgG4.

[0037] As used herein, the term "antibody" also includes antigen-binding fragments of whole antibody molecules. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques, including manipulating and expressing DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or directly available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced or chemically manipulated using molecular biotechnology techniques, for example, arranging one or more variable and / or constant domains into a suitable configuration, or introducing codons, generating cysteine residues, modifying, adding, or deleting amino acids, and so on.

[0038] Recombinant cell-based production systems can be used to produce biopharmaceutical products (such as target molecules, polypeptides, antibodies), such as insect baculovirus systems, yeast systems (such as Pichia sp.), or mammalian systems (such as CHO cells and CHO derivatives like CHO-K1 cells). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (such as strains of Escherichia coli, Bacillus sp., Streptomyces sp., etc.), mycobacterial cells, fungal cells, yeast cells (such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methylica, etc.), plant cells, insect cells (such as SF-9, SF-21, insect cells infected with bacculovirus, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the cells can be human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells can be eukaryotic cells and can be selected from the following cells: CHO (such as CHO K1, DXB-11CHO, Veggie-CHO), COS (such as COS-7), retinal cells, Vero, CV1, kidney (such as HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (such as BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Setle's cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the cells mentioned above. In some embodiments, the cells can contain one or more viral genes, such as retinal cells expressing viral genes (such as PER.C6 TM cells).

[0039] The term "target molecule" as used herein can be used to refer to a target polypeptide (e.g., an antibody, an antibody fragment, or other protein or protein fragment), or a molecule intended to be made, isolated, purified, and / or included in a pharmaceutical (e.g., an adeno-associated virus (AAV) or other molecule for therapeutic use). Although the methods according to the present disclosure can be related to target polypeptides, they can be applicable to other target molecules. For example, AAV can be prepared according to suitable methods (e.g., depth filtration, affinity chromatography, etc.), and mixtures comprising AAV can be subjected to the methods according to the present disclosure. Before or after following one or more of the methods according to the present disclosure, additional procedures (e.g., removing "empty cassettes" or AAVs that do not contain the target sequence) can be performed on mixtures comprising AAV.

[0040] 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 an 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.

[0041] In some embodiments, the target molecule (e.g., an antibody) is selected from the group consisting of an anti-Programmed Cell Death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication No. US2015 / 0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication No. US2015 / 0203580A1), an anti-Dll4 antibody, an anti-Angiopoetin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9402898), an anti-Angiopoetin-Like 3 antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9018356), an anti-platelet derived growth factor receptor antibody (e.g., the anti-PDGFR antibody described in U.S. Patent No. 9265827), an anti-Prolactin Receptor antibody (e.g., the anti-PRLR antibody described in U.S. Patent No. 9302015), an anti-Complement 5 antibody (e.g., the anti-C5 antibody described in U.S. Patent Application Publication No. US2015 / 0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., the anti-EGFR antibody described in U.S. Patent No. 9132192 or the anti-EGFRvIII antibody described in U.S. Patent Application Publication No. US2015 / 0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g., the anti-PCSK9 antibody described in U.S. Patent No. 8062640 or U.S. Patent Application Publication No. US2014 / 0044730A1), an anti-Growth And DifferentiationFactor-8 antibody (such as the anti-GDF8 antibody described in U.S. Patent No. 8,871,209 or 9,260,515, also known as anti-myostatin antibody), anti-glucagon receptor (such as the anti-GCGR antibody described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibody, anti-IL1R antibody, interleukin 4 receptor antibody (such as the anti-IL4R antibody described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibody (such as the anti-IL6R antibody described in U.S. Patent No. 7,582,298, 8,043,617 or 9,173,880), anti-interleukin 33 (such as the anti-IL33 antibody described in U.S. Patent Application Publication No. US2014 / 0271658A1 or US2014 / 0271642A1), anti-Respiratory syncytial virus antibody (such as the anti-RSV antibody described in U.S. Patent Application No. US2014 / 0271653A1), anti-Cluster of differentiation 3 (such as the anti-CD3 antibody described in U.S. Patent Application Publication No. US2014 / 0088295A1, US20150266966A1 and U.S. Patent Application No. 62 / 222,605), anti-Cluster of differentiation 20 (such as the anti-CD20 antibody described in U.S. Patent Application Publication No. US2014 / 0088295A1, US2015,026,6966A1 and U.S. Patent No. 7,879,984), anti-Cluster of Differentiation-48 (such as the anti-CD48 antibody described in U.S. Patent No. 9,228,014), anti-Fel d1 antibody (such as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virusvirus) (such as anti-MERS antibody), anti-Ebola virus antibody (such as REGN-EB3 of Regeneron), 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 (such as anti-LAG3 antibody or anti-CD223 antibody), and anti-Activin A antibody. Each of the U.S. patents and U.S. patent publications mentioned in this paragraph is incorporated by reference in its entirety.

[0042] In some embodiments, the target molecule (such as a bispecific antibody) is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibody, anti-CD3 × anti-mucin 16 bispecific antibody, and anti-CD3 × anti-prostate specific membrane antigen bispecific antibody. In some embodiments, the target molecule is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab.

[0043] 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 that comprises one or more extracellular domains of a receptor coupled to the 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 contains two or more different receptor chains that bind a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as IL-1 trap (e.g., rilonacept, which comprises the IL-1RAcP ligand-binding region fused to the extracellular region of Il-1R1 fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, the entire contents of which are incorporated herein by reference), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which comprises the Ig domain 2 of VEGF receptor Flk1 fused to the Ig domain 3 of VEGF receptor Fltl fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, both of which are incorporated herein by reference in their entireties). In other embodiments, the Fc fusion protein is a ScFv-Fc-fusion protein that comprises one or more of the antigen-binding domains, such as the variable heavy chain fragment and variable light chain fragment of an antibody coupled to the Fc portion.

