Chromatographic media with multiple affinities and methods for the use and production thereof

Abstract

Chromatographic media for chromatographic purification of a target biomolecule includes a substrate material and copolymers grafted to the substrate material. The copolymers include: an aliphatic proportion of aliphatic hydrophobic monomers; an aromatic proportion of aromatic hydrophobic monomers; a hydrophilic proportion of neutral hydrophilic monomers; and at least one of a positive proportion of positively charged monomers and a negative proportion of negatively charged monomers. The copolymers exhibit: a hydrophobic-hydrophilic ratio of the aliphatic proportion and the aromatic proportion to the hydrophilic proportion, the hydrophobic -hydrophilic ratio selected based on a relative surface hydrophilicity of the target biomolecule and a relative surface hydrophobicity of the target biomolecule; and an aliphatic-aromatic ratio of the aliphatic proportion to the aromatic proportion, the aliphatic-aromatic ratio selected based on a relative surface aliphatic nature of the target biomolecule and a relative surface aromatic nature of the target biomolecule.

Description

CHROMATOGRAPHIC MEDIA WITH MULTIPLE AFFINITIES AND METHODS FOR THE USE AND PRODUCTION THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Provisional Application No. 63 / 582,423, filed on 13-SEP-2023, which is incorporated in its entirety by this reference.

[0002] This Application is related to U.S. Patent Application No. 18 / 036,678, filed on 12-MAY-2023, which is incorporated in its entirety by this reference.TECHNICAL FIELD

[0003] This invention relates generally to the field of target biomolecule production and purification via chromatography and more specifically to new and useful targeted chromatographic media and methods for the production and use thereof in the field of target biomolecule production and purification via chromatography.BRIEF DESCRIPTION OF THE FIGURES

[0004] FIGURE 1 is a schematic representation of one variation of the targeted chromatographic media;

[0005] FIGURE 2A is a flowchart representation of one variation of the production process for the targeted chromatographic media;

[0006] FIGURE 2B is a flowchart representation of another variation of the production process for the targeted chromatographic media; and

[0007] FIGURE 3 is a flowchart representation of one variation of a purification process utilizing the targeted chromatographic media.DESCRIPTION OF THE EMBODIMENTS

[0008] The following description of embodiments of the invention is not intended to limit the invention to these embodiments but rather to enable a person skilled in the art to make and use this invention. Variations, configurations, implementations, example implementations, and examples described herein are optional and are not exclusive to the variations, configurations, implementations, example implementations, and examples they describe. The invention described herein can include any and all permutations of these variations, configurations,implementations, example implementations, and examples. Additionally, the terms “ratio” and “proportion” as utilized herein refer to molar ratios and molar proportions unless otherwise specified. Furthermore, the adjective phrases “positive,” “positively charged,” and “cationic,” are interchangeable when referencing monomers or proportions herein. Likewise, the adjective phrases “negative,” “negatively charged,” and “anionic” are also interchangeable when references monomers or proportions herein.1. Biomolecule-Targeted Chromatographic Media

[0009] As shown in FIGURE 1, the targeted chromatographic media 100 includes a substrate material 110 and copolymers 120 grafted to the substrate material 110. The copolymer chains 120 include: an aliphatic proportion 130 of aliphatic hydrophobic monomers; an aromatic proportion 140 of aromatic hydrophobic monomers; a hydrophilic proportion 150 of neutral hydrophilic monomers; and at least one of a positive proportion 160 of positively charged monomers and a negative proportion 170 of negatively charged monomers. Additionally, the copolymers exhibit: a hydrophobic-hydrophilic ratio 180 of the aliphatic proportion 130 and the aromatic proportion 140 to the hydrophilic proportion 150, the hydrophobic-hydrophilic ratio 180 selected based on a relative surface hydrophilicity of the target biomolecule 200 and a relative surface hydrophobicity of the target biomolecule 200; and an aliphatic-aromatic ratio 190 of the aliphatic proportion 130 to the aromatic proportion 140, the aliphatic-aromatic ratio 190 selected based on a relative surface aliphatic nature of the target biomolecule 200 and a relative surface aromatic nature of the target biomolecule 200.2. Method for Targeted Chromatographic Media Production

