Attorney Docket No.00166-0147-0304 METHODS AND SYSTEMS FOR SCALED CHROMATOGRAPHY CROSS-REFERENCE TO RELATED APPLICATION(S) [0001] This patent application claims priority to U.S. Provisional Patent Application No.63/659,381 filed on June 13, 2024, the entirety of which is incorporated herein by reference. FIELD OF DISCLOSURE [0002] The present disclosure relates to systems and methods for developing chromatography protocols. Aspects of the present disclosure includes methods and systems for developing mechanistic chromatography models and applying those models to bench-scale chromatography protocols. INTRODUCTION [0003] Biopharmaceutical products (e.g., antibodies, antibody-drug conjugates, fusion proteins, adeno-associated viruses (AAVs), proteins, tissues, cells, polypeptides, or other therapeutic products of biological origin) are increasingly being used in the treatment and prevention of infectious diseases, genetic diseases, autoimmune diseases, and other ailments. Production of the biopharmaceutical products requires chromatography to purify, characterize, and validate the products. [0004] Developing and/or adjusting chromatography protocols can be time and labor intensive. For example, developing and/or adjusting chromatography protocols may involve the use of investigatory chromatography runs. In addition to costs associated with the time and labor of conducting investigatory chromatography runs, investigatory chromatography runs may waste reagents and biopharmaceutical products. The use of non-optimized chromatography protocols themselves can also lead to waste of reagents and/or products, which increases the costs associated with the production of biopharmaceutical products. [0005] Reduced scale chromatography (e.g., lab scale, bench scale, and/or microscale chromatography) may be used in process development to optimize chromatography protocols and study chromatography operation robustness while reducing the use of time, reagents, and biopharmaceutical products. Due to differences of scale, chromatography parameters traditionally used for bench scale or manufacturing scale chromatography may not be suitable for microscale chromatography. Similarly, data values measured during a microscale chromatography operation may not be representative of the same data values measured for bench scale or manufacturing scale chromatography. Therefore, there exists a need for systems and methods of utilizing microscale chromatography operations to develop and/or adjust chromatography protocols. SUMMARY [0006] Aspects of the present disclosure may be directed to a method of developing a hydrophobic interaction chromatography mechanistic model. The method may include determining a total
Attorney Docket No.00166-0147-0304 available porosity and a column porosity of a chromatography system including a column comprising hydrophobic interaction chromatography media. The method may further include generating a generalized mechanistic model based on the total available porosity and the column porosity, determining mass transfer parameters of the mechanistic model, and/or determining binding isotherm parameters of the mechanistic model. [0007] In some aspects of the present disclosure, determining mass transfer parameters includes conducting a first blue dextran pulse test at a first flow rate, and a second blue dextran pulse test at a second flow rate. Determining mass transfer parameters may include regressing the mass transfer parameters using data generated from the first and second blue dextran pulse tests. The generalized mechanistic model may include a binding isotherm term, and regressing the mass transfer parameters may include regressing the mass transfer parameters using data generated from the first and second blue dextran pulse tests, while ignoring the binding isotherm term of the generalized mechanistic model. Determining binding isotherm parameters may include conducting a plurality of laboratory- scale chromatography runs. Determining binding isotherm parameters may include regressing the isotherm parameters based on data generated from the plurality of laboratory-scale chromatography runs. Methods of generating a hydrophobic interaction chromatography mechanistic model may further include characterizing system hold-ups within the chromatography system. Characterizing retention of the mobile phase within the chromatography system may include conducting a column- less acetone pulse test, a column-less salt pulse test, or both. The generalized mechanistic model may be based on the total available porosity, the column porosity, and a pulse test retention volume measured during the characterization of system hold-ups within the chromatography system. [0008] Aspects of the present disclosure may be directed to a method of developing an anion exchange chromatography mechanistic model. The method may include characterizing system hold- ups within a chromatography system including a column comprising anion exchange chromatography media and/or determining a porosity of the chromatography system. The method may further include generating a generalized mechanistic model based on the porosity, wherein the generalized mechanistic model comprises a binding isotherm including a term defined by Equation 7. The method may also include determining mass transfer parameters of the mechanistic model and/or determining binding isotherm parameters of the mechanistic model. [0009] In some aspects of the present disclosure, the generalized mechanistic model further includes a term defined by Equation 5, a term defined by Equation 6, or both. Determining a porosity of the chromatography system may include determining a total available porosity and determining a column porosity. Characterizing system hold-ups within the chromatography system may include conducting a column-less acetone pulse test and a column-less salt pulse test. Determining the porosity may include conducting an unretained monoclonal antibody pulse test and a blue dextran pulse test. Generating the generalized mechanistic model based on the porosity may include generating the generalized mechanistic model based on retention volumes calculated from the
Attorney Docket No.00166-0147-0304 column-less acetone pulse test, the column-less salt pulse test, the unretained monoclonal antibody pulse test, and the blue dextran pulse test. Determining isotherm parameters may include conducting a plurality of laboratory-scale chromatography runs, and regressing the isotherm parameters based on data generated from the plurality of laboratory-scale chromatography runs. The laboratory-scale chromatography runs may each be conducted using a column having a total column volume of approximately 1 milliliter (mL) to approximately 20 mL. [0010] In addition or alternatively, aspects of the present disclosure may be directed to a chromatography method including generating a chromatography mechanistic model, and/or conducting a plurality of microscale chromatography runs, thereby generating microscale data. The method may further include determining predicted manufacturing scale data based on the microscale data, using the chromatography mechanistic model. [0011] In some aspects of the present disclosure, the chromatography mechanistic model describes a hydrophobic interaction exchange chromatography operation or an anion exchange chromatography operation. The method may further comprise using the chromatography mechanistic model to generate a predicted laboratory scale elution peak width, conducting a laboratory scale chromatography run to generate an experimental laboratory scale elution peak width, using the chromatography mechanistic model to generate a predicted microscale elution peak width, conducting a microscale chromatography run to generate an experimental microscale elution peak width, and calculating a laboratory scale elution peak width delta and a microscale elution peak width delta. The laboratory scale chromatography run may be conducted using a first column having a total column volume of approximately 1 mL to approximately 20 mL, and the microscale chromatography run may be conducted using a second column having a total column volume of less than or equal to approximately 1 mL. BRIEF DESCRIPTION OF THE DRAWINGS [0012] 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 features of an embodiment or example described herein, e.g., composition, formulation, method, etc., may be combined with any other embodiment or example, and all such combinations are encompassed by the present disclosure. Moreover, the described systems and methods are neither limited to any single aspect nor embodiment thereof, nor to any combinations or permutations of such aspects and embodiments. For the sake of brevity, certain permutations and combinations are not discussed and/or illustrated separately herein. [0013] FIG.1 is a generalized diagram depicting movement of a mobile phase through a chromatography column, according to aspects of the present disclosure; [0014] FIG.2 depicts, in flow-chart form, an exemplary process for developing a chromatography mechanistic model, according to aspects of the present disclosure;
Attorney Docket No.00166-0147-0304 [0015] FIG.3 is a chromatogram generated from an acetone pulse test, according to aspects of the present disclosure; [0016] FIG.4 is a chromatogram generated from an acetone pulse test, according to aspects of the present disclosure; [0017] FIG.5 is a chromatogram generated from a salt pulse test, according to aspects of the present disclosure; [0018] FIG.6 is a chromatogram generated from an unretained antibody pulse test, according to aspects of the present disclosure; [0019] FIG.7 is a chromatogram generated from a blue dextran pulse test, according to aspects of the present disclosure; [0020] FIG.8 is a chromatogram generated from a salt pulse test, according to aspects of the present disclosure; [0021] FIG.9 is a chromatogram generated from blue dextran pulse test, according to aspects of the present disclosure; [0022] FIGs.10A and 10B are chromatograms generated from unretained antibody pulse tests, according to aspects of the present disclosure; [0023] FIGs.11A and 11B are chromatograms generated from blue dextran pulse tests, according to aspects of the present disclosure; [0024] FIGs.12A, 12B, and 12C are chromatograms generated from reduced scale chromatography runs, according to aspects of the present disclosure; [0025] FIGs.13A, 13B, and 13C are chromatograms generated from reduced scale chromatography runs, according to aspects of the present disclosure; [0026] FIGs.14A and 14B depict plots of reduced scale chromatography runs and chromatography mechanistic model predictions, according to aspects of the present disclosure; [0027] FIGs.15A and 15B depict bar graphs comparing peak widths determined from reduced-scale chromatography runs and peak widths predicted by a chromatography mechanistic model, according to aspects of the present disclosure; [0028] FIGs.16A and 16B depict plots of reduced scale chromatography runs, according to aspects of the present disclosure; [0029] FIG.17 depicts a plot of reduced scale chromatography runs and chromatography mechanistic model predictions, according to aspects of the present disclosure; [0030] FIGs.18A and 18B depict bar graphs comparing peak widths determined from reduced-scale chromatography runs and peak widths predicted by a chromatography mechanistic model, according to aspects of the present disclosure; and [0031] FIG.19 depicts a plot including results from anion exchange chromatography pulse tests, according to aspects of the present disclosure.