[0044] As used herein, the term "chromatography" refers to any process of separating the 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 of the mobile phase pass through the medium at different rates, including but 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, reverse phase chromatography, hydrophobic interaction chromatography (HIC), fast protein liquid chromatography, high performance liquid chromatography, countercurrent chromatography, periodic countercurrent chromatography, chiral chromatography, or mixed mode chromatography. Although the embodiments herein may be disclosed with reference to an exemplary type of chromatography or apparatus, such as column chromatography, the embodiments disclosed herein are applicable to any type of chromatography.

[0045] The systems and methods of the present disclosure include methods and systems for studying the impact of column integrity on the production of biopharmaceutical products. Chromatography columns compatible with the methods and systems herein include any column suitable for separating and / or purifying the components of a mobile phase.

[0046] The chromatography column may comprise a chromatography medium. For example, the chromatography column may comprise 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 may be in the form of a resin, beads, particles bound in a packed bed column, a membrane, or any form that can accommodate a mixture including a biopharmaceutical product or other liquids. The medium may comprise a support structure such as agarose beads (e.g., agarose gel (sepharose)), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, or other compactable synthetic structures.

[0047] The chromatography medium may comprise one or more ligands configured to interact with one or more components of the mobile phase, and a support structure that supports the one or more ligands. For example, the chromatography medium may comprise ligands that include a quaternary amine, a protein A-derived group, a protein L-derived group, a phenyl group, a sulphopropyl group, a triazabicyclodecene (TBD) group, a trimethylammoniumethyl (TMAE) group, a dimethylaminoethyl (DMAE) group, a sulfoethyl group, or a combination thereof. The support structure may include cross-linked agarose, highly cross-linked agarose, silica, alumina, methacrylate, glass, polyvinyl ether, or a combination thereof.

[0048] The chromatography column for manufacturing a biopharmaceutical product may be configured such that the medium has a depth (e.g., bed height) of about 15 centimeters (cm) to about 30 cm. In some embodiments, the chromatography column may be configured such that the inner diameter of the chromatography column is about 15 cm to about 200 cm. In some embodiments, the chromatography column has a total volume of about 25 liters (L) to about 277 L (e.g., the total capacity for accommodating a mixture, a mobile phase, or other substances).

[0049] In addition to one or more columns, the chromatography system may further comprise a detector. The detector may be any type of detector suitable for detecting one or more characteristics at the column outlet. Such characteristics may include, for example, column outlet conductivity, pH, optical density, and / or ultraviolet (UV) or visible light absorbance. In some embodiments, the detector may comprise 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 related to the protein concentration of the mobile phase exiting the column.

[0050] Chromatographic operations typically can include one or more steps, including, for example, one or more pre-equilibration steps, equilibration steps, loading steps, washing steps, elution steps, strip steps, and / or regeneration steps. Data collected from a detector at the outlet of a chromatography column can be used to track and / or record chromatographic operations. For chromatographic operations relevant to biopharmaceutical product manufacturing, it is necessary to monitor the quality, consistency, and integrity of chromatographic operations to ensure that the manufactured biopharmaceutical products meet internal quality assurance metrics and the standards of applicable regulatory agencies.

[0051] Generally, column integrity can be determined by the characteristics of how the mobile phase flows through the stationary phase (e.g., chromatographic medium) of the column. Signals detected by a detector can be plotted against the time elapsed and / or volume elapsed for a chromatographic operation. These plots are called chromatograms and can be used to monitor the process of a chromatographic operation and to determine whether the chromatographic operation is proceeding within acceptable operating parameters. For example, the presence of abnormal features in a chromatogram may indicate a decrease in column integrity.

[0052] Column integrity refers to the ability of a chromatography column to operate at maximum efficiency. Changes in the axial dispersion of the chromatographic medium within the column may affect the ability of the column to operate at maximum efficiency. Additionally or alternatively, changes in the radial dispersion of the chromatographic medium within the column may affect the ability of the column to operate at maximum efficiency. Depending on the type of chromatography column (e.g., the type of chromatographic medium in the column), a decrease in column integrity may result in a lack of binding of mobile phase components to the column, a lack of separation between components of the mobile phase, and / or the introduction of impurities into the mobile phase. When a chromatography column does not operate at maximum efficiency, the column integrity of the chromatography column is decreased.

[0053] The degree of decrease in column integrity of a chromatography column 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 chromatography column operating at maximum efficiency, a chromatography column with decreased column integrity may increase the HETP and / or decrease the number of theoretical plates. Other methods of quantifying column integrity include transformation analysis, in which the peaks of a chromatogram are analyzed relative to historical or expected chromatogram peaks. Changes in the peaks of a chromatogram identified in transformation analysis may indicate that the chromatography column used to generate the chromatogram has decreased in column integrity.

[0054] A decrease in column integrity may be caused by damage to the chromatographic medium within the column, and such damage may have a negative impact on the ability of substances to flow through the column. Repeated use of a chromatography column may result in a decrease in column integrity. Damage to the chromatographic medium may cause substances to flow through the column too quickly and not contact a sufficient amount of the chromatographic medium to effectively separate the components of the introduced substances. Additionally or alternatively, the damage may obstruct areas where substances flow through the column, which may also result in a decrease in the efficiency of the column.

[0055] One type of disruption within a chromatographic medium, namely channeling, refers to the presence of voids within the chromatographic medium, which can cause the mobile phase to advance through the chromatographic medium at a faster rate than the average flow rate of the chromatographic operation. A chromatographic medium that has become too dense may crack, resulting in the formation of channels and voids within the chromatographic medium. Channeling can lead to a lack of separation of components of the mobile phase because the preferential flow of the mobile phase through the voids of the chromatographic medium reduces the interaction between the components of the mobile phase and the chromatographic medium.

[0056] Peak broadening and peak fronting in a chromatogram can indicate channeling in the chromatographic column used to generate the chromatogram. Additionally or alternatively, peak splitting, incorrect peaks, shifted peak elution times, and / or the absence of expected peaks can indicate channeling.