[0010] As shown in FIGURE 2A, a method for producing chromatographic media 100 targeted for purification of a target biomolecule 200 (hereinafter “the production process SI 00) includes: selecting a hydrophobic-hydrophilic ratio 180 based on a relative surface hydrophilicity of the target biomolecule 200 and a relative surface hydrophobicity of the target biomolecule 200 in Step SI 10; and selecting an aliphatic-aromatic ratio 190 based on a relative surface aliphatic nature of the target biomolecule 200 and a relative surface aromatic nature of the target biomolecule 200 in Step SI 12. The production process SI 00 includes preparing a polymerization precursor mixture 102 including: an aliphatic proportion 130 of aliphatic hydrophobic monomers, an aromatic proportion 140 of aromatic hydrophobic monomers, a hydrophilic proportion 150 of neutral hydrophilic monomers, and at least one of a positive proportion 160 ofpositively charged monomers and a negative proportion 170 of negatively charged monomers; and exhibiting the hydrophobic -hydrophilic ratio 180 of the aliphatic proportion 130 and the aromatic proportion 140 to the proportion of the neutral hydrophilic monomers and the aliphatic-aromatic ratio 190 of the proportion of the aliphatic hydrophobic monomers to the proportion of the aromatic hydrophobic monomers in Step SI 20. The production process SI 00 additionally includes producing the chromatographic media 100 by: polymerizing the polymerization precursor mixture 102 to yield copolymer chains 120 comprising the aliphatic hydrophobic monomers, the aromatic hydrophobic monomers, the neutral hydrophilic monomers, the positive charged monomers, and the negative charged monomers in Step SI 30; and grafting the copolymer chains 120 to a substrate material 110 in Step SI 32.3. Method for Targeted Chromatographic Purification

[0011] As shown in FIGURE 3, a method for targeted chromatographic purification of a target biomolecule 200 (hereinafter “the purification process S200”) includes obtaining chromatographic media 100 targeted to the target biomolecule 200 comprising copolymer chains 120 grafted to a substrate material 110, the copolymer chains 120 comprising: an aliphatic proportion 130 of aliphatic hydrophobic monomers; an aromatic proportion 140 of aromatic hydrophobic monomers; a hydrophilic proportion 150 of neutral hydrophilic monomers; and at least one of a positive proportion 160 of positively charged monomers and a negative proportion 170 of negatively charged monomers in Step S210. The purification process S200 also includes: preparing a loading buffer 300 as an interpolation between a canonical hydrophobic interaction chromatography loading buffer 300 and a canonical ion-exchange chromatography loading buffer 300 based on the aliphatic proportion 130, the aromatic proportion 140, and at least one of the positive proportion 160 and the negative proportion 170 in Step S220; preparing a loading solution 310 comprising the loading buffer 300 and a quantity of the target biomolecule 200 in Step S230; preparing an elution buffer 400 as an interpolation between a canonical hydrophobic interaction chromatography elution buffer 400 and a canonical ion-exchange chromatography elution buffer 400 based on the aliphatic proportion 130, the aromatic proportion 140, and at least one of the positive proportion 160 and the negative proportion 170 in Step S240; exposing the loading solution 310 to the chromatographic media 100 causing selective binding of the quantity of the target biomolecule 200 to the chromatographic media 100 in Step S250; exposing the quantity of the target biomolecule 200 bound to the chromatographic media 100 to the elution buffer 400 causing elution of the quantity of the target biomolecule 200 from thechromatographic media 100, yielding an elution solution 410 in Step S260; and extracting the quantity of the target biomolecule 200 from the elution solution 410 in Step S270.4. Applications

[0012] Generally, the production process SI 00 is executed to generate biomolecule-targeted chromatographic media (hereinafter “the targeted chromatographic media 100”), which enables execution of the purification process S200. More specifically, the targeted chromatographic media 100 is produced according to the method SI 00 to exhibit strong affinity for a target biomolecule 200 (e.g., proteins, enzymes, nucleic acids, hormones) across multiple chromatographic modalities including at least hydrophobicity, aliphatic or aromatic nature, and charge. Thus, when utilized in the purification process S200, the targeted chromatographic media 100 can, in a single stationary phase, achieve higher yield and material efficiency than multi-phase, single-modality chromatographic purification, which relies on separate stationary phases for each modality of the target biomolecule 200 (e.g., sequential ion exchange and hydrophobic interaction phases).

[0013] In particular, the targeted chromatographic media 100 can consist of a polymer brush, including a set of copolymer chains 120 grafted to a substrate material 110. The copolymer chains 120 can include a set of functionalized monomers, each of which exhibits a predominant affinity interaction with the target biomolecule 200, such as an aliphatic hydrophobic monomer, an aromatic hydrophobic monomer, a hydrophilic monomer, a positively charged monomer (i.e., a cationic monomer or positive monomer), and a negatively charged monomer (i.e., an anionic monomer or negative monomer). The copolymer chains 120 include the set of functionalized monomers in proportions selected according to localized and / or bulk surface properties of the target biomolecule 200, such as the size, intensity, and aliphatic-aromatic nature of hydrophobic patches, the size and charge intensity of charged patches of the target biomolecule 200, and / or the net charge of the target biomolecule 200. Thus, the targeted chromatographic media 100 exhibits highly specific affinity for the target biomolecule 200 relative to other molecules in a loading solution 310 for chromatographic purification while the end- tethered conformation of the copolymer chains 120 to the substrate material 110.