Attorney Docket No.00166-0147-0304 DETAILED DESCRIPTION [0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is 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 the present disclosure, particular example methods are now described. All publications mentioned are hereby incorporated by reference. [0033] As used herein, the terms “comprises,” “comprising,” 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 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 grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. [0034] As used herein, the term “approximately” is meant to account for variations due to experimental error. When applied to numeric values, the term “approximately” may indicate a variation of +/- 5% from the disclosed numeric 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. Further, all ranges are understood to be inclusive of endpoints, e.g., from 1 mm to 5 mm would include 1 mm and 5 mm and all distances or lengths between 1 mm and 5 mm. [0035] It should be noted that all numeric values disclosed or claimed herein (including all disclosed values, limits, and ranges) may have a variation of +/- 5% from the disclosed numeric value unless a different variation is specified. [0036] The term “polypeptide” as used herein refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide may be a protein, and a protein may contain multiple polypeptides to form a single functioning biomolecule. [0037] Post-translational modifications may modify or alter the structure of a polypeptide. For example, disulfide bridges (e.g., S–S bonds between cysteine residues) may be formed post- translationally in some proteins. Some disulfide bridges are essential to proper structure, function, and interaction of polypeptides, immunoglobulins, proteins, co-factors, substrates, and the like. In addition to disulfide bond formation, proteins may be subject 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 glycosyl groups at arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and/or tryptophan), and phosphorylation (i.e., the addition of a phosphate group to serine, threonine, tyrosine, and/or histidine). Post-translational modifications may affect the hydrophobicity, electrostatic surface
Attorney Docket No.00166-0147-0304 properties, or other properties which determine the surface-to-surface interactions participated in by the polypeptide. [0038] As used herein, the term “protein” includes biotherapeutic 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, and the like. A protein of interest (POI) may include any polypeptide or protein that is desired to be isolated, purified, or otherwise prepared. POIs may include polypeptides produced by a cell, including antibodies. [0039] The term “antibody,” as used herein, includes immunoglobulins comprised of four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Typically, antibodies have a molecular weight of over 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 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, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). 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 be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3. [0040] A class of immunoglobulins called Immunoglobulin G (IgG), for example, is common in human serum and comprises 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 bound to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, four subclasses exist: IgG 1, IgG 2, IgG 3, and IgG 4. Each subclass differs in their constant regions, and as a result, may 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 IgG 4. [0041] The term “antibody,” as used herein, also includes antigen-binding fragments of full 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 an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody
Attorney Docket No.00166-0147-0304 molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. [0042] Biopharmaceutical products (e.g., target molecules, polypeptides, antibodies) may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), or mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells). The term “cell” includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria 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, bacculovirus-infected insect cells, Trichoplusiani, etc.), non- human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments a cell may be a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a cell may be eukaryotic and may be selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, 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 cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, a cell may comprise one or more viral genes, e.g. a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell). [0043] The term “target molecule” may be used herein to refer to target polypeptides (e.g., antibodies, antibody fragments, or other proteins or protein fragments), or to other molecules intended to be produced, isolated, purified, and/or included in drug products (e.g., adeno-associated viruses (AAVs) or other molecules for therapeutic use). While methods according to the present disclosure may refer to target polypeptides, they may be as applicable to other target molecules. AAVs, for example, may be prepared according to suitable methods (e.g., depth filtration, affinity chromatography, and the like), and mixtures including AAVs may be subjected to methods according to the present disclosure. Before or after following one or more methods of the present disclosure, mixtures including AAVs may be subjected to additional procedures (e.g., to the removal of “empty cassettes” or AAVs that do not contain a target sequence). [0044] In some embodiments, the target molecule is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecif c antibody, a bispecific
Attorney Docket No.00166-0147-0304 antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or 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. [0045] In some embodiments, a target molecule (e.g., an antibody) is selected from a group consisting of an anti-Programmed Cell Death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., an anti-PD-L1 antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580A1), an anti-Dll4 antibody, an anti-Angiopoetin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti- Angiopoetin-Like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Pat. No.9,018,356), an anti-platelet derived growth factor receptor antibody (e.g., an anti- PDGFR antibody as described in U.S. Pat. No.9,265,827), an anti-Prolactin Receptor antibody (e.g., anti-PRLR antibody as described in U.S. Pat. No.9,302,015), an anti-Complement 5 antibody (e.g., an anti-C5 antibody as described in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody as described in U.S. Pat. No.9,132,192 or an anti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No. US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No.8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1), an anti-Growth And Differentiation Factor-8 antibody (e.g., an anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos.8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g., anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, an interleukin 4 receptor antibody (e.g., an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat. Nos.8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Pat. Nos.7,582,298, 8,043,617, or 9,173,880), an anti-interleukin 33 (e.g., anti- IL33 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0271658A1 or US2014/0271642A1), an anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271653A1), an anti- Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Application No.62/222,605), an anti- Cluster of differentiation 20 (e.g., an anti-CD20 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Pat. No.7,879,984), an anti- Cluster of Differentiation-48 (e.g., anti-CD48 antibody as described in U.S. Pat. No.9,228,014), an anti-Fel d1 antibody (e.g., as described in U.S. Pat. No.9,079,948), an anti-Middle East Respiratory Syndrome
Attorney Docket No.00166-0147-0304 virus (e.g., an anti-MERS antibody), an anti-Ebola virus antibody (e.g., Regeneron’s REGN-EB3), an anti-CD19 antibody, an anti-CD28 antibody, an anti-IL1 antibody, an anti-IL2 antibody, an anti- IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-Erb3 antibody, an anti-Zika virus antibody, an anti-Lymphocyte Activation Gene 3 (e.g., anti-LAG3 antibody or anti-CD223 antibody) and an anti-Activin A antibody. Each U.S. patent and U.S. patent publication mentioned in this paragraph is incorporated by reference in its entirety. [0046] In some embodiments, a target molecule (e.g., a bispecific antibody) is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody, an anti-CD3 x anti-Mucin 16 bispecific antibody, and an anti-CD3 x 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. [0047] In some embodiments, the target molecule is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein). In some embodiments, an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety. In some embodiments, the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG. In some embodiments, the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands. For example, an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular region fused to Fc of hIgG1; see U.S. Pat. No.6,927,004, which is incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hIgG1; see U.S. Pat. Nos.7,087,411 and 7,279,159, both of which are incorporated by reference in their entireties). In other embodiments, an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety. [0048] The term “chromatography,” as used herein, refers to any process which separates components of a mobile phase (e.g., a mixture or solution containing multiple constituents) by passing the mobile phase through a medium such that the constituents 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), cation exchange chromatography, anion exchange chromatography, size-exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography (HIC), fast protein liquid chromatography, high- performance liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or mixed-mode chromatography. While examples herein may be disclosed with respect to an exemplary type of chromatography process or apparatus, for
Attorney Docket No.00166-0147-0304 example, column chromatography, examples disclosed herein may be applicable to any type of chromatography. Methods and systems herein may be described as being applicable to certain modes of chromatography (e.g., hydrophobic interaction chromatography and anion exchange chromatography), however, those of ordinary skill in the art will appreciate the methods and systems described herein may be applicable to all modes of chromatography. [0049] Chromatography techniques may be used to separate components of a mixture, purify biopharmaceutical products, and/or characterize the content of a mixture. Reduced scale chromatography techniques allow for the collection of larger amounts of data with less time and resources compared to traditional laboratory scale techniques. Therefore, reduced scale chromatography can be useful for the development, improvement, optimization, and/or validation of manufacturing scale chromatography operations. If a first chromatography operation is a reduced scale (e.g., microscale) chromatography operation used to evaluate, investigate, inform, and/or study a second manufacturing scale chromatography operation, then the first and second chromatography operations may be considered corresponding chromatography operations. [0050] Chromatography operations may include a sequence of one or more steps, including, for example, one or more pre-equilibration steps, equilibration steps, loading steps, wash steps, elution steps, strip steps, and/or regeneration steps. One or more steps of a chromatography operation may be tracked and/or recorded using performance data collected from a detector within the chromatography system (e.g., at the outlet of a chromatographic column). For example, the signal received by the detector may be plotted as a function of volume passed through the detector. In some examples, the signal received by the detector may be plotted as a function of time elapsed. A plot of a signal received by the detector versus volume passed through the detector or time elapsed may be referred to as a chromatogram. Performance data collected from the detector, including plots generated from signals received by the detector, may be used to monitor, track, and/or validate a chromatography operation. [0051] As described herein, parameters relating to a manufacturing scale chromatography operation may not be identical to parameters relating to the corresponding microscale chromatography operation. For example, a chromatography protocol may include certain parameters that affect the outcome of a chromatography operation, such as, for example, column geometry (e.g., a column volume, a bed height, an inner diameter), a flow rate, a resin bead size, a column porosity, a particle porosity, an axial dispersion, a film diffusion, an adsorption rate, a desorption rate, a pH, a pH change, a maximum binding capacity, a conductivity, a conductivity change, a characteristic charge, a maximum ionic capacity, and/or a steric binding factor. A chromatography protocol may include defined values for one or more parameters. The value of a parameter used in a manufacturing scale chromatography protocol may be different than the value of the parameter used in a corresponding microscale chromatography protocol. As one example, column geometry varies between a manufacturing chromatography operation and the corresponding microscale chromatography
Attorney Docket No.00166-0147-0304 operation (e.g., the microscale chromatography operation utilizes a column with a smaller column volume). The difference in scale also affects other properties of the operations. For example, due to decreased column sizes, flowrates associated with manufacturing scale chromatography operations are typically larger than the flowrates used in corresponding microscale chromatography operations. [0052] A conductivity, an absorbance, a pH, or one or more metrics derived from a conductivity, an absorbance, and/or a pH may be measured during a chromatography operation. These data, derived from one or more measurements made during a chromatography operation, may be referred to as performance data. Performance data may be generated based on measurements at the inlet of a chromatography column, measurements through the length of the column, and/or measurements at the outlet of a chromatography column. Exemplary performance data may include a conductivity, an absorbance, a pH, and/or a metric derived from one or more conductivity, absorbance, and/or pH measurements. For example, a concentration of a target molecule may be determined based on an absorbance, and performance data may include a concentration of the target molecule. In addition or alternatively, performance data may include a conductivity, an absorbance, a pH, or one or more metrics derived from a conductivity, an absorbance, and/or a pH, associated with a point in a chromatography operation (e.g., at a given time in the operation or at a given volume passed of the operation). Performance data may include a data set including performance parameter values and points in a chromatography operation associated with the performance parameter values. The performance parameter values may be plotted against their associated points in the chromatography operation (e.g., as time or volume values) to generate a chromatogram. [0053] Performance data derived from chromatography operations may be dependent on scale. For example, because of differences in flow rate and column volume in chromatography operations, signals detected (e.g., pHs, conductivities, absorbances) during a chromatography operation may vary in amplitude and may occur at different points (e.g., times or volumes) in the operation. Elution pool volume may also be affected by the differences in scale between microscale and manufacturing scale operations. As a result of the differences in data received from operations at different scale, a chromatogram resulting from a microscale technique may have a wider elution peak than a chromatogram resulting from a corresponding manufacturing scale technique. [0054] Because parameters and performance data of a microscale chromatography operation may not be identical to parameters and performance data of a corresponding manufacturing scale chromatography operation, scale differences must be accounted for in order to utilize microscale chromatography operations to investigate or inform manufacturing scale chromatography operations. The present disclosure includes methods and system of generating a model that describes the relationship between parameters and performance data of a microscale chromatography operation to parameters and performance data of a corresponding manufacturing scale chromatography operation. The model (e.g., a mechanistic model) may include a mathematical representation of the physical and chemical dynamics within a chromatography column. In some aspects, a mechanistic model may