[0057] Another type of disruption within a chromatographic medium, namely fouling, refers to the blocking of the pores or flow paths of the chromatographic medium. For example, the support structure of the chromatographic medium can be embedded in a screen within the chromatographic column. The embedded medium can prevent the mobile phase from flowing through a portion of the chromatographic column. Poor flow caused by the blockage can lead to the formation of biofilms or other growths, further disrupting the movement of the mobile phase through the chromatographic column. Fouling can cause the first portion of the mobile phase to flow more slowly than the second portion of the mobile phase. The resulting delay in the elution of the mobile phase through the chromatographic column can lead to poor separation of the components of the mobile phase. The delay in elution can also lead to peak broadening and peak tailing. Therefore, the detection of peak broadening, peak tailing, and / or poor peak resolution can indicate a fouled situation.

[0058] Reduced column integrity can be detected by the presence of asymmetric chromatogram peaks. For example, a chromatogram plotted based on chromatographic operation using a column with reduced column integrity may contain fronting peaks and / or tailing peaks. Peak fronting refers to an asymmetric peak where the front half of the chromatographic peak is wider than the rear half of the chromatographic peak. Peak tailing refers to a symmetric peak where the rear half of the chromatographic peak is wider than the front half of the chromatographic peak.

[0059] Peak asymmetry can be quantified by one or more peak symmetry metrics such as the asymmetry factor. The asymmetry factor (a) of a peak can be calculated according to Equation 1, where b h％ is the width of the rear half of the peak at h% of the peak height, and f h％ is the width of the front half of the peak at h% of the peak height.

[0060]

[0061] Figure 1 An example of the asymmetry calculation at 5% of the peak height is shown. Figure 1 The peak shown is symmetric. Therefore, the width f of the front half of the peak at 5% of the peak height5％h is equal to the width b of the rear half of the peak at 5% of the peak height 5％h . The asymmetry factor (a) of a symmetric peak is 1.0. The asymmetry factor of a front peak will be less than 1.0, and the asymmetry factor of a tail peak will be greater than 1.0.

[0062] Figure 2 Depicts an exemplary chromatogram depicting the chromatographic operation using a hydrophobic interaction chromatography column. Figure 2 The chromatographic operation shown is a flow-through process, where the eluate is collected while introducing the mobile phase (e.g., a mixture containing a biopharmaceutical product) and the wash buffer into the column. The collected eluate contains the desired biopharmaceutical product, while the unwanted molecules (e.g., host cell proteins) remain bound to the column. Subsequently, the wash and stripping steps remove the unwanted molecules bound to the column so that it can be regenerated and reused. The ultraviolet absorbance (black line), conductivity (gray line), and pH (dashed line) of the solution leaving the column are measured by one or more detectors and plotted against the volume passing through the column to produce Figure 2 the chromatogram shown.

[0063] Events related to the chromatographic operation are marked along the x-axis. At T0, the pre-equilibration step begins, followed by the equilibration step starting at T1. A mixture containing a biopharmaceutical product is introduced at T2, and the collection of the eluate begins. After introducing the mixture into the column, the wash buffer is introduced at T3. The collection of the eluate ends at T4. One or more stripping buffers are introduced into the column at T5, T6, and T7.

[0064] Figure 3 The chromatogram shown is an example of a chromatogram generated during a chromatographic operation using a hydrophobic interaction chromatography column with reduced column integrity. Similar to Figure 2 the chromatogram shown, at T0, the pre-equilibration step begins, followed by the equilibration step starting at T for T1. A mixture containing a biopharmaceutical product is introduced at T2, and the collection of the eluate begins. After introducing the mixture into the column, the wash buffer is introduced at T3. The collection of the eluate 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 the ultraviolet absorbance, the gray line represents the conductivity, and the dashed line represents the pH value.

[0065] The characteristics of the chromatogram can be monitored or analyzed to determine whether the chromatography column is operating within the expected parameters or whether the integrity of the column has been reduced. For example, referring to Figure 2 , after introducing a mixture containing a biopharmaceutical product into the column, the absorbance increases almost immediately. Compared with Figure 2 the chromatogram shown, Figure 3The absorbance peak of the chromatogram shown has a more gradual increase near T2. Additionally, compared to the absorbance peak of the chromatogram in Figure 2 , the absorbance peak of the chromatogram in Figure 3 is less symmetric. The gradual increase in the absorbance peak and the asymmetric peak distribution indicate a decrease in column integrity.

[0066] Figure 4 The exemplary chromatogram depicts the chromatographic operation using an affinity column. The black line represents the ultraviolet absorbance, the gray line represents the conductivity, the dashed line represents the pH, and the events of the affinity chromatographic operation are marked on the x-axis. At T0, a mixture containing a biopharmaceutical product is introduced into the column, which can be referred to as the loading step. At T1 and T2, one or more wash buffers are introduced into the column. At T3, the elution buffer is introduced into the column and the eluate collection is started. One or more wash and / or stripping buffers can be introduced into the column after the elution buffer. The collection of the eluate is stopped at T4, and the equilibration buffer is introduced into the column at T5.

[0067] As Figure 5 shown, the chromatogram is an example of a chromatogram generated during the chromatographic operation of an affinity column with reduced column integrity. Similar to the chromatogram in Figure 4 , the black line represents the ultraviolet absorbance, the gray line represents the conductivity, the dashed line represents the pH, and the events of the affinity chromatographic operation are marked on the x-axis. At T0, a mixture containing a biopharmaceutical product is introduced into the column, which can be referred to as the loading step. At T1 and T2, one or more wash buffers are introduced into the column. At T3, the elution buffer is introduced into the column and the eluate collection is started. One or more wash and / or stripping buffers can be introduced into the column after the elution buffer. The collection of the eluate is stopped at T4, and the equilibration buffer is introduced into the column at T5.