[0014] Prior attempts at targeted polymer brush chemistry, such as in related U.S. Patent Application No. 18 / 036,678, utilized a smaller number of functional monomers focused particularly on the localized surface hydrophobicity of a target enzyme for the application of enzyme stabilization in harsh environments. In the enzyme stability application of a targetedpolymer brush, enzyme stability improvements were found based on the inclusion of hydrophobic monomers exhibiting a degree of amphiphilicity in the targeted polymer brush (potentially due to interactions proposed by Wyman Theory). However, the targeted chromatographic media 100 improves upon this prior disclosure with respect to selective affinity for a target biomolecule 200 (as opposed to stabilization of a target enzyme) via the inclusion of additional monomers functionalized according to aliphatic-aromatic nature and lacking amphiphilic characteristics. Additionally, in some implementations, the targeted chromatographic media 100 includes cationic monomers and anionic monomers leveraging charge-based interactions with the target biomolecule 200 to further increase the selectivity of the targeted chromatographic media 100 for the target biomolecule 200.

[0015] The purification process S200 includes the preparation and application of targeted loading buffers 300 and targeted elution buffers 400 to facilitate binding between the targeted biomolecule and the targeted chromatographic media 100 and elution from the targeted chromatographic media 100, respectively. The purification process S200 includes preparation of the compositions of the targeted loading buffer 300 and the target elution buffers 400 based on an interpolation between canonical buffers for hydrophobic interaction and ionic exchange chromatography that are effectively combined into a single phase according to the purification process S200. Thus, in addition to leveraging the targeted chromatographic media 100, the purification process S200 includes buffers specific to the properties of the target biomolecule 200 and the corresponding targeted chromatographic media 100.5. Biomolecule Analysis

[0016] As shown in FIGURE 2, the production process SI 00 includes a biomolecule analysis stage and a synthesis stage to calculate the proportions of the various functionalized monomers included in the copolymer chains 120 of the targeted chromatographic media 100 based on the surface properties of the target biomolecule 200. More specifically, the production process can include hydrophobic patch surface analysis of the target biomolecule 200, aliphatic-aromatic classification of identified hydrophobic patches of the target biomolecule 200, and electrostatic surface analysis of the target biomolecule 200, each of which informs the selection of the proportions of the functionalized monomers corresponding to the analysis. Thus, the production process SI 00 results in target chromatographic media characterized by increased affinity between the targeted chromatographic media 100 and the target biomolecule 200 by matching the distribution of monomers to the surface properties of the target biomolecule 200.

[0017] Generally, the analysis stage of the production process SI 00 can be performed by a computer system executing a combination of specialized applications. The computer system can include a single computational device or multiple computational devices communicating over a local- and / or wide-area network such as the internet. Some steps of the production process SI 00 involve analyzing complex molecular surface geometries for target biomolecule 200s, such as Steps SI 10, SI 12, and SI 14, which benefit greatly from computational execution and cannot be executed accurately within the human mind.

[0018] For example, the production process SI 00 can leverage a protein database, such as the Protein Data Bank, to obtain accurate geometries for the target biomolecule 200. The production process SI 00 leverages the surface geometry of the target biomolecule 200 to calculate the total solvent accessible surface area (hereinafter “SASA”) of the target biomolecule 200. In Steps SI 10, SI 12, and SI 14, the production process can classify “patches” or “regions” of the SASA of the target biomolecule 200 that exhibit characteristics corresponding to the functionalized monomers, such as hydrophobicity and hydrophilicity, aliphatic and aromatic natures, and / or positive and negative charges. The production process SI 00 can include calculating the relative contributions of the above-described surface patches based at least in part on the proportion of the SASA occupied by a set of patches exhibiting similar characteristics in addition to the intensity of these characteristics, which can be calculated theoretically or empirically. The calculation of SASA is described in detail in U.S. Patent Application No. 18 / 036,678.5.1 Hydrophobic Patch Surface Analysis

[0019] Generally, the production process SI 00 includes hydrophobic patch surface analysis to characterize the surface hydrophobicity of the target biomolecule 200 and adjust the proportion of hydrophobic monomers relative to hydrophilic monomers in the polymerization precursor mixture 102. More specifically, the production process can include: selecting a hydrophobic -hydrophilic ratio 180 based on a relative surface hydrophilicity of the target biomolecule 200 and a relative surface hydrophobicity of the target biomolecule 200 in Step SI 10. Additionally, the production process S100 can include: calculating the relative surface hydrophilicity of the target biomolecule 200 and the relative surface hydrophobicity of the target biomolecule 200 by calculating the free energy of solvation of each atom on the SASA of the target biomolecule 200; identifying a set of hydrophobic patches characterized by greater than a threshold free energy of solvation (e.g., a free energy of solvation greater than zero); and selecting the proportion of hydrophobic monomers and the proportion of hydrophilic monomersbased on the free energies of solvation of the set of hydrophobic patches. Thus, the production process SI 00 leverages the surface properties of the target biomolecule 200 on an atomic scale to identify the hydrophobic and hydrophilic properties of the target biomolecule 200.