Attorney Docket No.00166-0147-0304 be determined that relates microscale chromatography operations to manufacturing scale chromatography operations. The mechanistic model may identify and account for scale effects, including, for example, validating differences in data resulting from chromatography operations at different scales. In some aspects, the mechanistic model may account for differences in elution peak width between microscale chromatography operations and corresponding manufacturing and/or laboratory scale chromatography operations. [0055] A mechanistic model may describe the movement of a mobile phase during a chromatography operation at different varying levels of detail (e.g., bulk movement, molecular movement, ionic movement). Mechanistic models may include systems of partial differential equations that describe a combination of fluid flow through a packed bed and interaction between molecules of the mobile phase (e.g., a target molecule) and the chromatography media (e.g., ligands within the media). [0056] Chromatography media may include one or more ligands configured to interact with one or more components of a mobile phase, and a support structure supporting the one or more ligands. For example, chromatography media may include ligands including 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 comprise cross-linked agarose, highly-linked agarose, silica, aluminum oxide, methacrylate, glass, polyvinyl ether, or a combination thereof. [0057] Exemplary modes of movement of a mobile phase through a chromatography column are shown in FIG.1. FIG 1. includes an enlarged cross-sectional slice 103 of an exemplary chromatography column 101. FIG.1 also shows an enlarged area 105 including a unit (e.g., a resin bead 108) of chromatography media 106 and pores 104 within the chromatography media 106. Arrow 221 shows the general direction of flow of the mobile phase through the column 101. The example shown in FIG.1 includes several target molecules 102. The movement of a mobile phase within the column 101 includes the movement of target molecules 102. While examples described herein may refer to movement of the target molecule 102 as one component of the mobile phase, those of ordinary skill in the art will understand that other components of the mobile phase exhibit one or more of the same modes of movement as target molecule 102. [0058] The enlarged cross-sectional slice 103 is enlarged for the purposes of illustration and is intended to represent a portion of the cross-sectional area of column 101. Slice 103 shows exemplary means of movement (e.g., mass transfer) of target molecules 102 within chromatography media 106 (e.g., hydrophobic interaction media and/or anion exchange media). For example, arrows 203 illustrate movement of target molecules 102 due to dispersive mass transfer. Dispersive mass transfer may include the movement of matter from areas including a relatively high concentration of matter to areas including a relatively low concentration of matter. Arrows 201 denote movement of target
Attorney Docket No.00166-0147-0304 molecules 102 due to convection. Movement of target molecules 102 due to convection may include longitudinal movement of the target molecule 102 within the column. In the context of FIG.1, arrows 201 show movement of molecules into and out of the cross-sectional slice 103. For example, mass transfer via convection may include movement of the target molecule 102 from a longitudinal position in column 101 above cross-sectional slice 103 to cross-sectional slice 103 and/or movement of the target molecule 102 from cross-sectional slice 103 to a longitudinal position within column 101 below cross-sectional slice 103. In addition or alternatively, mass transfer via convection may include movement of the target molecule laterally (e.g., axially) within cross-sectional slice 103. [0059] Still referring to FIG.1, movement of target molecule 102 longitudinally through chromatography media 106 (e.g., due to convection and/or dispersion) may include target molecule 102 passing through a pore 104 (or other type of aperture) within chromatography media 106. Interactions between components of the mobile phase (e.g., target molecule 102) and the chromatography media 106 may affect the movement of the mobile phase through column 101. In particular, the magnitudes of the interactions between components of the mobile phase and the chromatography media 106 may depend on the properties of each mobile phase component. The differences in magnitudes of these interactions contributes to the operative separation of mobile phase components achieved in chromatography. [0060] Referring to the enlarged area 105, arrows 209 represents movement of target molecule 102 due to adsorption (e.g., association of target molecule 102 from media 106 to bead 108) and desorption (e.g., disassociation of target molecule 102 from bead 108 to media 106). Arrows 205 represent movement of target molecule 102 due to surface diffusion, and arrows 207 represent movement of target molecule due to pore diffusion. [0061] Surface diffusion and pore diffusion may refer to similar modes of mass transfer through the bead 108 of the media 106. Portions of the mobile phase that are farther from a wall of the bead 108 (e.g., portions that are closer to the center of the bead 108) may have fewer interactions (e.g., adsorptions and/or desorptions) with media 106 than portions of the mobile phase that are closer to the wall of the bead 108 (e.g., portions that are farther from the center of the bead 108). Portions of the mobile phase that have relatively fewer interactions with the media 106 may travel through column 101 at a faster rate than portions of the mobile phase that have relatively more interactions with media 106. Movement of the portions of the mobile phase that have relatively fewer interactions with the media 106 may be referred to as surface diffusion, and movement of the portions of the mobile phase that have relatively more interactions may be referred to as pore diffusion. Mechanistic models described herein may include terms that relate to pore diffusion and/or terms that relate to surface diffusion. [0062] The bulk mass transfer of the mobile phase (e.g., target molecule 102) through chromatography column 101 including chromatography media 106 may be a product of several forces, including, but not limited to, dispersion, convection, gravity, pore diffusion, electrostatic
Attorney Docket No.00166-0147-0304 driving forces (e.g., in ion-exchange chromatography operations), surface diffusion (e.g., film diffusion), and adsorption. [0063] One mechanistic model may not necessarily be applicable to all chromatography operations, due to differences in principle, physics, means of elution, and molecular interactions involved. In particular, methods and systems for assisting the development of chromatography operations using bind-and-elute modes of chromatography (e.g., protein affinity and cation exchange chromatography) may not be applicable to the development of chromatography operations using flowthrough modes of chromatography (e.g., hydrophobic interaction and anion exchange chromatography). Further, one mechanistic model may be developed for a particular target molecule and/or chromatography medium, based on the interactions between the molecule and medium. Different target molecules and chromatography media may interact differently, and a mechanistic model for one combination of target molecule and chromatography media may not be applicable to a different combination of target molecule and media. The methods and systems described herein may allow for the rapid development of mechanistic models for multiple combinations of target molecules and chromatography media. [0064] As described herein, a mechanistic model may be generated that relates microscale chromatography operations to chromatography operations of other scales (e.g., manufacturing scale and/or laboratory scale chromatography operations). Microscale chromatography operations may refer to operations including a chromatography column or membrane having a total volume of less than or equal to approximately 600 μL. For example, microscale chromatography operations may include a chromatography column or membrane having a total volume of approximately 50 μL to approximately 600 μL, such as, for example, approximately 589 μL. Chromatography columns used in microscale chromatography operations may have a bed height of approximately 25 millimeters (mm) to approximately 30 mm. Microscale chromatography operations may include columns having an inner diameter of approximately 5 mm to approximately 10 mm. [0065] Laboratory scale operations may refer to operations including a chromatography column or membrane having a total volume of greater than or equal to 3.0 mL and less than or equal to approximately 50 mL. For example, laboratory scale chromatography operations may include a chromatography column or membrane having a total volume of approximately 3.5 mL to approximately 20 mL. Chromatography columns used in laboratory scale chromatography operations may have a bed height of approximately 18 centimeters (cm) to approximately 22 cm. Laboratory scale chromatography operations may include columns having an inner diameter of approximately 0.5 cm to approximately 3.5 cm. [0066] Manufacturing scale chromatography operations may refer to operations including a chromatography column or membrane having a total volume greater than or equal to approximately 0.1 L. For example, manufacturing chromatography operations may include a chromatography column or membrane having a total volume of approximately 0.1 L to approximately 157 L.
Attorney Docket No.00166-0147-0304 Chromatography columns used in manufacturing scale chromatography operations may have an inner diameter of approximately 5 cm to approximately 100 cm. [0067] Aspects of systems and methods described herein may be implemented by a chromatography system. Exemplary chromatography systems may include one or more chromatographic columns, mobile phase supply reservoirs, components configured to facilitate introduction of material to the columns (e.g., pumps, conduits, valves, automated fluid handling devices), one or more detectors, and/or components configured to control, determine, and monitor operations of the chromatography system (e.g., controllers, integrated circuit chips, processors, or other suitable computing devices). [0068] Detectors that may be included within the chromatography systems may include any type of detector suitable for measuring or determining performance data at an inlet, body, or outlet of a column as described herein. In some aspects, an eluate of a column may be collected in a vessel (e.g., a well of a well plate), and the detector may measure or determine performance data from the eluate within the vessel. According to some aspects of the present disclosure, an exemplary detector may include an electrical conductivity detector, an ultraviolet (UV) detector, a fluorescence detector, a refractive detector, a pH detector, and/or a pressure gauge. For example, an exemplary detector may measure an absorbance of UV light (e.g., a wavelength of 280 nm, 275 nm, etc.), and the measured absorbance may correlate to a concentration of a component of a mobile phase (e.g., a target molecule). [0069] In some aspects, columns used in a microscale chromatography operation may be configured for use with an automated fluid handling device. Such devices may include robotic arms or other components configured to hold and/or move one or more chromatographic columns between different locations within a chromatography system. In some examples, an automated fluid handling device may include a robotic dispensing device configured to hold, move, and/or actuate a fluid dispensing instrument (e.g., an injector, a pipette, a syringe, or other suitable device) such that the fluid dispensing instrument introduces a fluid into a column. In addition or alternatively, fluid dispensing instruments may be configured to receive (e.g., withdraw) fluid from one or more reagent reservoirs. In some aspects, an automated fluid handling device may be configured to conduct multiple chromatography operations in parallel (e.g., eight parallel chromatography runs). For example, a fluid dispending instrument may include multiple outlets (e.g., injectors) that are configured to introduce fluid into a plurality of columns simultaneously. [0070] In a chromatography system including multiple chromatographic columns, an automated fluid handling device may be configured to perform operations on each column simultaneously, sequentially, randomly, and/or in an order that is algorithmically determined. Automated fluid handling devices as described herein may be operated and controlled by one or more computing devices. Such computing devices may be embodied by a centralized device having one or more processors, a memory, and/or storage. Computing devices may include or have access to instructions that are stored in or otherwise accessible from the memory and/or storage. The instructions may be
Attorney Docket No.00166-0147-0304 read and executed by the one more processors to perform the operations mentioned herein with respect to one or more chromatographic columns. [0071] As described herein, values of parameters and performance data associated with a reduced scale chromatography operation may not be equivalent to values of parameters and performance data associated with a corresponding manufacturing scale chromatography operation. For example, because reduced scale chromatography operations utilize columns with reduced dimensions (e.g., column volume, inner diameter, and/or bed height), other parameters of the reduced scale chromatography operation may require scale-based adjustments in order to accurately correspond to chromatography operations at other scales. In some aspects, to ensure residence times of the mobile phase in the reduced scale operations match those of larger scale operations (e.g., manufacturing scale), the reduced scale chromatography protocol may include a slower flow rate, compared to the larger scale chromatography operation. The reduced flow rate may result in pool elution volumes that depend on the scale of the chromatography operation. The pool elution volume of a chromatography operation can be characterized by the elution peak width of the chromatogram generated by the operation. Therefore, the chromatogram of a reduced scale chromatography operation may have a reduced elution peak width compared to a corresponding larger scale chromatography operation. Mechanistic models described herein can account for the scale differences in elution peak width, and characterize the relationship between elution peak width and scale. In addition or alternatively, mechanistic models described herein can account for scale differences of other parameters or properties of a chromatography operation. For example, mechanistic models described herein may account for differences in column loading (e.g., mass of target molecule per volume of chromatography media) and/or residence time between corresponding microscale, laboratory scale, and manufacturing scale chromatography operations. [0072] Developing a mechanistic model may include characterizing retention of a mobile phase within a chromatography system, determining mass transfer parameters, and/or determining isotherm parameters. Characterizing retention of a mobile phase within a column may include identifying system hold-ups. System hold-ups of a chromatography operation may include limitations on the flow or analysis of a mobile phase due to the structure of the column or an automated fluid handling device utilized in the operation. Examples of system hold-ups include, but are not limited to, a dead volume of a column (e.g., the volume of mobile phase retained in the column after the chromatography operation), dead volumes within the automated fluid handling system (e.g., volumes of mobile phase retained within conduits or other components of the automated fluid), and/or volume limitations of one or more components of the automated fluid handling device (e.g., a maximum volume of a pipette). In addition or alternatively, identifying system hold-ups may include determining a volume between the inlet and a chromatography column and/or determining a volume between the chromatography column and a detector (e.g., an absorbance detector).