[0068] The absorbance peak corresponding to the elution (i.e., the peak between T3 and T4) of Figure 5 is wider than the absorbance peak corresponding to the elution (i.e., the peak between T3 and T4) of Figure 4 . Additionally, the absorbance peak corresponding to the elution of Figure 5 is more asymmetric than the absorbance peak corresponding to the elution of Figure 4 . Figure 6 The absorbance peak corresponding to the elution shown in

[0069] Figure 6An exemplary chromatogram depicting chromatographic operation using a cation exchange column is shown. The black line represents ultraviolet absorbance, the gray line represents conductivity, the dashed line represents pH, and events of the ion exchange chromatographic operation are marked on the x-axis. At T0, a mixture containing a biopharmaceutical product is introduced into the column, which can 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. Collection of the eluate begins at T3 and ends at T4. At T5 and T6, one or more equilibration buffers can be introduced into the column.

[0070] Figure 7 The chromatogram shown is an example of a chromatogram generated during chromatographic operation of a cation exchange column with reduced column integrity. Similar to Figure 6 the chromatogram shown, the black line represents ultraviolet absorbance, the gray line represents conductivity, the dashed line represents pH, and events of the ion exchange chromatographic operation are marked on the x-axis. At T0, a mixture containing a biopharmaceutical product is introduced into the column, which can 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. Collection of the eluate begins at T3 and ends at T4. At T5 and T6, one or more equilibration buffers can be introduced into the column.

[0071] Compared to Figure 6 the absorbance peak corresponding to elution (i.e., the peak between T2 and T4) shown, Figure 7 the absorbance peak corresponding to elution (i.e., the peak between T2 and T4) shown is broader. Additionally, Figure 7 the absorbance peak shown represents breakthrough loading at approximately T1. In other words, during the loading step, the protein containing the biopharmaceutical product flows through the column rather than binding to the column as expected. Breakthrough loading is a result of loss of column integrity.

[0072] As described above, a reduction in column integrity can be determined by monitoring signals from the detector (e.g., plotting conductivity, absorbance, and / or pH as a function of the volume through the column). Certain characteristics of the chromatogram peaks may indicate a reduction in column integrity. For example, broad elution peaks, fronting peaks, tailing peaks, and breakthrough loading can all indicate a reduction in column integrity.

[0073] During the manufacture of biopharmaceutical products, signals generated by a detector during a chromatography run can be monitored to evaluate the column integrity of the column used in the chromatography run. For example, it can be monitored whether a chromatogram plotted based on the signals received from the detector exhibits an indication of reduced column integrity. If a loss of column integrity is detected, the column may be considered unfit for use. The product that has come into contact with the column considered unfit for use may become unusable. In addition, production must be stopped when regenerating, repairing, or replacing an inappropriate column. The unusable product and the production downtime increase the costs and time required for the manufacture of biopharmaceutical products.

[0074] Since little is known about the impact of reduced column integrity, a larger quantity of biopharmaceutical products may be considered unusable due to quality control and regulatory standards compared to the biopharmaceutical products actually affected by reduced column integrity. If the relationship between loss of column integrity and the quality of the manufactured biopharmaceutical products is better understood, more tightly customized chromatography process control can be implemented. Compared to traditional chromatography process control, tightly customized chromatography process control will reduce product waste and production interruptions.

[0075] Factors limiting the understanding of the relationship between loss of column integrity and the quality of the manufactured biopharmaceutical products include: the cost of studying the relationship and the lack of an assessment of the precision of a bad column. For example, there is currently no model or commercially available analog for a column with reduced column integrity. Traditional studies of the impact of column integrity require columns that naturally lose column integrity during multiple chromatography runs. Therefore, the control of the columns used in such studies is poor, and the destruction patterns within the chromatographic medium are rarely consistent. In addition, the quantity of biopharmaceutical products and the operation of manufacturing-scale equipment for studying the impact and mechanism of reduced column integrity can be costly.

[0076] The interaction between a biopharmaceutical product and a chromatographic medium can depend on the characteristics, structure, and features of the biopharmaceutical product. For example, the distribution and size of hydrophobic regions within a biopharmaceutical product may 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 features of the biopharmaceutical product within the mobile phase. Therefore, the relationship between column integrity and the quality of the manufactured biopharmaceutical products can be unique for each product and chromatography run.

[0077] Accordingly, there is a need for systems and methods for modeling a chromatography system with a column exhibiting reduced column integrity. In particular, there is a need for a chromatographic column analog with reduced column integrity, and a system for studying the impact of column integrity on a processed biopharmaceutical product.

[0078] The present disclosure includes analogs for columns with reduced column integrity. Additionally, the present disclosure includes methods for analyzing the impact of column integrity on the quality of processed biopharmaceutical products, as well as methods for developing chromatographic process control. For example, a system may include an analog configured to cause the mobile phase passing through the analog to behave similarly to the mobile phase passing through a column with reduced column integrity.

[0079] 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 regions of the chromatographic column with disrupted chromatographic medium. For example, the analog may include regions that simulate, model, and / or mimic channels. Additionally or alternatively, the analog may include regions that simulate, model, and / or simulate fouling.

[0080] One or more regions of the analog that simulate, model, and / or mimic a region of the chromatographic column and have disrupted chromatographic medium may include one or more of disks, blocks, voids, lumens, tubes, or other structures that can disrupt the chromatographic medium.

[0081] The analog may be configured to be compatible with a standard chromatographic system. For example, a chromatographic system including a pump, an inlet, a detector, and one or more chromatographic columns may have a removable, interchangeable, and / or replaceable column. The analogs of the present disclosure may be configured to be usable to replace the chromatographic column within the chromatographic system.

[0082] Referring to Figure 8A and 8B , the channel analog 110 may 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 may be formed as the lumen of a tube, a channel, or other space formed within a three-dimensional structure inserted into the analog 110. For example, a tube including 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 may include the space within a tube or other three-dimensional structure within the chromatographic medium 115 of the analog 110. The void 225 may include one or more screens permeable to the mobile phase. For example, the top opening of the void 225 may include a first screen and the bottom opening of the void 225 may include a second screen.