[0020] In one implementation, the production process SI 00 executes machine-leaming-based clustering algorithms to identify hydrophobic patches on the SASA of the target biomolecule 200 and select the hydrophobic-hydrophilic ratio 180 based on the identified hydrophobic patches. For example, the production process SI 00 can execute Density-Based Spatial Clustering and Noise (hereinafter “DBSCAN”) to differentiate hydrophobic atoms on the SASA of the target biomolecule 200 that contribute to a hydrophobic patch from hydrophobic atoms on the SASA of the target biomolecule 200 that can be classified as noise in view of the properties of neighboring atoms. Thus, the production process SI 00 can accurately identify surface regions of the target biomolecule 200 that contribute to hydrophobic versus hydrophilic affinity.

[0021] In another implementation, the production process SI 00 includes calculating a hydrophobic intensity for each hydrophobic patch on the SASA of the target biomolecule 200 and selecting the hydrophobic-hydrophilic ratio 180 based on the hydrophobic intensities of the set of hydrophobic patches. In this implementation, the production process can include calculating the hydrophobic intensity of each hydrophobic patch based on the maximum free energy of solvation of the hydrophobic patch and / or the total free energy of solvation of the hydrophobic patch.

[0022] In yet another implementation, the production process SI 00 includes selecting the hydrophobic -hydrophilic ratio 180 based on the free energy of solvation per SASA of the target biomolecule 200. In this implementation, the production process SI 00 can include accessing a mapping between the free energy of solvation per SASA of the target biomolecule 200 and hydrophobic -hydrophilic ratios and selecting the hydrophobic-hydrophilic ratio 180 based on the mapping. Thus, in this implementation, the production process SI 00 considers the bulk hydrophobicity / hydrophilicity of the surface of the target biomolecule 200 as opposed to a patch-based approach.

[0023] In each of the above-described implementations, the production process SI 00 can include calculating the proportion of hydrophobic monomers and the proportion of hydrophilic monomers based on the hydrophobic -hydrophilic ratio 180 according to basic arithmetic. Generally, the proportion of aromatic monomers and the proportion of aliphatic monomers contribute or sum to the proportion of hydrophobic monomers, while the proportion ofhydrophilic monomers can also include the proportion of anionic monomers and the proportion of cationic monomers for the purposes of the hydrophobic-hydrophilic ratio 180.5.2 Aliphatic-Aromatic Classification

[0024] Generally, the production process SI 00 includes classification of hydrophobic patches of the SASA of the target molecule according to the aromatic nature or aliphatic nature of each hydrophobic patch. More specifically, the production process SI 00 includes selecting an aliphatic-aromatic ratio 190 based on a relative surface aliphatic nature of the target biomolecule 200 and a relative surface aromatic nature of the target biomolecule 200 in Step SI 12. In particular, the production process SI 00 can include: classifying each hydrophobic patch identified on the SASA of the target biomolecule 200 in a binary manner as either aliphatic or aromatic; calculating a free energy of solvation of each classified hydrophobic patch; and selecting the aliphatic-aromatic ratio 190 based on a ratio of total free energy of solvation per SASA for the aliphatic hydrophobic patches to total free energy of solvation per SASA for the aromatic hydrophobic patches. Thus, the production process SI 00 can adjust the aliphatic-aromatic ratio 190 according to the relative contributions of the aromatic and aliphatic hydrophobic patches to the overall hydrophobicity of the target biomolecule 200.