Attorney Docket No.00166-0147-0304 [0073] Identifying system hold-ups may include conducting one or more runs without a chromatography column. For example, when developing a mechanistic model for chromatography operations that include an automated fluid handling system, identifying system hold-ups may include passing a mobile phase through the automated fluid handling system, without a column. In some aspects, identifying system hold-ups may include conducting multiple runs with mobile phases of different compositions. For example, identifying system hold-ups may include conducting one or more acetone pulse tests and/or one or more salt pulse tests. The one or more acetone pulse tests may be used to determine a system hold-up from an inlet of the fluid handling system to an absorbance detector. The one or more salt pulse tests may be used to determine a system hold-up from an inlet to a conductivity detector. [0074] An acetone pulse test may include passing an amount of an acetone solution through an automated fluid handling device without a column. The amount of the acetone solution may be approximately 8 mL to approximately 20 mL. The acetone solution may have an acetone concentration of approximately 1 volume percent (vol.%) to approximately 3 vol.%, based on the total volume of the acetone solution. While the acetone solution is being passed, a signal (e.g., an absorbance) detected by the detector of the automated fluid handling device may be plotted against the volume elapsed of the acetone pulse test to generate a chromatogram. The chromatogram may be used to calculate a retention volume associated with the acetone pulse test, and the retention volume may be used to determine system hold-ups. As described herein, the retention volume may be equal to a volume of a mobile phase (e.g., an acetone solution, a salt solution, a blue dextran solution) that passes through the fluid handling system prior to a maximum signal is measured by a detector. [0075] A salt pulse test may include passing a first amount of a first salt solution through an automated fluid handling device without a column, passing an amount of a second salt solution through the device, and then passing a second amount of the first salt solution through the device. The first amount of the first salt solution may include approximately 1.5 CVs to approximately 5 CVs, such as, for example, approximately 12 mL. The second amount of the first salt solution may include approximately 1.5 CVs to approximately 5 CVs, such as, for example, approximately 12 mL. In some examples, the second amount of the first salt solution is approximately the same as the first amount of the first salt solution. The amount of the second salt solution may be approximately 0.0005 CVs to approximately 0.01 CVs, such as, for example, approximately 0.08 mL. [0076] The second salt solution may include a higher concentration of salt than the first salt solution. The first and second salt solutions may include the same salt, such as, for example, sodium chloride, or another suitable salt with a detectable conductivity. The first salt solution may comprise approximately 50mM to approximately 250 mM of a salt, such as, for example, 150 mM sodium chloride. The second salt solution may comprise approximately 1.0M to approximately 3.0M of a salt, such as, for example, 2.0M sodium chloride. In some aspects, the first and second salt solutions may include different salts.
Attorney Docket No.00166-0147-0304 [0077] In some aspects, characterizing retention of a mobile phase within a column may include determining a column porosity, a total available porosity of the chromatography column, and/or a particle porosity of the column. [0078] Column porosity refers to a volume of void within the column that is outside or in between resin beads. Particle porosity refers to a volume of void within resin beads of the column. Total available porosity, also referred to herein as total porosity, is a volume of void within the entire column, including the void outside resin beads, between resin beads, and within resin beads. In some examples total porosity of a column is a sum of the column porosity and the particle porosity of the column. [0079] Determining a porosity of a chromatography operation including a column may include conducting one or more runs. For example, an unretained monoclonal antibody pulse test, a blue dextran pulse test, a salt pulse test, or a combination thereof, may be conducted to determine a porosity of the chromatography operation. [0080] An unretained monoclonal antibody pulse test may include passing a first amount of a first salt solution through an automated fluid handling device, passing an amount of a second solution through the device, and then passing a second amount of the first salt solution through the device. The first amount may include approximately 1.5 CVs to approximately 5 CVs, such as, for example, approximately 3 CVs. The second amount may include approximately 0.5% to approximately 5% of the column volume, such as, for example 0.02 column volumes. The second solution may include a protein of interest, such as, for example, a monoclonal antibody. In some examples, the protein of interest used in the unretained monoclonal antibody pulse test is the same as the protein of interest for the chromatography operation being studied. The first salt solution may comprise sodium chloride, or another suitable salt with a detectable conductivity. The first salt solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. The second solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. [0081] In some aspects, a plurality of antibody pulse tests with identical conditions and test parameters may be conducted to obtain a plurality of retention volumes. The plurality of retention volumes may be averaged to obtain an average retention volume. [0082] A retention volume determined from an unretained antibody test (e.g., an average retention volume) may be used to determine a total porosity ( t), according to Equation 1. Equation 1 [0083] Referring to Equation 1, the total porosity ( t) may be calculated as a quotient of a difference of the system absorbance hold-up (e.g., determined from one or more acetone pulse tests) subtracted from the unretained antibody retention volume (e.g., average retention volume), divided by the
Attorney Docket No.00166-0147-0304 volume of the column used in the antibody pulse test. Equation 1 may be applicable to systems including anion-exchange chromatography. [0084] In some aspects, an unretained antibody pulse test may not be used to determine the total porosity. For example, for some chromatography operations, retention of the target molecule within the column may invalidate results from an unretained antibody pulse test. For such chromatography operations, a salt pulse test may be used to determined total porosity. [0085] A retention volume determined from a salt pulse test (e.g., an average retention volume) may be used to determine a total porosity ( t), according to Equation 2. Equation 2 [0086] Referring to Equation 2, the total porosity ( t) may be calculated as a quotient of a difference of the system conductivity hold-up (e.g., determined from salt pulse tests) subtracted from the salt pulse test retention volume (e.g., average retention volume), divided by the volume of the column used in the salt pulse test. Equation 2 may be applicable to systems including hydrophobic interaction chromatography. [0087] A blue dextran pulse test may include passing a first amount of a first salt solution through an automated fluid handling device, passing an amount of a second solution through the device, and then passing a second amount of the first salt solution through the device. The first amount may include approximately 1.5 CVs to approximately 10 CVs, such as, for example, approximately 5 CVs. The second amount may include approximately 0.5% to approximately 5% of the column volume, such as, for example 0.02 column volumes. The second solution may include blue dextran, such as, for example, a solution comprising approximately 10 grams per liter of blue dextran in water. The first salt solution may comprise sodium chloride, or another suitable salt with a detectable conductivity. The first salt solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. [0088] In some aspects, a plurality of blue dextran pulse tests with identical conditions and test parameters may be conducted to obtain a plurality of retention volumes. The plurality of retention volumes made be averaged to obtain an average retention volume. [0089] A retention volume determined from an unretained antibody test (e.g., an average retention volume) may be used to determine a column porosity ( c), according to Equation 3. Equation 3 [0090] Referring to Equation 3, the column porosity ( c) may be calculated as a quotient of a difference of the system absorbance hold-up (e.g., determined from one or more acetone pulse tests) subtracted from the blue dextran retention volume (e.g., average retention volume), divided by the volume of the column used in the blue dextran pulse test.
Attorney Docket No.00166-0147-0304 [0091] A particle porosity ( p) for a chromatography operation may be calculated from the total porosity ( t) and column porosity ( c), according to Equation 4. Equation 4 [0092] Referring to Equation 4, the particle porosity ( p) may be calculated as a quotient of a difference of the column porosity ( c) subtracted from the total porosity ( t), divided by a difference of the column porosity ( c) subtracted from one. [0093] In some aspects, a generalized mechanistic model may be generated from the characterization of retention of the mobile phase. For example, a generalized mechanistic model may be generated based on retention volumes determined in one or more runs conducted during the characterization of retention of the mobile phase. The generalized mechanistic model may include a component that describes characteristics of the general flow of a mobile phase through the column. [0094] System hold-ups determined and porosities determined for a chromatography system, column configuration, and resin type may be used for multiple chromatography operations including the chromatography system, column configuration, and resin type. Therefore, once a generalized mechanistic model is determined, it may be used in the development of various mechanistic models to describe chromatography operations including different target molecules. [0095] In some aspects, the generalized mechanistic model may include a component described by Equation 5. Equation 5
[0096] Referring to Equation 5, represents the change in concentration of a species i (e.g., the target molecule) as a function of time. Equation 5 may characterize the change in concentration of species i at the bulk solution scale. Equation 5 includes terms that describe movement of the species due to dispersion, convection, and mass transfer from the bulk solution of the mobile phase to the chromatography media. Still referring to Equation 5, x is the axial distance along the column, Dax represents the axial dispersion coefficient, vsup represents the superficial fluid velocity, c represents the effective extra-particle column porosity, and rp represents the resin bead radius. The combined film mass transfer coefficient (keff) lumps the true film mass transfer and pore diffusion. The concentration difference [ci - cp,i] is calculated as the difference between the concentration of species i in the bulk fluid (ci) and the concentration of species i within the pores of the media (cp,i). [0097] In addition or alternatively, the generalized mechanistic model may include a component described by Equation 6. Equation 6
Attorney Docket No.00166-0147-0304 [0098] Referring to Equation 6,
represents the change in concentration of a species i (e.g., the target molecule) as a function of time, at the particle level. Equation 6 includes terms that describe movement of the species due to mass transfer from the bulk solution of the mobile phase to the chromatography media and adsorption of the species to the media. Similar to Equation 5, c represents the effective extra-particle column porosity, rp represents the resin bead radius, and keff represents the combined film mass transfer coefficient. [0099] In some examples, Equation 5 and Equation 6 may be solved simultaneously (e.g., using a mathematical solver) at each point in time t and axial distance x to produce values for the concentration of species i in the bulk fluid (ci), the concentration of species i within the pores of the media (cp,i), and the concentration of species i bound to the resin (qi). [0100] Still referring to Equation 6, represents the binding isotherm, which describes the relationship between species i in the mobile phase and species i adsorbed on the surface of the media. The definition of the binding isotherm depends on the mode of chromatography being utilized. For example, mechanistic models used to describe chromatography operations utilizing anion exchange chromatography may include a component described by Equation 7.