[0083] The simulator 110 can be configured to have the chromatographic medium 115 with a bed height 107 (e.g., depth) of about 15 cm to about 30 cm. The simulator 110 can have an inner diameter 103 of about 1 cm to about 14 cm. In some embodiments, the simulator 110 can have an overall volume of about 15 milliliters (mL) to 4600 mL (e.g., the total capacity for containing a mixture, a mobile phase, or other substances).

[0084] The void 225 can have an elongated shape (e.g., cylindrical shape). The longitudinal axis of the void 225 can be parallel to the longitudinal axis of the simulator 110. For example, the longitudinal axis of the void 225 can be parallel to the sidewall of the simulator 110. The void 225 can have a width 223 (e.g., diameter) of about 0.5 cm to about 1.0 cm. The void 225 can have a height 227 of about 1.0 cm to about 10 cm.

[0085] The width 223 of the void 225 can be about 10% to about 40% of the inner diameter 103 of the simulator 110. The height 227 of the void 225 can be about 10% to about 30% of the bed height 107 of the simulator 110.

[0086] The void 225 can be located at about 1 cm to about 29 cm from the bottom edge of the chromatographic medium 115. In some embodiments, the void 225 can be located at about 1 cm to about 29 cm from the top edge of the chromatographic medium 115. The void can be located at about 1 cm to about 6 cm from the sidewall of the simulator 110.

[0087] Although Figure 8A and 8B the illustrated channel simulator 110 includes one void 225, it is only an example. The channel simulator 110 can include two, three, four, or more voids 225, depending on the degree of reduced column integrity being simulated. For example, to simulate or model a chromatographic column with a severe loss of column integrity, the simulator 110 can include more than one void 225. Additionally or alternatively, the size of one or more voids 225 can be adjusted to achieve the desired level of disruption within the chromatographic medium 115. Larger and more voids 225 simulate a greater reduction in column integrity.

[0088] Referring to Figure 9A and 9B , the fouling simulator 120 can include the chromatographic medium 115 and one or more blocks 235, 235'.

[0089] The simulator 120 can be configured to have the chromatographic medium 115 with a bed height 107 (e.g., depth) of about 15 cm to about 30 cm. The simulator 120 can have an inner diameter 103 of about 1 cm to about 14 cm. In some embodiments, the simulator 120 can have an overall volume of about 15 mL to about 4600 mL (e.g., the total capacity for containing a mixture, a mobile phase, or other substances).

[0090] The blocks 235, 235' are structures inserted into the mock-up 120, which do not contain the chromatographic medium 115 and block and / or prevent the mobile phase from passing through the space occupied by the blocks 235, 235'. The block 235 may comprise stainless steel, glass, polystyrene, plastic or other materials with suitable properties. For example, the block 235 may comprise a material that is impermeable to the chromatographic medium. Additionally or alternatively, a suitable material for the block 235 may be impermeable to the mobile phase and strong enough not to deform during filling the mock-up 120 with the chromatographic medium.

[0091] Still referring to Figure 9A and Figure 9B , the block 235 may have a disc shape, including a thickness 237 between a circular top surface and a circular bottom surface. In some embodiments, the top surface and the bottom surface of the block 235 may have a triangular, oval, rectangular or other configuration. Although Figure 9A and 9B show that the width 233 of the blocks 235, 235' is greater than the thickness 237, this is only an example. In other embodiments, the block 235 may have a thickness 237 greater than the width 233.

[0092] 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% to about 90% of the inner diameter 103 of the mock-up 120. The thickness 237 of the block 235 may be about 0.001% to about 0.1% of the bed height 107 of the mock-up 120.

[0093] The block 235 closest to the bottom edge of the chromatographic medium 115 may be located at least about 0.5 cm to about 1.0 cm from the bottom edge of the chromatographic medium 115. In some embodiments, the block 235 closest to the top edge of the chromatographic medium 115 may be located at least about 0.5 cm to about 1.0 cm from the top edge of the chromatographic medium 115. The block may be located about 0.5 cm to about 5 cm from the nearest side wall of the mock-up 120.

[0094] Although Figure 9A and 9B show that the fouling mock-up 120 contains two blocks, this is only an example. The fouling mock-up 110 may contain 1, 3, 4, 5, 6, 7, 8, 9, 10 or more blocks 235, depending on the degree of column integrity reduction being simulated. Additionally or alternatively, the size of one or more blocks 235 may be adjusted to achieve the desired level of disruption within the chromatographic medium 115. Larger and more blocks 235 simulate a greater reduction in column integrity.

[0095] Referring toFigure 9A and Figure 9B In embodiments where the analog 120 includes a plurality of blocks 235, 235', these blocks may be positioned parallel to each other. In some embodiments, the plurality of blocks 235, 235' may be located within the analog 120 such that at least two of the blocks 235, 235' are not parallel to each other. Each of the plurality of blocks 235, 235' may have the same shape and size. In some embodiments, at least one of the plurality of blocks 235, 235' has a different shape and / or size from at least one other of the plurality of blocks 235, 235'. Each of the blocks 235, 235' may be coaxial with each other (i.e., the center points of each of the blocks 235, 235' lie along a single axis of the analog 120). In some embodiments, at least one of the plurality of blocks 235, 235' is located at a different position within the chromatographic medium relative to the central axis of the analog 120 than at least one other of the plurality of blocks 235, 235'.

[0096] As described above, the analogs (e.g., the channel analog 110 and the fouling analog 120) allow the study of the impact of column integrity of a chromatographic column on the purity, yield, and quality of biopharmaceutical products processed using the chromatographic column. The analogs can be configured to simulate a desired level of column integrity by, for example, varying the size, shape, and / or number of the voids 225 and / or the blocks 235. The analogs are capable of allowing the mobile phase to pass through the analogs in a consistent and repeatable manner similar to how the mobile phase passes through a chromatographic column with reduced column integrity. The consistent and repeatable nature of the mobile phase flow through the analogs allows for the study of the mechanisms and impact of column integrity on biopharmaceutical product processing.