[0025] In one implementation, the production process can additionally apply an aromatic factor (e.g., empirically calculated) to the total free energy of solvation per SASA of the aromatic hydrophobic patches to account for the higher intensity of aromatic hydrophobic interactions per unit solvation energy per SASA.5.3 Charged Patch Surface Analysis

[0026] Generally, the production process SI 00 can include identifying cationic (positively charged) patches and anionic (negatively charged) patches on the SASA of the target biomolecule 200. More specifically, the production process SI 00 can include selecting a charge ratio based on: a target pH for chromatographic purification of the target biomolecule 200; a negative surface charge area of the target biomolecule 200 at the target pH; a negative surface charge intensity of the target biomolecule 200 at the target pH; a positive surface charge area of the target biomolecule 200 at the target pH; and a positive surface charge intensity of the target biomolecule 200 at the target pH in Step SI 14. In particular, the production process SI 00 can include: simulating (or empirically identifying) an electrostatic map of the target biomolecule 200; calculating a threshold potential of the target biomolecule 200; identifying a positive SASAof the target biomolecule 200 exhibiting a charge above the threshold potential; identifying a negative SASA of the target biomolecule 200 exhibiting a charge below the threshold potential; calculating an intensity of the positive SASA; calculating an intensity of the negative SASA; and selecting the charge ratio based on the positive SASA, the intensity of the positive SASA, the negative SASA, and the intensity of the negative SASA.

[0027] In one implementation, the production process SI 00 can include simulating an electrostatic state of the target biomolecule 200 at the target pH for chromatographic purification of the target biomolecule 200 by simulating the presence or absence of protons on the SASA of the target biomolecule 200. Additionally, the production process SI 00 can include simulating conformational changes of the biomolecule based on the target pH. Thus, the production process SI 00 considers the binding conditions of the chromatographic process when selecting the charge ratio.

[0028] In addition to calculating the charge ratio, the production process SI 00 can also calculate the total proportion of charged monomers relative to the neutral hydrophilic monomer. In one implementation, the production process SI 00 can include calculating the proportion of positive monomers based on the proportion of the SASA of the target biomolecule 200 occupied by positively charged regions weighted by the intensity of the charge within those regions. Likewise, the production process SI 00 can include calculating the proportion of negative monomers based on the proportion of the SASA of the target biomolecule 200 occupied by negatively charged regions weighted by the intensity of the charge within those regions. Thus, in this implementation, the production process SI 00 calculates proportions of both positively charged monomers and negatively charged monomers, which results in a counterintuitive increase in selectivity for the target biomolecule 200 based on matching the charge ratio of the proportion of positive monomers to the proportion of negative monomers to the specific electrostatic distribution on the SASA of the target biomolecule 200.

[0029] In another implementation, the production process SI 00 can select either a positive monomer or a negative monomer opposite the net charge of the SASA of the target biomolecule 200. In this implementation, the production process SI 00 can disregard local charge distribution on the SASA of the target biomolecule 200 in favor of the net charge exhibited across the entire surface. In one example of this implementation, the production process SI 00 can select between targeted chromatographic media 100 including both positive and negative monomers or a monomers characterized by either a positive or a negative charge based on the sizes and distribution of charged regions on the SASA of the target biomolecule 200. The productionprocess SI 00 can select the single-charged monomer approach in response to a noisy charge distribution exhibiting few (e.g., less than a threshold number of) distinct charged patches on the SASA of the biomolecule as measured by a patch detection algorithm similar to those described above with respect to hydrophobic patches.

[0030] Upon calculating the proportion of negatively charged monomers and a proportion of positively charged monomers, the production process SI 00 can include calculating a proportion of neutral hydrophilic monomers by subtracting the proportion of negatively charged monomers and the proportion of positively charged monomers from the proportion of hydrophilic monomers. Thus, the production process SI 00 treats the charged monomers as contributing hydrophilic interactions.6. Targeted Chromatographic Media Synthesis

[0031] Generally, the production process SI 00 includes a synthesis stage during which the monomeric proportions calculated during the analysis stage are utilized in preparation of a polymerization precursor mixture 102, which is grafted onto a substrate material 110 to form the targeted chromatographic media 100 that is characterized by a polymer brush conformation. More specifically, one variation of the synthesis stage utilizes a “grafted from” approach where copolymer chains 120 are grown directly from the substrate material 110. Alternatively, a second variation of the synthesis stage utilizes a “grafted to” approach where copolymer chains 120 are grown prior to introduction to the substrate material 110, at which point the copolymer chains 120 attach to the substrate material 110. Each of these variations is described in further detail below.6.1 Polymerization Precursor Mixture Preparation

[0032] Generally, the production process SI 00 includes preparing a polymerization precursor mixture 102, including the constituent functionalized monomers of the targeted chromatographic media 100 in solution. More specifically, the production process SI 00 includes preparing a polymerization precursor mixture 102 including an aliphatic proportion 130 of aliphatic hydrophobic monomers, an aromatic proportion 140 of aromatic hydrophobic monomers, a hydrophilic proportion 150 of hydrophilic monomers, and at least one of a positive proportion 160 of positively charged monomers and a negative proportion 170 of negatively charged monomers. Additionally, the production process can include preparing a polymerization precursor mixture 102 containing crosslinking agents (e.g., an amine-reactive crosslinking agentsuch as methacrylic acid N-hydroxysuccinimide ester) or other solutes that promote binding between the functionalized monomers and / or between the functionalized monomers and the substrate material 110. Furthermore, the polymerization precursor mixture 102 includes a solvent sufficient to dissolve the aforementioned constituents of the polymerization precursor mixture 102.