Equation 7 [0101] Referring to Equation 7, represents the ionic capacity of the column, vi represents the characteristic charge of species i, kads,i represents the adsorption rate constant for species i, kdes,i represents the desorption rate constant for species i, i represents the steric factor of species i, qi represents the concentration of species i bound to the resin, and cs represents the salt concentration of the system. [0102] The ionic capacity of the column is correlated to the total number of binding sites available for adsorption within the media. The characteristic charge is correlated to the number of charged sites in species i that are available for binding to the media. The steric factor is correlated to the number of binding sites within the media that are blocked when a formula unit of species i adsorbs to the media. [0103] Mechanistic models used to describe chromatography operations utilizing hydrophobic interaction chromatography may include a component described by Equation 8. Equation 8
[0104] Referring to Equation 8, kads,i represents the adsorption rate constant for species i, kdes,i represents the desorption rate constant for species i, qi represents the concentration of species i bound to the resin, cx,i represents the concentration of species i at an axial distance x, and qmax,i is the binding capacity for species i.
Attorney Docket No.00166-0147-0304 [0105] In a hydrophobic interaction chromatography operation, the adsorption constant may vary as a function of the pH change ( pH) as the operation progresses from an equilibration pH to an elution pH. In such chromatography operations, the adsorption constant (kads,i) may be defined by Equation 9.
Equation 9 [0106] Referring to Equation 9, pH represents a difference between an equilibration pH and an elution pH (e.g., the pH of the eluate in proximity to the detector), kads0,i represents to the adsorption constant of species i at a pH of zero (i.e., where the current pH equals the equilibration pH), and represents a tuning parameter that is proportional to a rate of change of the pH during an elution phase. The tuning parameter is dependent on interactions between species i (e.g., the target molecule) and the chromatography media (e.g., resin), and is specific to the combination of target molecule and chromatography media. [0107] In some aspects, after a particle porosity, a column porosity, and/or a total porosity are determined, a generalized mechanistic model may be generated. Generating the generalized mechanistic model may include substituting determined porosity values (e.g., a particle porosity, a column porosity, and/or a total porosity) into Equations 5–9, described above. For example, generating the generalized mechanistic model for a hydrophobic interaction chromatography protocols may include inserting experimentally determined porosity values into Equations 5, 6, 8, and 9. Generating the generalized mechanistic model for anion exchange chromatography may include inserting experimentally determined porosity values into Equations 5, 6, and 7. [0108] As described herein, methods of developing a mechanistic model may include determining mass transfer parameters (e.g., Dax and/or keff) of the generalized mechanistic model. Determining mass transfer parameters may include fitting parameters of the generalized mechanistic model, while ignoring the terms of the generalized mechanistic model that describe binding mechanisms (e.g., the binding isotherm). For example, fitting parameters of the generalized mechanistic model may include conducting unretained monoclonal antibody pulse tests at various flow rates. [0109] An unretained monoclonal antibody pulse test may include passing a first amount of a first salt solution through a column within an automated fluid handling device, passing an amount of a second solution through the column, and then passing a second amount of the first salt solution through the device. The first amount may include approximately 1.5 CVs to approximately 5 CVs, such as, for example, approximately 3 CVs. The second amount may include approximately 0.5% to approximately 5% of the column volume, such as, for example 0.02 column volumes. The second solution may include a protein of interest, such as, for example, a monoclonal antibody. In some examples, the protein of interest used in the unretained monoclonal antibody pulse test is the same as the protein of interest for the chromatography operation being studied. The first salt solution may comprise sodium chloride, or another suitable salt with a detectable conductivity. The first salt
Attorney Docket No.00166-0147-0304 solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. The second solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. [0110] In some aspects, an unretained antibody pulse test may not be used to generate data for the fitting of the mass transfer parameters. For example, for some chromatography operations, retention of the target molecule within the column may invalidate results from an unretained antibody pulse test. For such chromatography operations, data from a blue dextran pulse test may be used for the fitting of the mass transfer parameters. The blue dextran pulse test may include passing a first amount of a first salt solution through a column of an automated fluid handling device, passing an amount of a second solution through the column, and then passing a second amount of the first salt solution through the column. The first amount may include approximately 1.5 CVs to approximately 10 CVs, such as, for example, approximately 5 CVs. The second amount may include approximately 0.5% to approximately 5% of the column volume, such as, for example 0.02 column volumes. The second solution may include blue dextran, such as, for example, a solution comprising approximately 10 grams per liter of blue dextran in water. The first salt solution may comprise sodium chloride, or another suitable salt with a detectable conductivity. The first salt solution may comprise approximately 0.5M to approximately 5M of a salt, such as, for example, 2M sodium chloride. [0111] As described herein, methods of developing a mechanistic model may include determining values for one or more mass transfer parameters (e.g., Dax and/or keff). In some aspects, the mass transfer parameters are determined by performing an inverse fit between the generalized mechanistic model and pulse test data (e.g., unretained antibody pulse test data or blue dextran pulse test data) for one or more flow rates. While the mass transfer parameters are determined, the binding mechanisms of the generalized mechanistic model may be turned off. For example, may be set as equal to zero. In addition or alternatively, one or more boundary conditions may be set for evaluating the equations of the mechanistic model. Exemplary boundary conditions are described by Equations 10, 11, and 12. , 0 , 0 ; for all t greater than 0 Equation 10 than 0 Equation 11
all x between 0 and L Equation 12 [0112] Referring to Equations 10, 11, and 12, u represents the quotient of vsup divided by c, and L represents a length of the column. Referring to Equation 12, the boundary condition is for all values of x between 0 and L, inclusively. [0113] Determining mass transfer parameters for hydrophobic interaction chromatography protocols may include solving Equations 5, 6, 8, and 9, using the boundary conditions set by Equations 10–12.
Attorney Docket No.00166-0147-0304 Determining mass transfer parameters for anion exchange chromatography may include solving Equations 5, 6, and 7, using the boundary conditions set by Equations 10–12. [0114] Methods of developing a mechanistic model may include determining isotherm parameters. Determining isotherm parameters may include conducting laboratory-scale chromatography runs, and using data from the chromatography runs to fit parameters of the binding isotherm. [0115] One or more equation solvers or parameter fitting algorithms may be utilized to solve the equations of the mechanistic model (e.g., while fitting mass transfer parameters or isotherm parameters). In one aspect, a MATLAB function (e.g., lsqnonlin) may be used for parameter fitting. The one or more equation solvers or parameter fitting algorithms may utilize an objective function to compare experimental data to output data of a candidate generalized mechanistic model. In one aspect, the objective function is a residual sum of squares, defined by Equation 13. Residual Sum of Squares
Equation 13 [0116] FIG.2 depicts, in flowchart form, an exemplary method 1000 of developing a mechanistic model. Method 1000 may include characterizing system hold-ups and porosities within a chromatography system (step 1010). Characterizing system hold-ups within the chromatography system may include performing an acetone pulse test and/or a salt pulse test, without a column. During the acetone pulse test and/or the salt pulse test, measurements of retention volume may be obtained. The acetone pulse test and/or the salt pulse test may also identify system hold-ups. Characterizing porosities within the chromatography system may include determining a total porosity and a column porosity. For example, characterizing porosities the chromatography system may include performing an unretained antibody pulse test and/or a salt pulse test to determine the total porosity and/or performing a blue dextran pulse test to determine the column porosity. As described herein, the determined total porosity and column porosity may be used as parameters of a mechanistic model. [0117] In some aspects, method 1000 may include determining mass transfer parameters (step 1020). Determining mass transfer parameters may include performing unretained antibody pulse tests at various flow rates. For example, an axial dispersion coefficient and/or a lumped film diffusion coefficient may be determined from the unretained antibody pulse tests, and used as parameters of a mechanistic model. [0118] In addition or alternatively, method 1000 may include determining isotherm parameters (step 1030). Determining isotherm parameters may include performing laboratory-scale chromatography runs. During the laboratory-scale chromatography runs, a relationship between antibody concentration of the eluate and time elapsed of the chromatography operation is determined. In addition or alternatively, an adsorption rate, a desorption rate, a maximum binding capacity, a tuning parameter, an ionic capacity, a characteristic charge, and/or a steric factor may be determined during the laboratory-scale chromatography runs.