[0097] Example

[0098] Prior to using a chromatographic column to manufacture a biopharmaceutical product, a pre-use assessment of the column can be performed to determine whether the mobile phase flows through the column as expected. The pre-use assessment can include generating a baseline chromatogram using the column and generating a chromatogram based on monitoring the conductivity of the eluate containing the salt strip that exits the column. By comparing the baseline chromatogram with the monitored conductivity of the eluate containing the salt strip, it can be determined whether the mobile phase flows through the chromatographic column as expected. A chromatographic column capable of comparison can be used to compare the characteristics of the peaks corresponding to the salt strip with historical or expected data.

[0099] For example, the peak start point (e.g., the point at which the signal first reaches at least 5% of the maximum peak height), the peak maximum, and / or the peak end point (e.g., the point after the maximum peak height where the signal reaches 5% of the peak height) can be compared between the peaks generated from the pre-use evaluation criteria and historical peak data (e.g., peaks generated by an unused chromatographic column or a chromatographic column with confirmed performance efficiency). If one or more of the peak start point, peak maximum, or peak end point of the peak generated from the pre-use evaluation criteria is different from the historical or expected peak data (e.g., differs by at least about 0.1 column volumes), the column used to generate the deviated peak may be considered unfit for use.

[0100] During the manufacture of biopharmaceutical products, pre-use evaluation can be used to detect channeling and / or plugging caused by damage in the chromatographic medium exhibited by the chromatographic column. For example, compared to historical peak data (e.g., peak fronting), the characteristics of the chromatographic peak generated using a chromatographic column exhibiting channeling may appear earlier. Compared to historical peak data (e.g., peak tailing), the characteristics of the chromatographic peak generated using a chromatographic column exhibiting plugging may appear later. To demonstrate that the analogs of the present disclosure that simulate the flow of the mobile phase through the chromatographic column will exhibit channeling and / or plugging, pre-use evaluation is performed on analogs with various sizes and types of damage in the chromatographic medium.

[0101] An example of pre-use evaluation criteria, pre-use evaluation criteria A includes generating a baseline chromatogram by passing a buffer solution containing 0.1 M sodium chloride at a flow rate of 125 centimeters per hour (cm / hr) through the chromatographic column for 2.5 column volumes (CV), and monitoring the conductivity of the eluate exiting the column. Pre-use evaluation criteria A also includes generating a chromatogram by monitoring the conductivity of the eluate while performing: (a) introducing a buffer solution containing 1.0 M sodium chloride at 0.01 CV into the chromatographic column, and (b) washing the chromatographic 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 criteria A, the characteristics of the 0.01 CV peak corresponding to the buffer solution containing 1.0 M sodium chloride can be used to determine the relative efficiency of the chromatographic column. For example, the characteristics of the 0.01 CV peak corresponding to the buffer solution containing 1.0 M sodium chloride can be used to determine whether the chromatographic column contains damage (e.g., channeling or plugging) within the chromatographic medium.

[0102] Comparative example

[0103] Figure 10AA chromatogram generated according to Criteria A for pre-use evaluation of a chromatography column without introducing damage to the chromatography medium is shown. 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 conductivity measured from the baseline chromatogram, and the solid line represents the conductivity measured from the eluent containing salt streaks. The asymmetry of the eluted peak at 5% of the peak height, calculated according to Equation 1, was 1.17.

[0104] Example 1

[0105] Figure 10B A chromatogram generated from pre-use assessment criteria A of a channel mimic according to aspects of the present disclosure is shown. The channel mimic has a bed height of approximately 20 cm, an inner diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL, and comprises a stainless steel tube positioned parallel to the longitudinal axis of the column. The tube comprises a screen at the top opening and a screen at the bottom opening to prevent the chromatographic medium from penetrating into the tube. The space within the tube forms a void having a height of approximately 2 cm and an inner diameter of approximately 0.7 cm. Figure 10B The dashed line represents the conductivity measured from the baseline chromatogram, and the solid line represents the conductivity measured from the eluent containing the salt stripe. The asymmetry of the eluted peak at 5% of the peak height was calculated according to Equation 1 to be 0.85. This asymmetry is smaller than that of the comparative example, confirming that the channel mimetic causes a peak front shift.

[0106] Example 2

[0107] Figure 10C A chromatogram generated from pre-use assessment criteria A of a fouling simulant according to aspects of the present disclosure is shown. The fouling simulant has a bed height of approximately 20 cm, an inner diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL, and comprises three polystyrene blocks. Each polystyrene block has a generally circular shape, a thickness of approximately 0.2 mm, and a diameter of approximately 1.75 cm. The dotted line represents the conductivity measured from the baseline chromatogram, and the solid line represents the conductivity measured from the eluate containing salt streaks. The asymmetry of the eluate peak at 5% of the peak height is 1.53, calculated according to Equation 1. The asymmetry of 1.53 is greater than that of the comparative example, confirming that the fouling simulant causes peak tailing.

[0108] although Figures 10A to 10C The pre-use evaluation chromatogram shown includes a line for the baseline and the eluent containing salt stripes, but the pre-use evaluation chromatogram can be presented by plotting the difference between the baseline conductivity and the measured conductivity of the eluent containing salt stripes.

[0109] Additional channels and fouling simulants are prepared in accordance with aspects of the present disclosure and are described in Examples 3 - 10. All of the simulants described in Examples 3 - 10 include an affinity chromatography medium and / or an ion exchange medium. The simulants described in Examples 3 - 10 have a bed height of approximately 20 cm, an inner diameter of approximately 2.5 cm, and a volume of approximately 98.175 mL.

[0110] Example 3

[0111] A channel simulant is prepared that includes a void having a height of approximately 2.0 cm and a width of approximately 0.7 cm. A tube including stainless steel is inserted into the chromatography medium to form the void. The tube includes a screen at the top opening and a screen at the bottom opening to prevent the chromatography medium from invading the void formed within the tube.