[0033] In one implementation, the polymerization precursor mixture 102 includes functionalized monomers configured to form a methacrylate copolymer with a linear conformation anchored at one end to the substrate material 110. In this implementation, the aliphatic hydrophobic monomers can include isopropyl methacrylate, the aromatic hydrophobic monomers can include ethylene glycol phenyl ether methacrylate, the (neutral) hydrophilic monomers can include zwitterionic 2-methacryloyloxyethyl phosphorylcholine, the positively charged monomers can include cationic [3 -(methacryloylamino)propyl] trimethylammonium chloride, and the negatively charged monomers can include anionic 3-sulfopropyl methacrylate. Generally, the inclusion of a zwitterionic hydrophilic monomers as the hydrophilic monomers aids in resisting non-specific adsorption of molecules other than the target biomolecule 200 by the targeted chromatographic media 100 based on the strong affinity of the zwitterionic monomers to the mobile phase without contributing to net charge.6.2 Substrate Material

[0034] Generally, the production process S100 can include obtaining a substrate material 110 to which the functionalized monomers can bind to form the targeted chromatographic media 100 with a polymer brush conformation. More specifically, the production process SI 00 can include obtaining a substrate material 110 commonly employed as a stationary phase in chromatography, such as silica microspheres, silicon-based polymers, silica gel, cellulose, agarose, dextran, polyacrylamide, or any other similar stationary phase material.6.3 “Grafted From” Variation

[0035] As shown in FIGURE 2A, the production process S100 can include a “grafted from” approach wherein copolymer chains 120, including the functionalized monomers, are grown from the surface of the substrate material 110. More specifically, the production process S100 can include: binding polymerization initiators to the surface of the substrate material 110; exposing the substrate material 110 to the polymerization precursor mixture 102; and catalyzing a reaction between the functionalized monomers contained by the polymerization precursormixture 102, causing assembly of copolymers from the surface of the substrate material 110 in Step S132. Thus, in this variation of the production process S100, formation of the targeted chromatographic media 100 is accomplished in a single- step reaction.6.4 “Grafted To” Variation

[0036] As shown in FIGURE 2B, the production process S100 can include a “grafted to” approach wherein the copolymer chains 120, including the functionalized monomers, are grown separately from the substrate material 110 before being grafted to the substrate material 110 in a subsequent step. More specifically, the production process S100 can include: synthesizing copolymers from the functionalized monomers in solution; and exposing the copolymers to the substrate material 110, causing attachment of the copolymers to the substrate material 110 in Step S134. In one implementation, the production process S100 causes non-covalent attachment of the copolymers to the substrate material 110. Alternatively, the production process S100 causes covalent attachment between reactive groups on the copolymers and anchor groups on the substrate material 110.7. Targeted Chromatographic Media Composition

[0037] Generally, the production process S100 results in a targeted chromatographic media 100 that exhibits selective affinity for the target biomolecule 200 over other dissolved molecules within a loading solution 310. As described above, the targeted chromatographic media 100 includes an aliphatic proportion 130, an aromatic proportion 140, a hydrophilic proportion 150, and at least one of a cationic (positive) proportion and an anionic (negative) proportion all attached to a substrate material 110 and exhibiting the selected hydrophobic-hydrophilic ratio 180, the aliphatic-aromatic ratio 190, and, optionally, the charge ratio. Due to the stochastic nature of the polymerization and substrate attachment interactions of the production process S100, the precise ratios and proportions of the targeted chromatographic media 100 may vary from nominal proportions and ratios or those prepared within the polymerization precursor mixture 102 within a distribution which may be experimentally determined.8. Purification Process

[0038] Upon production of the targeted chromatographic media 100, the purification process S200 employs the targeted chromatographic media 100 in a chromatographic purification process that combines hydrophobic interaction and ion exchange chromatography into a single- stepprocess. In addition to the targeted chromatographic media 100, the purification process S200 leverages loading buffers 300 and elution buffers 400 with compositions selected based on the composition of the targeted chromatographic media 100. The preparation of the loading buffer 300 and elution buffer 400 is further described below.8.1 Loading Buffer Preparation

[0039] Generally, the purification process S200 includes preparing a loading buffer 300 that increases the affinity of the target biomolecule 200 for the targeted chromatographic media 100. More specifically, the purification process S200 can include: preparing a loading buffer 300 as an interpolation between a canonical hydrophobic interaction chromatography loading buffer 300 and a canonical ion-exchange chromatography loading buffer 300 based on the aliphatic proportion 130, the aromatic proportion 140, and at least one of the positive proportion 160 and the negative proportion 170. Thus, because the purification process S200 effectively combines at least two forms of chromatography, the purification process S200 incorporates elements from each type of chromatography to maximize the combined affinity between the target biomolecule 200 and the targeted chromatographic media 100.