Attorney Docket No.00166-0147-0304 [0119] After the parameters for the mechanistic model are determined, the mechanistic model may be validated at multiple scales. For example, chromatography runs may be conducted at multiple scales (e.g., microscale, laboratory scale, and/or manufacturing scale), and the results of the chromatography runs may be compared to predictions generated by the mechanistic model. Validation of the mechanistic model may include confirming that the predictions generated by the mechanistic model are within acceptable deviations from the results observed in the chromatography runs. [0120] In some aspects, validation of the mechanistic model may include comparing elution peak widths generated from the chromatography runs to predicted elution peak widths generated by the mechanistic model. One of the goals of developing a mechanistic model may be to characterize the effect of reduced flow rate in reduced scale chromatography, compared to manufacturing scale chromatography. For example, flow rate of reduced scale chromatography operations may be reduced compared to the corresponding manufacturing scale chromatography operations, in order to ensure the residence times of the mobile phase within the chromatography column for the reduced scale operations are approximately equivalent to the residence times of the mobile phase within the column for the corresponding manufacturing scale chromatography operations. Comparison of the predicted peak widths generated by the mechanistic model to the observed peak widths generated from the chromatography runs can validate that the model functions to demonstrate that peak width is a function of axial dispersion at the lower flow rate, and accounted for. [0121] In some aspects, predicted elution peak heights generated by the mechanistic model are not indicative of the strength of the fit of the mechanistic model. For example, reduced scale chromatography operations may result in residual product being retained on the column. The residual product may not be accounted for by the mechanistic model, resulting in differences in peak height. Regardless of peak height discrepancies, the mechanistic model may still be a useful tool in utilizing reduced scale chromatography to study manufacturing scale chromatography. [0122] After a developed mechanistic model is validated, it can be used to inform chromatography processes at microscale, laboratory scale, and manufacturing scale. For example, the mechanistic model can be used to optimize parameters of a chromatography operation (e.g., flow rates, initial concentrations, loading volumes). In some aspects, microscale chromatography runs may be conducted with varying initial concentrations, flow rates, and loading volumes. Performance data from the microscale chromatography runs (e.g., yield) can be used to inform optimized parameters of a manufacturing scale chromatography operation, using the mechanistic model to confirm the manufacturing scale performance data correlates to the microscale performance data. [0123] Mechanistic models developed by methods of the present disclosure may be used to understand the impact of chromatography operation parameters on performance data of the operation. For example, process inputs (e.g., flow rate, bed height, total available porosity, column porosity, wash length, mobile phase composition, and/or column load) can be varied and the
Attorney Docket No.00166-0147-0304 mechanistic model can be used to determine the impact of the variations of process inputs on the performance data of the operation. Use of a mechanistic model to explore the interactions of the process inputs can provide a high-throughput analysis of these interactions, compared to traditional chromatography investigations. [0124] Mechanistic models developed by methods of the present disclosure may be used to investigate process or system related effects on the performance of a chromatography operation. For example, traditional chromatography models assume highly efficient flow-through. Depending on the mobile phase composition and chromatography media, the efficiency of the flow-through may be reduced, and result in traditional models not accurately characterizing the movement of the mobile phase through the media. Mechanistic models of the present disclosure can be used to investigate and account for such process or system related effects on the performance of the chromatography operation. [0125] In some aspects, reduced scale chromatography operations (e.g., microscale) can be used to inform manufacturing scale chromatography operations, using the mechanistic model to verify that performance data received from reduced scale operations corresponds to desired performance data at a manufacturing scale operation. EXAMPLES Example 1 [0126] An acetone pulse test was performed with two laboratory scale automated fluid handling devices. The first automated fluid handling device is used for chromatography operations, including anion exchange chromatography operations. The second automated fluid handling device is used for chromatography operations, including hydrophobic interaction chromatography operations. The acetone pulse tests were performed without a chromatography column, in order to determine a system hold-up from the inlet to the absorbance detector, associated with the automated fluid handling devices. [0127] A first acetone pulse test was performed by passing 12 mL of an acetone solution through a first automated fluid handling device, at a flow rate of 231 cm/hr. The acetone solution had an acetone concentration of approximately 1–3 vol.%, based on the total volume of the solution. A chromatogram was generated from the first pulse test and is shown in FIG.3. [0128] A second acetone pulse test was performed by passing 12 mL of an acetone solution through a second automated fluid handling device, at a flow rate of 200 cm/hr. The acetone solution had an acetone concentration of approximately 1–3 vol.%, based on the total volume of the solution. A chromatogram was generated from the second pulse test and is shown in FIG.4. Example 2 [0129] A salt pulse test was performed for the second laboratory scale automated fluid handling device described in Example 1. The salt pulse test was performed without a chromatography column, in order to determine a system hold-up from the inlet to the conductivity detector. The salt pulse test
Attorney Docket No.00166-0147-0304 included establishing a baseline by passing 12 mL of a first solution including 150 mM sodium chloride through the automated fluid handling device, and then passing 0.08 mL of a second solution including 2M sodium chloride through the automated fluid handling device. The conductivity of the solution exiting the fluid handling device was measured and plotted as a function of the volume passed through the fluid handling device to generate a chromatogram. [0130] A first salt pulse test was performed on the second automated fluid handling device, at a flow rate of 200 cm/hr. The chromatogram generated from the first salt pulse test is shown in FIG.5. [0131] Based on the chromatogram in FIG.3, a volumetric system hold-up from the inlet to the absorbance detector of 0.595 mL was calculated for the laboratory scale automated fluid handling device configured for anion exchange chromatography operations, i.e., the first automated fluid handling device. [0132] Based on the chromatogram in FIG.4 a volumetric system hold-up from the inlet to the absorbance detector of 0.837 mL was calculated for the laboratory scale automated fluid handling device configured for hydrophobic interaction chromatography operations, i.e., the second automated fluid handling device. [0133] Based on the chromatogram in FIG.5, a volumetric system hold-up from the inlet to the conductivity detector of 1.019 mL was calculated for the laboratory scale automated fluid handling device configured for hydrophobic interaction chromatography operations, i.e., the second automated fluid handling device. [0134] As described herein, for some chromatography systems (e.g., those configured for microscale chromatography operations), the flow path volume outside of the column is negligible, and the volumetric system hold-ups can be assumed to be zero. Example 3 [0135] An unretained antibody pulse test was conducted with the first automated fluid handling device described in Example 1, utilizing a Q Sepharose Fast Flow column, to characterize total porosity of the anion-exchange column. [0136] The unretained antibody pulse test included introducing three column volumes (approximately 11.8 mL) of a first solution including 2M sodium chloride into the column. Next, a pulse of 0.02 column volumes of a second solution, including a target molecule and 2M sodium chloride, was introduced into the column. Finally, three column volumes of the first solution including 2M sodium chloride was introduced into the column. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatogram shown in FIG.6. The pulse test was carried out at flow rates of 22.5 cm/hr and 150 cm/hr. Referring to FIG.6, the chromatogram generated from the 22.5 cm/hr pulse test is represented by the solid line and the chromatogram generated from the 150 cm/hr pulse test is represented by the dashed line. Retention volumes for the chromatograms shown in FIG.6 were averaged to determine an average retention volume of 2.683 mL.
Attorney Docket No.00166-0147-0304 [0137] A blue dextran pulse test was conducted with the first automated fluid handling device described in Example 1, utilizing a Q Sepharose Fast Flow column, to characterize column porosity of the anion-exchange chromatography column. [0138] The blue dextran pulse test included introducing five column volumes (approximately 19.6 mL) of a first solution including 2M sodium chloride into the column. Next, a pulse of 0.02 column volumes of a second solution, including 10 g/L of blue dextran in water, was introduced into the column. Finally, five column volumes of the first solution including 2M sodium chloride was introduced into the column. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatogram shown in FIG.7. The pulse test was carried out at flow rates of 22.5 cm/hr and 150 cm/hr. Referring to FIG.7, the chromatogram generated from the 22.5 cm/hr pulse test is represented by the solid line and the chromatogram generated from the 150 cm/hr pulse test is represented by the dashed line. Retention volumes for the chromatograms shown in FIG.7 were averaged to determine an average retention volume of 1.747 mL. [0139] Total porosity of the chromatography operation was calculated based on the unretained antibody pulse test retention volume, and according to Equation 1. The column porosity of the chromatography operation was calculated based on the blue dextran pulse test retention volume, and according to Equation 3. The particle porosity of the chromatography operation was calculated based on the total porosity and the column porosity, and according to Equation 4. The total, column, and particle porosities calculated for the anion-exchange chromatography operation described in Example 3 are summarized in Table 1. Example 4 [0140] An unretained antibody pulse test was conducted with the second automated fluid handling device described in Examples 1 and 2, utilizing a CaptoPhenyl high-sub column, to characterize total porosity of the hydrophobic interaction chromatography column. During the antibody pulse test, it was determined that the target molecule was being retained on the column. Therefore, the results from the unretained antibody pulse test were not used in the development of a mechanistic model. Instead, salt pulse tests were conducted to determine the total porosity of the hydrophobic interaction chromatography operation. [0141] A first salt pulse test included establishing a baseline by passing 12 mL of a first solution including 150 mM sodium chloride through the second automated fluid handling device, and then passing 0.08 mL of a second solution including 2M sodium chloride at a flow rate of 30 cm/hr through the automated fluid handling device. A second salt pulse test included establishing a baseline by passing 12 mL of a first solution including 150 mM sodium chloride through the second automated fluid handling device, and then passing 0.08 mL of a second solution including 2M sodium chloride at a flow rate of 200 cm/hr through the automated fluid handling device. For both first and second salt pulse tests, the conductivity of the eluate exiting the fluid handling device was
Attorney Docket No.00166-0147-0304 measured and plotted as a function of the volume passed through the fluid handling device to generate a chromatogram. [0142] For each salt pulse test, the conductivity of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatogram shown in FIG.8. Referring to FIG.8, the chromatogram generated from the 30 cm/hr pulse test is represented by the solid line and the chromatogram generated from the 200 cm/hr pulse test is represented by the dashed line. Retention volume for the chromatograms shown in FIG.8 were averaged to determine an average retention volume of 4.782 mL. [0143] A blue dextran pulse test was conducted with the second automated fluid handling device described in Examples 1 and 2, utilizing a CaptoPhenyl high-sub column, to characterize column porosity of the hydrophobic interaction column. [0144] The blue dextran pulse test included introducing three column volumes (approximately 11.8 mL) of a first solution including 40mM tris and 80mM sodium citrate into the column. Next, a pulse of 0.02 column volumes of a second solution, including 10 g/L of blue dextran in water, was introduced into the column. Finally, ten column volumes (approximately 39.3 mL) of the first solution including 40mM tris and 80mM sodium citrate was introduced into the column. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatogram shown in FIG.9. The pulse test was carried out at flow rates of 30 cm/hr and 200 cm/hr. Referring to FIG.9, the chromatogram generated from the 30 cm/hr pulse test is represented by the solid line and the chromatogram generated from the 200 cm/hr pulse test is represented by the dashed line. Retention volumes for the chromatograms shown in FIG.9 were averaged to determine an average retention volume of 3.008 mL. [0145] Total porosity of the chromatography operation was calculated based on the salt pulse test retention volume, and according to Equation 2. The column porosity of the chromatography operation was calculated based on the blue dextran pulse test retention volume, and according to Equation 3. The particle porosity of the chromatography operation was calculated based on the total porosity and the column porosity, and according to Equation 4. The total, column, and particle porosities calculated for the hydrophobic interaction chromatography operation described in Example 4 are summarized in Table 1. Table 1
Attorney Docket No.00166-0147-0304 Example 5 [0146] Unretained antibody pulse tests were conducted with the first automated fluid handling device described in Example 1, utilizing a Q Sepharose Fast Flow column, in order to provide data that can be used to determine mass transfer parameters of a mechanistic model. [0147] The unretained antibody pulse test included introducing three column volumes (approximately 11.8 mL) of a first solution including 2M sodium chloride. Next, a pulse of 0.02 column volumes of a second solution, including a target molecule (e.g., a monoclonal antibody), was introduced into the column. Finally, three column volumes (approximately 11.8 mL) of the first solution including 2M sodium chloride was introduced into the column. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatograms shown in FIGs.10A and 10B. The pulse test was carried out at flow rates of 22.5 cm/hr and 150 cm/hr. The chromatogram generated from the 22.5 cm/hr pulse test is represented by the solid line in FIG.10A, and the chromatogram generated from the 150 cm/hr pulse test is represented by the solid line in FIG.10B. [0148] As described herein, mass transfer parameters of a generalized mechanistic model may be determined from the unretained antibody pulse test. The mass transfer parameters of a generalized mechanistic model were determined from the pulse test chromatograms shown in FIGs.10A and 10B. A summary of the determined mass transfer parameters, including the associated sum of squared errors for each regression fit, is provided in Table 2. The dashed lines in FIGs.10A and 10B represent the mechanistic model, including the determined mass transfer parameters, with the binding mechanisms set to zero. Example 6 [0149] An unretained antibody pulse test was conducted with the second automated fluid handling device described in Examples 1 and 2, utilizing a CaptoPhenyl high-sub column, in order to provide data that can be used to determine mass transfer parameters of a mechanistic model. During the antibody pulse test, it was determined that the target molecule was being retained on the column. Therefore, the results from the unretained antibody pulse test were not used in the development of a mechanistic model. Instead, blue dextran pulse tests were conducted to determine the mass transfer parameters for the hydrophobic interaction chromatography mechanistic model. [0150] The blue dextran pulse test included introducing three column volumes (approximately 11.8 mL) of a first solution including 40mM tris and 80mM sodium citrate into the column. Next, a pulse of 0.02 column volumes of a second solution, including 10 g/L of blue dextran in water, was introduced into the column. Finally, ten column volumes (approximately 39.3 mL) of the first solution including 40mM tris and 80mM sodium citrate was introduced into the column. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatograms shown in FIGs.11A and 11B. The pulse test was carried out
Attorney Docket No.00166-0147-0304 at flow rates of 30 cm/hr and 200 cm/hr. The chromatogram generated from the 30 cm/hr pulse test is represented by the solid line in FIG.11A, and the chromatogram generated from the 200 cm/hr pulse test is represented by the solid line in FIG.11B. [0151] As described herein, mass transfer parameters of a generalized mechanistic model may be determined from the blue dextran pulse test. The mass transfer parameters of a generalized mechanistic model were determined from the pulse test chromatograms shown in FIGs.11A and 11B. A summary of the determined mass transfer parameters, including the associated sum of squared errors for each regression fit, is provided in Table 2. The dashed lines in FIGs.11A and 11B represent the mechanistic model, including the determined mass transfer parameters, with the binding mechanisms set to zero. Table 2
Example 7 [0152] Laboratory scale chromatography runs were conducted utilizing a Q Sepharose Fast Flow column, in order to generate data that can be used to determine isotherm parameters of the mechanistic model for an anion exchange chromatography operation. [0153] The laboratory scale chromatography runs each had a different load volume and load concentration. A first chromatography run included a load volume of 126.27 mL and a load concentration of 9.050 g/L. A second chromatography run included a load volume of 95.69 mL and a load concentration of 11.942 g/L. A third chromatography run included a load volume of 120.38 mL and a load concentration of 12.657 g/L. [0154] The chromatography runs included a flush with 2.5 CVs of water, a strip with 2.0 CVs of a solution including 0.5 M acetic acid, and a equilibration step including 3.0 CVs of a solution including 10 mM sodium phosphate and 0.5 M sodium chloride. After the equilibration step, the load was introduced. After the load was introduced, the chromatography runs included a first washing step, a second washing step, and an elution step. The first washing step included 3 CVs of a solution comprising 10 mM sodium phosphate and 0.5 sodium chloride. The second washing step included 2 CVs of a solution comprising 20 mM sodium phosphate. The elution step included 3 CVs of a solution comprising 40 mM acetic acid.