[0112] Example 4

[0113] A channel simulant is prepared that includes a void having a height of 3.0 cm and a width of approximately 0.7 cm. A tube including stainless steel is inserted into the chromatography medium to form the void. The tube includes a screen at the top opening and a screen at the bottom opening to prevent the chromatography medium from invading the void formed within the tube.

[0114] Example 5

[0115] A channel simulant is prepared that includes a void having a height of 3.5 cm and a width of approximately 0.7 cm. A tube including stainless steel is inserted into the chromatography medium to form the void. The tube includes a screen at the top opening and a screen at the bottom opening to prevent the chromatography medium from invading the void formed within the tube.

[0116] Example 6

[0117] A channel simulant is prepared that includes a void having a height of 4.0 cm and a width of approximately 0.7 cm. A tube including stainless steel is inserted into the chromatography medium to form the void. The tube includes a screen at the top opening and a screen at the bottom opening to prevent the chromatography medium from invading the void formed within the tube.

[0118] Example 7

[0119] A fouling simulant is prepared that includes three blocks, each block having a generally circular shape, a thickness of approximately 0.2 mm, and a diameter of approximately 1.75 cm. The blocks include polystyrene disks inserted into the chromatography medium.

[0120] Example 8

[0121] A fouling simulant is prepared that includes four blocks, each block having a generally circular shape, a thickness of approximately 0.2 mm, and a diameter of approximately 1.88 cm. The blocks include polystyrene disks inserted into the chromatography medium.

[0122] Example 9

[0123] Prepare fouling analogs comprising six blocks, each block having a generally circular shape, a thickness of about 0.2 mm, and a diameter of about 2.00 cm. The blocks comprise polystyrene disks inserted into a chromatographic medium.

[0124] Example 10

[0125] Prepare fouling analogs comprising eight blocks, each block having a generally circular shape, a thickness of about 0.2 mm, and a diameter of about 2.13 cm. The blocks comprise polystyrene disks inserted into a chromatographic medium.

[0126] Figures 11A to 11H Chromatograms generated from pre-use evaluation criterion A of the analogs of Examples 3 to 10 according to aspects of the present disclosure are shown. For Figures 11A to 11H each of the figures in, the dashed line represents the conductivity measured from the baseline chromatogram, and the solid line represents the conductivity measured from the eluate containing the salt strip. For each chromatogram, the start point, maximum, and end point of each peak are calculated based on the column volume of the analog. The peak start point is the first point where the conductivity reaches 5% of its maximum. The peak end point is the last point where the conductivity is 5% of its maximum. The peak maximum is the point where the conductivity reaches its maximum. The asymmetry factor at 5% peak height of each chromatogram is also calculated according to Equation 1. Table 1 summarizes the peak start points, peak maxima, peak end points, and asymmetry factors of Examples 3 to 10.

[0127] Table 1

[0128]

[0129] The channel analog has a front elution peak with an asymmetry factor less than 1. The fouling analog has a tail elution peak with an asymmetry factor greater than 1.

[0130] Analogs of 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 chromatographic operations. For example, chromatographic operations can be run using analogs that incorporate various conditions of loss of integrity. The quality of biopharmaceutical products processed using the analogs can be analyzed to study the effect of column integrity on the quality of the resulting biopharmaceutical products.

[0131] 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 include analyzing the test article by non-reducing and reducing microchip capillary electrophoresis using a GXII instrument to estimate the purity and impurity levels of the product pool sample (with an emphasis on determining the fragmentation level). SE-UPLC purity analysis includes size exclusion chromatography and ultra-performance liquid chromatography for separating protein species based on molecular weight. iCIEF involves an imaging capillary isoelectric focusing method to determine the relative abundance of charge variants of a biopharmaceutical product. Glycan analysis includes determining the fucosylated glycan content in the product using reversed-phase high-performance liquid chromatography. Host cell DNA analysis includes detecting DNA from host cells (such as Chinese hamster ovary cells) in a product sample using real-time quantitative polymerase chain reaction (PCR). Host cell protein analysis includes quantifying the presence of host cell proteins (HCP) in the product using enzyme-linked immunosorbent assay (ELISA).

[0132] Based on biopharmaceutical product quality testing, chromatographic 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 detrimentally affect the quality of a biopharmaceutical product. The determined threshold can be used to establish pre-use evaluation criteria to effectively screen column integrity during biopharmaceutical product manufacturing.

[0133] The present disclosure is further described by the following non-limiting items.

[0134] Item 1: A chromatographic column analog, comprising:

[0135] A chromatographic medium; and

[0136] One or more of the following:

[0137] A void configured to create a region of preferential flow within the chromatographic medium; or

[0138] A block configured to create a region of reduced flow within the chromatographic medium.

[0139] Item 2: The analog according to Item 1, wherein the analog comprises the void and a tube, the tube includes a top opening and a bottom opening, and the void is located between the top opening and the bottom opening of the tube.

[0140] Item 3: The analog according to Item 2, wherein the void has a length of about 1.0 cm to about 10 cm, and the void has a width of about 0.5 cm to about 1.0 cm.

[0141] Item 4: The analog according to Item 2, further comprising a first screen in contact with the top opening and a second screen in contact with the bottom opening, wherein the first screen and the second screen are impermeable to the chromatographic medium.

[0142] Item 5: The analog according to Item 2, wherein the tube comprises a wall between the top opening and the bottom opening, and the wall is in contact with the chromatographic medium.

[0143] Item 6: The analog according to Item 2, wherein the tube comprises stainless steel, glass, or other materials impermeable to water.

[0144] Item 7: The analog according to Item 1, wherein the analog comprises the block, and the block comprises a top surface, a bottom surface, and a thickness between the top surface and the bottom surface.

[0145] Item 8: The analog according to Item 7, wherein the bottom surface has a width of about 0.5 cm to about 2.5 cm.