[0040] In one example, the purification process S200 includes preparation of a loading buffer 300 as an interpolation of: a canonical hydrophobic interaction chromatography loading buffer 300, including 100 millimolar sodium phosphate 1 molar sodium chloride, and exhibiting a pH of 8.2; and a canonical ion-exchange chromatography loading buffer 300, including 10 millimolar sodium phosphate, lo millimolar magnesium chloride, and 10 millimolar sodium sulfate. In this example, the purification process S200 can include preparing a loading buffer 300 as an interpretation based on the nominal proportion of hydrophobic monomers relative to the nominal proportion of charged monomers of the targeted chromatographic media 100. For example, for a targeted chromatographic media 100 characterized by a 1:1 ratio of hydrophobic monomers to charged monomers, the production process S200 includes preparing a loading buffer 300, including concentrations halfway between the two canonical solutions. However, in other examples and example implementation, the purification process S200 can include preparing a loading buffer 300 based on a non-linear interpolation function.

[0041] In one implementation, the purification process S200 includes: preparing a loading buffer 300 as a combination of a buffer salt contributing to ionic strength and maintaining stable pH (sodium phosphate), a divalent salt that includes a divalent anion or cation depending on the desired charge interaction (magnesium chloride or sodium sulfate), a monovalent salt thatimproves hydrophobic interactions (sodium chloride). In this implementation, the purification process S200 can include selecting the concentration of the divalent salt based on the anionic proportion and / or the cationic proportion of the targeted chromatographic media 100. Additionally, in this implementation, the purification process S200 can include selecting the concentration of the monovalent salt based on the proportion of hydrophobic monomers (i.e., the aliphatic proportion 130 plus the aromatic proportion 140).

[0042] Upon preparation of the loading buffer 300, the purification process S200 also includes preparing a loading solution 310 comprising the loading buffer 300 and a quantity of the target biomolecule 200, usually in the form of a cellular lysate or cell culture medium resulting from synthesis of the biomolecule.8.2 Elution Buffer Preparation

[0043] Generally, the purification process S200 includes preparing an elution buffer 400 that causes elution of the target biomolecule 200 from the targeted chromatographic media 100. More specifically, the purification process S200 can include preparing an elution buffer 400 as an interpolation between a canonical hydrophobic interaction chromatography elution buffer 400 and a canonical ion-exchange chromatography elution buffer 400 based on the aliphatic proportion 130, the aromatic proportion 140, and at least one of the positive proportion 160 and the negative proportion 170. Thus, like the preparation of the loading buffer 300, the purification process S200 includes preparing an elution buffer 400 that combines ideal properties from hydrophobic interaction chromatography and ion-exchange chromatography.

[0044] In one implementation, the purification process S200 includes: preparing an elution buffer 400 as a combination of a buffer salt contributing to ionic strength and maintaining stable pH (sodium phosphate), a divalent salt that includes a divalent anion or cation depending on the desired charge interaction (magnesium chloride or sodium sulfate), a monovalent salt (sodium chloride). In this implementation, the purification process S200 can include selecting the concentration of the divalent salt based on the anionic proportion and / or the cationic proportion of the targeted chromatographic media 100. Additionally, in this implementation, the purification process S200 can include selecting the concentration of the monovalent salt based on the proportion of hydrophobic monomers (i.e., the aliphatic proportion 130 plus the aromatic proportion 140). Additionally or alternatively, the purification process S200 can include competitive binders to improve elution from the targeted chromatographic media 100.Furthermore, the purification process S200 can include a low concentration of acetonitrile to disrupt hydrophobic interaction for solvent- tolerant target biomolecule 200s.8.3 Chromatography Process

[0045] Generally, the purification process S200 can utilize any stationary phase liquid chromatography apparatus to expose the targeted chromatographic media 100 to the loading buffer 300 and, subsequently, the elution buffer 400, thereby yielding a purified quantity of the target biomolecule 200. More specifically, the purification process S200 can utilize a chromatography column for liquid chromatography. Alternatively, the purification process S200 can include mixing the targeted chromatographic media 100 in the form of a loose resin into the loading solution 310 and agitating the mixture. However, the purification process S200 can utilize any apparatus for liquid chromatography for use with a stationary phase.