Attorney Docket No.00166-0147-0304 [0155] The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatograms shown in FIGs.12A, 12B, and 12C. Chromatogram generated from the first chromatography run is shown in FIG.12A, the chromatogram generated from the second chromatography run is shown in FIG.12B, and the chromatogram generated from the third chromatography run is shown in FIG.12C. The chromatograms generated from chromatography runs are represented by the solid lines in FIGs.12A, 12B, and 12C. [0156] As described herein, isotherm parameters of the generalized mechanistic model may be determined from the laboratory scale chromatography runs. The isotherm parameters of the mechanistic model for the anion exchange chromatography operation were determined from the chromatograms shown in FIGs.12A, 12B, and 12C. A summary of the determined isotherm parameters, including the associated sum of squared errors for each regression fit, is provided in Table 3. The dashed lines in FIGs.12A, 12B, and 12C represent the mechanistic model, including the determined parameters, where the binding mechanisms are not equal to zero. Table 3
[0157] Additionally, laboratory scale chromatography runs were conducted utilizing a CaptoPhenyl high-sub column, in order to generate data that can be used to determine isotherm parameters of the mechanistic model for a hydrophobic interaction chromatography operation. The absorbance of the eluate was measured and plotted as a function of the volume passed through the column to generate the chromatograms shown in FIGs.13A, 13B, and 13C. The chromatograms generated from chromatography runs are represented by the solid lines in FIGs.13A, 13B, and 13C. [0158] As described herein, isotherm parameters of the generalized mechanistic model may be determined from the laboratory scale chromatography runs. The isotherm parameters of the mechanistic model for the hydrophobic interaction chromatography operation were determined from the chromatograms shown in FIGs.13A, 13B, and 13C. A summary of the determined isotherm parameters, including the associated sum of squared errors for each regression fit, is provided in Table 4. The dashed lines in FIGs.13A, 13B, and 13C represent the mechanistic model, including the determined parameters, where the binding mechanisms are not equal to zero.
Attorney Docket No.00166-0147-0304 Table 4
Example 8 [0159] A mechanistic model was generated to describe movement of a mobile phase in a hydrophobic interaction chromatography operation. The hydrophobic interaction chromatography operation includes separating components of the mobile phase, including a monoclonal antibody target molecule. The mechanistic model included terms from Equations 5, 6, and 8. Additionally, the mechanistic model included the porosity values shown in Table 1, the mass transfer parameters shown in Table 2, and the binding isotherm parameters shown in Table 4. [0160] Validation of the mechanistic model included comparing experimental performance data from microscale and laboratory scale chromatography runs to predicted performance data generated by the model. Microscale experimental and predicted performance data were generated for two lots (i.e., Lot 1 and Lot 2) of the target molecule. Performance data included UV absorbance (e.g., at a wavelength of 280nm), and concentration of the target molecule determined from the UV absorbance data. For each lot, eight microscale chromatography runs were performed simultaneously using a chromatography system including an automated fluid handling device. The performance data for each of the eight runs were averaged to determine the experimental mean. [0161] FIG.14A includes a plot of microscale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 1 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.14A, the load volume was 8.7 mL and the load concentration was 4.3 g/L. Target molecule concentrations (in grams per liter) are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.14A includes line 301 which represents the experimental mean performance data. Each point along line 301 shown in FIG.14A represents a mean of the eight experimentally determined performance data measurements. The plot of FIG.14A also includes line 302 that represents the predicted performance data generated by the mechanistic model. [0162] FIG.14B includes a plot of microscale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 2 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.14B, the load volume was 8.2 mL and the load concentration was 4.5 g/L. Target molecule concentrations (in grams per liter) are plotted versus the volume passed through the column for the chromatography operation (in
Attorney Docket No.00166-0147-0304 column volumes). The plot of FIG.14B includes line 311 which represents the experimental mean performance data. Each point along line 311 shown in FIG.14B represents a mean of the eight experimentally determined performance data measurements. The plot of FIG.14B also includes line 312 that represents the predicted performance data generated by the mechanistic model. [0163] As seen in FIGs.14A and 14B, the measured target molecule concentration begins to decrease at around 7 CVs. This is due to a collection pause while an injector of the automated fluid handling device refills with the remaining load. While the collection pause affects the height of the elution peak, it does not impact the width of the elution peak. As described herein, validation of the mechanistic model may include comparison of peak widths between the chromatogram of the experimentally generated performance data and the chromatogram of predicted performance data generated by the mechanistic model. Therefore, differences in the height of the elution peak between microscale experimentally determined chromatograms and mechanistic model predicted chromatograms may not be indicative of the fit of the model. Comparisons of the elution peaks widths between microscale experimentally determined chromatograms and mechanistic model predicted chromatograms are shown below in Table 5. [0164] Laboratory scale experimental and predicted performance data were generated for three lots (i.e., Lots 1, 2, and 3) of the target molecule. Performance data included UV absorbance (e.g., at a wavelength of 280nm). [0165] FIG.13B includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 1 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.13B, the load volume was 216 mL and the load concentration was 4.1 g/L. Measured and predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.13B includes line 401 which represents the experimental performance data, and line 402 that represents the predicted performance data generated by the mechanistic model. [0166] FIG.13C includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 2 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.13C, the load volume was 189 mL and the load concentration was 4.7 g/L. Measured and predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.13C includes line 411 which represents the experimental performance data, and line 412 that represents the predicted performance data generated by the mechanistic model. [0167] FIG.13A includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 3 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.13A, the load
Attorney Docket No.00166-0147-0304 volume was 213 mL and the load concentration was 4.2 g/L. Measured and predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.13A includes line 421 which represents the experimental performance data, and line 422 that represents the predicted performance data generated by the mechanistic model. [0168] Comparisons of the elution peaks widths between laboratory scale experimentally determined chromatograms and mechanistic model predicted chromatograms are shown below in Table 5. The peak width delta is defined as the quotient of the absolute value of the difference between the experimental peak width and the predicted peak width divided by the experimental peak width. Table 5
[0169] The peak widths shown in Table 5 for the microscale chromatography operations are also shown graphically in the bar graphs of FIG.15A. The peak widths shown in Table 5 for the laboratory scale chromatography operations are also shown graphically in the bar graphs of FIG. 15B. In both FIGs.15A and 15B, the black bars represent experimentally determined peak widths and the white bars represent the predicted peak widths generated by the mechanistic model. As can be seen from the data in Table 5 and the plots in FIGs.15A and 15B, the model accurately predicts elution peak widths for microscale and laboratory scale chromatography operations. Therefore, the model captures the main mechanisms of the hydrophobic interaction chromatography operation. Example 9 [0170] A mechanistic model was generated to describe movement of a mobile phase in an anion exchange chromatography operation. The anion exchange chromatography operation includes separating components of the mobile phase, including a monoclonal antibody target molecule. The mechanistic model included terms from Equations 5, 6, and 7. Additionally, the mechanistic model included the porosity values shown in Table 1, the mass transfer parameters shown in Table 2, and the binding isotherm parameters shown in Table 3. [0171] Validation of the mechanistic model included comparing experimental performance data from microscale and laboratory scale chromatography runs to predicted performance data generated by the model. Microscale experimental and predicted performance data were generated for a lot (i.e., Lot 1) of the target molecule. Performance data included UV absorbance (e.g., at a wavelength of
Attorney Docket No.00166-0147-0304 280nm), and concentration of the target molecule determined from the UV absorbance data. Seven microscale chromatography runs were performed simultaneously using a chromatography system including an automated fluid handling device. [0172] FIGs.16A and 16B includes plots of microscale experimentally determined performance data for Lot 1 of the target molecule. For the chromatography runs shown in FIGs.16A and 16B, the load volume was 4.8 mL and the load concentration was 11.64 g/L. Target molecule concentrations (in grams per liter) are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.16A includes lines for each of four columns used in the seven parallel microscale chromatography runs, the plot of FIG.16B includes lines for the three remaining columns used in the seven parallel microscale chromatography runs. Referring to FIG. 16A, the line with triangular data points represents the chromatography run conducted on the first column, the line with square data points represents the chromatography run conducted on the second column, the line with circular data points represents the chromatography run conducted on the third column, and the line with data points marked by an ‘*’ represents the chromatography run conducted on the fourth column. Referring to FIG.16B, the line with triangular data points represents the chromatography run conducted on the fifth column, the line with square data points represents the chromatography run conducted on the sixth column, and the line with circular data points represents the chromatography run conducted on the seventh column. [0173] The performance data for each of the seven runs were averaged to determine the experimental mean. FIG.17 includes a plot of microscale experimentally determined performance data (e.g., the experimental mean) and predicted performance data generated by the anion exchange mechanistic model for Lot 1 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.17, the load volume was 4.8 mL and the load concentration was 11.64 g/L. Target molecule concentrations (in grams per liter) are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.17 includes line 501 which represents the experimental mean performance data. Each point along line 501 shown in FIG.17 represents a mean of the seven experimentally determined performance data measurements. The plot of FIG.17 also includes line 502 that represents the predicted performance data generated by the mechanistic model. Comparisons of the elution peaks widths between microscale experimentally determined chromatograms and mechanistic model predicted chromatograms are shown below in Table 6. [0174] Laboratory scale experimental and predicted performance data were generated for three lots (i.e., Lots 1, 2, and 3) of the target molecule. Performance data included UV absorbance (e.g., at a wavelength of 280nm). FIG.12A includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 1 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG. 12A, the load volume was 126.27 mL and the load concentration was 9.05 g/L. Measured and