[0146] Item 9: The analog according to Item 1, wherein the analog has an overall volume of about 15 mL to about 4600 mL.

[0147] Item 10: A chromatographic column analog, comprising:

[0148] A chromatographic medium; and

[0149] One or more of the following:

[0150] A void that does not contain the chromatographic medium, wherein the void is permeable to water; or

[0151] A block that does not contain the chromatographic medium, wherein the block is impermeable to water.

[0152] Item 11: The analog according to Item 10, wherein the analog comprises the void and an inner cavity, the inner cavity includes a top opening and a bottom opening, and the void is located between the top opening and the bottom opening of the inner cavity.

[0153] Item 12: The analog according to Item 11, wherein the inner cavity is substantially parallel to the longitudinal axis of the analog.

[0154] Item 13: The analog according to Item 11, wherein a first portion of the chromatographic medium is located above the top opening and a second portion of the chromatographic medium is located below the bottom opening.

[0155] Item 14: The analog according to Item 11, wherein the analog comprises the block, and the block is located below the top opening of the inner cavity and above the bottom opening of the inner cavity.

[0156] Item 15: The analog according to Item 10, wherein the analog comprises the block, the width of the block is greater than or equal to the thickness of the block, and the thickness of the block is substantially parallel to the longitudinal axis of the analog.

[0157] Item 16: The analog according to Item 15, wherein the width of the block is about 50% to about 90% of the inner diameter of the analog.

[0158] Item 17: The analog according to Item 15, wherein the block is a first block and the analog further comprises a second block.

[0159] Item 18: A method for developing pre-use evaluation criteria, the method comprising:

[0160] Performing a first iteration of a chromatographic operation using a chromatographic column to generate a first chromatogram and a first product pool;

[0161] Performing a second iteration of the chromatographic operation using a channel analog to generate a second chromatogram and a second product pool;

[0162] Performing a third iteration of the chromatographic operation using a blocking analog to generate a third chromatogram and a third product pool;

[0163] Analyzing the product quality of the first product pool, the second product pool, and the third product pool;

[0164] Determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram;

[0165] Determining the relationship between the product quality and the one or more peak characteristics.

[0166] Item 19: The method according to 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.

[0167] Item 20: The method according to item 18, wherein the chromatographic operation comprises introducing a mobile phase comprising a salt strip;

[0168] Determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram comprises:

[0169] Determining a first peak start point, a first peak maximum, and a first peak end point of a first peak of the first chromatogram;

[0170] Determining a second peak start point, a second peak maximum, and a second peak end point of a second peak of the second chromatogram; and

[0171] Determining a third peak start point, a third peak maximum, and a third peak end point of a third peak of the third chromatogram; and

[0172] wherein the first peak, the second peak, and the third peak correspond to the elution of the salt strip.

[0173] Those skilled in the art will understand that the concepts on which this disclosure is based can be readily used as a basis for designing other methods and systems for carrying out several purposes of this disclosure. Accordingly, the claims should not be regarded as limited to the foregoing description.

Claims

1. A chromatographic column mimic, comprising: a chromatographic medium; and one or more of the following: a void configured to create a region of preferential flow within the chromatographic medium; or a block configured to create a region of reduced flow within the chromatographic medium.

2. The mimic according to claim 1, wherein the mimic comprises the void and a tube, the tube comprising a top opening and a bottom opening, and the void being located between the top opening and the bottom opening of the tube.

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

4. The mimic according to claim 2, further comprising a first screen in contact with the top opening and a second screen in contact with the bottom opening, wherein the first screen and the second screen are impermeable to the chromatographic medium.

5. The mimic according to claim 2, wherein the tube comprises a wall located between the top opening and the bottom opening, and the wall is in contact with the chromatographic medium.

6. The mimic according to claim 2, wherein the tube comprises stainless steel, glass, or other material impermeable to water.

7. The mimic according to claim 1, wherein the mimic comprises the block, and the block comprises a top surface, a bottom surface, and a thickness between the top surface and the bottom surface.

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

9. The mimic according to claim 1, wherein the mimic has an overall volume of about 15 mL to about 4600 mL.

10. A chromatographic column mimic, comprising: a chromatographic medium; and one or more of the following: a void that does not contain the chromatographic medium, wherein the void is permeable to water; or a block that does not contain the chromatographic medium, wherein the block is impermeable to water.

11. The mimic according to claim 10, wherein the mimic comprises the void and a lumen, the lumen comprising a top opening and a bottom opening, and the void being located between the top opening and the bottom opening of the lumen.

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

13. The mimic according to claim 11, wherein a first portion of the chromatographic medium is located above the top opening and a second portion of the chromatographic medium is located below the bottom opening.

14. The mimic according to claim 11, wherein the mimic comprises the block, and the block is located below the top opening of the lumen and above the bottom opening of the lumen.

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

16. The mimic according to claim 15, wherein the width of the block is about 50% to about 90% of the inner diameter of the mimic.

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

18. A method for developing pre-use evaluation criteria, the method comprising: Performing a first iteration of a chromatography operation using a chromatography column to generate a first chromatogram and a first product pool; Performing a second iteration of the chromatography operation using a channel analog to generate a second chromatogram and a second product pool; Performing a third iteration of the chromatography operation using a blockage analog to generate a third chromatogram and a third product pool; Analyzing the 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 the relationship between the product quality and the one or more peak characteristics.

19. The method according to 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 method according to claim 18, wherein the chromatography operation comprises introducing a mobile phase comprising a salt strip; Determining one or more peak characteristics of the first chromatogram, the second chromatogram, and the third chromatogram comprises: Determining a first peak start point, a first peak maximum value, and a first peak end point of a first peak of the first chromatogram; Determining a second peak start point, a second peak maximum value, and a second peak end point of a second peak of the second chromatogram; and Determining a third peak start point, a third peak maximum value, and a third peak end point of a third peak of the third chromatogram; and wherein the first peak, the second peak, and the third peak correspond to the elution of the salt strip.

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