Attorney Docket No.00166-0147-0304 predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.12A includes line 601 which represents the experimental performance data, and line 602 that represents the predicted performance data generated by the mechanistic model. [0175] FIG.12B includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 2 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.12B, the load volume was 95.69 mL and the load concentration was 11.94 g/L. Measured and predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.12B includes line 611 which represents the experimental performance data, and line 612 that represents the predicted performance data generated by the mechanistic model. [0176] FIG.12C includes a plot of laboratory scale experimentally determined performance data and predicted performance data generated by the mechanistic model for Lot 3 of the target molecule. For the chromatography runs and mechanistic model predictions shown in FIG.12C, the load volume was 120.38 mL and the load concentration was 12.66 g/L. Measured and predicted UV absorbances are plotted versus the volume passed through the column for the chromatography operation (in column volumes). The plot of FIG.12C includes line 621 which represents the experimental performance data, and line 622 that represents the predicted performance data generated by the mechanistic model. [0177] As seen in FIGs.12A, 12B, 12C, and 17 the mechanistic model overestimates peak heights due to assumptions regarding highly efficient flowthrough of the mobile phase. In some chromatography runs, a portion of the target molecule from the load is retained in the chromatography system (e.g., within the media), and is released in the wash step. This release of the target molecule during the wash step is evidenced by the shoulder at approximately 8 CVs in the experimentally determined chromatograms shown in FIGs.12A, 12B, 12C, and 17. As described herein, validation of the mechanistic model may include comparison of peak widths between the chromatogram of the experimentally generated performance data and the chromatogram of predicted performance data generated by the mechanistic model. Therefore, differences in the height of the elution peak between reduced scale experimentally determined chromatograms and mechanistic model predicted chromatograms may not be indicative of the fit of the model. [0178] Comparisons of the elution peaks widths between reduced scale experimentally determined chromatograms and mechanistic model predicted chromatograms are shown below in Table 6. The peak width delta is defined as the quotient of the absolute value of the difference between the experimental peak width and the predicted peak width divided by the experimental peak width.
Attorney Docket No.00166-0147-0304
[0179] The peak widths shown in Table 6 for the microscale chromatography operations are also shown graphically in the bar graphs of FIG.18A. The peak widths shown in Table 6 for the laboratory scale chromatography operations are also shown graphically in the bar graphs of FIG. 18B. In both FIGs.18A and 18B, the black bars represent experimentally determined peak widths and the white bars represent the predicted peak widths generated by the mechanistic model. As can be seen from the data in Table 6 and the plots in FIGs.18A and 18B, the model accurately predicts elution peak widths for microscale and laboratory scale chromatography operations. Therefore, the model captures the main mechanisms of the anion exchange chromatography operation. Example 10 [0180] As discussed above in Example 9, a portion of the load may remain bound to the column after an anion exchange chromatography operation. Pulse tests were conducted under pre-strip conditions and equilibration/load/wash, using a Q Sepharose fastflow column, to examine the retention of the target molecule within the chromatography system. Pre-strip conditions included passing two column volumes (approximately 7.85 mL) of a first solution including 2M sodium chloride through the column, followed by two column volumes (approximately 7.85 mL) of a second solution including 50 mM tris and 50 mM sodium acetate. Equilibration/load/wash conditions included first passing a pulse (approximately 0.02 column volumes) including the target molecule (e.g., a monoclonal antibody) through the column. After the pulse, equilibration/load/wash conditions included passing two column volumes (approximately 7.85 mL) of a solution including 50 mM tris and 50 mM sodium acetate through the column, followed by passing two column volumes (approximately 7.85 mL) of a solution including 2M sodium chloride through the column. [0181] Chromatograms generated from the pulses tests are shown in FIG.19, where the dashed line represents the chromatogram generated from the pre-strip conditions pulse test, and the solid line represents the chromatogram generated from the equilibration/load/wash conditions pulse test. The retention volume for the pre-strip conditions pulse test was 2.48 mL and the retention volume for the equilibration/load/wash conditions pulse test was 4.61 mL. The larger retention volume during equilibration/load/wash conditions compared to pre-strip conditions demonstrates that some target molecules bind to the media during flowthrough of the mobile phase. This retention of the target molecules is not accounted for in the anion exchange binding isotherm (e.g., an isotherm including
Attorney Docket No.00166-0147-0304 Equation 6), and can account for some peak height difference observed between the mechanistic model and experimental results. [0182] The present disclosure is further described by the following non-limiting items. [0183] Item 1. A method of developing a hydrophobic interaction chromatography mechanistic model, the method comprising: determining a total available porosity and a column porosity of a chromatography system including a column comprising hydrophobic interaction chromatography media; generating a generalized mechanistic model based on the total available porosity and the column porosity; determining mass transfer parameters of the mechanistic model; and determining binding isotherm parameters of the mechanistic model. [0184] Item 2. The method of item 1, wherein determining mass transfer parameters includes conducting a first blue dextran pulse test at a first flow rate, and a second blue dextran pulse test at a second flow rate. [0185] Item 3. The method of item 2, wherein determining mass transfer parameters includes regressing the mass transfer parameters using data generated from the first and second blue dextran pulse tests. [0186] Item 4. The method of item 3, wherein the generalized mechanistic model includes a binding isotherm term, and regressing the mass transfer parameters includes regressing the mass transfer parameters using data generated from the first and second blue dextran pulse tests, while setting the binding isotherm term of the generalized mechanistic model equal to zero. [0187] Item 5. The method of item 1, wherein determining binding isotherm parameters includes conducting a plurality of laboratory-scale chromatography runs. [0188] Item 6. The method of item 5, wherein determining binding isotherm parameters includes regressing the isotherm parameters based on data generated from the plurality of laboratory-scale chromatography runs. [0189] Item 7. The method of item 1, further comprising characterizing system hold-ups within the chromatography system. [0190] Item 8. The method of item 7, wherein characterizing system hold-ups within the chromatography system includes conducting a column-less acetone pulse test, a column-less salt pulse test, or both. [0191] Item 9. The method of item 8, wherein the generalized mechanistic model is based on the total available porosity, the column porosity, and a pulse test retention volume measured during the characterization of the system hold-ups within the chromatography system. [0192] Item 10. A method of developing an anion exchange chromatography mechanistic model, the method comprising:
Attorney Docket No.00166-0147-0304 characterizing system hold-ups within a chromatography system including a column comprising anion exchange chromatography media; determining a porosity of the chromatography system; generating a generalized mechanistic model based on the porosity, wherein the generalized mechanistic model comprises a binding isotherm including a term defined by Equation 7; determining mass transfer parameters of the mechanistic model; and determining binding isotherm parameters of the mechanistic model. [0193] Item 11. The method of item 10, wherein the generalized mechanistic model further comprises a term defined by Equation 5, a term defined by Equation 6, or both. [0194] Item 12. The method of item 10, wherein determining a porosity of the chromatography system includes determining a total available porosity and determining a column porosity. [0195] Item 13. The method of item 10, wherein characterizing system hold-ups within the chromatography system includes conducting a column-less acetone pulse test and a column-less salt pulse test. [0196] Item 14. The method of item 13, wherein determining the porosity includes conducting an unretained monoclonal antibody pulse test and a blue dextran pulse test. [0197] Item 15. The method of item 14, wherein generating the generalized mechanistic model based on the porosity includes generating the generalized mechanistic model based on retention volumes calculated from the column-less acetone pulse test, the column-less salt pulse test, the unretained monoclonal antibody pulse test, and the blue dextran pulse test. [0198] Item 16. The method of item 10, wherein determining isotherm parameters includes conducting a plurality of laboratory-scale chromatography runs in parallel, and regressing the isotherm parameters based on data generated from the plurality of microscale chromatography runs. [0199] Item 17. The method of item 16, wherein the laboratory-scale chromatography runs are each conducted using a column having a total column volume of less than or equal to approximately 1 mL. [0200] Item 18. A chromatography method comprising: generating a chromatography mechanistic model; conducting a plurality of microscale chromatography runs, thereby generating microscale data; using the chromatography mechanistic model, determining predicted manufacturing scale data based on the microscale data. [0201] Item 19. The method of item 18, wherein the chromatography mechanistic model describes a hydrophobic interaction exchange chromatography operation or an anion exchange chromatography operation. [0202] Item 20. The method of item 18, further comprising:
Attorney Docket No.00166-0147-0304 using the chromatography mechanistic model to generate a predicted laboratory scale elution peak width; conducting a laboratory scale chromatography run to generate an experimental laboratory scale elution peak width; using the chromatography mechanistic model to generate a predicted microscale elution peak width; conducting a microscale chromatography run to generate an experimental microscale elution peak width; and calculating a laboratory scale elution peak width delta and a microscale elution peak width delta. [0203] Item 21. The method of item 20, wherein the laboratory scale chromatography run is conducted using a first column having a total column volume of approximately 1 mL to approximately 20 mL, and the microscale chromatography run is conducted using a second column having a total column volume of less than or equal to approximately 1 mL. [0204] Those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be used as a basis for designing other methods and systems for carrying out the several purposes of the present disclosure. Accordingly, the claims are not to be considered as limited by the foregoing description.