[0046] The purification process S200 can include exposing the loading solution 310 to the targeted chromatographic media 100, causing selective binding of the quantity of the target biomolecule 200 to the chromatographic media via a selected chromatography apparatus according to standard practice for the selected chromatography apparatus. Upon completion of the binding process, the purification process S200 can include rinsing the targeted chromatographic media 100 and bound quantity of the target biomolecule 200 to remove any remaining loading solution 310. Subsequently, the purification process S200 can include exposing the quantity of the target biomolecule 200 bound to the targeted chromatographic media 100 to the elution buffer 400, causing elution of the quantity of the targeted biomolecule from the chromatographic media, yielding an elution solution 410.

[0047] The purification process S200 can include extraction of the target biomolecule 200 from the elution solution 410 according to means appropriate for the specific target biomolecule 200 and application. Thus, the purification process S200 includes fewer steps, thereby resulting in increased material efficiency and improved purity and yield of the target biomolecule 200.

[0048] The methods described herein can be embodied and / or implemented at least in part as a machine configured to receive a computer-readable medium storing computer-readable instructions. The instructions can be executed by computer-executable components integrated with the application, applet, host, server, network, website, communication service, communication interface, hardware / firmware / software elements of a user computer or mobile device, wristband, smartphone, or any suitable combination thereof. Other systems and methods of the embodiment can be embodied and / or implemented at least in part as a machine configuredto receive a computer-readable medium storing computer-readable instructions. The instructions can be executed by computer-executable components integrated by computer-executable components integrated with apparatuses and networks of the type described above. The computer-readable medium can be stored on any suitable computer-readable media such as RAMs, ROMs, flash memory, EEPROMs, optical devices (CD or DVD), hard drives, floppy drives, or any suitable device. The computer-executable component can be a processor but any suitable dedicated hardware device can (alternatively or additionally) execute the instructions.

[0049] As a person skilled in the art will recognize from the previous detailed description and from the figures and claims, modifications and changes can be made to the embodiments of the invention without departing from the scope of this invention as defined in the following claims.

Claims

CLAIMS aim:

1. Chromatographic media for chromatographic purification of a target biomolecule comprising:• a substrate material; and• copolymers grafted to the substrate material, the copolymers: o comprising:■ an aliphatic proportion of aliphatic hydrophobic monomers;■ an aromatic proportion of aromatic hydrophobic monomers;■ a hydrophilic proportion of neutral hydrophilic monomers; and■ at least one of a positive proportion of positively charged monomers and a negative proportion of negatively charged monomers; and o exhibiting:■ a hydrophobic-hydrophilic ratio of the aliphatic proportion and the aromatic proportion to the hydrophilic proportion, the hydrophobic -hydrophilic ratio selected based on a relative surface hydrophilicity of the target biomolecule and a relative surface hydrophobicity of the target biomolecule; and■ an aliphatic-aromatic ratio of the aliphatic proportion to the aromatic proportion, the aliphatic-aromatic ratio selected based on a relative surface aliphatic nature of the target biomolecule and a relative surface aromatic nature of the target biomolecule.

2. The chromatographic media of Claim 1, wherein the copolymers:• further comprise: o the negative proportion; and o the positive proportion; and• further exhibit a charge ratio of the positive proportion to the negative proportion, the charge ratio selected based on a target pH for the chromatographic purification of the target biomolecule, a negative surface charge area of the target biomolecule at the target pH, a negative surface charge intensity of the target biomolecule at the target pH, apositive surface charge area of the target biomolecule at the target pH, and a positive surface charge intensity of the target biomolecule at the target pH.

3. The chromatographic media of Claim 1, wherein the copolymers further comprise one of the negative proportion or the positive proportion, opposite in charge to a net charge of the target biomolecule, and exhibiting a magnitude based on a net surface charge intensity of the target biomolecule at a target pH for the chromatographic purification of the target biomolecule.

4. The chromatographic media of Claim 1, wherein the copolymers define the aliphatic-aromatic ratio based on:• a solvent-accessible-surface-area-normalized free energy of solvation of aliphatic hydrophobic patches of the target biomolecule; and• solvent-accessible-surface-area-normalized free energy of solvation of aromatic hydrophobic patches of the target biomolecule.

5. The chromatographic media of Claim 1, wherein the copolymers define the hydrophobic -hydrophilic ratio based on:• a solvent-accessible-surface-area-normalized free energy of solvation of hydrophilic regions of the target biomolecule; and• a solvent-accessible-surface-area-normalized free energy of solvation of hydrophobic patches of the target biomolecule.

6. A method for producing chromatographic media targeted for purification of a target biomolecule comprising:• selecting a hydrophobic-hydrophilic ratio based on: o a relative surface hydrophilicity of the target biomolecule; and o a relative surface hydrophobicity of the target biomolecule;• selecting an aliphatic-aromatic ratio based on: o a relative surface aliphatic nature of the target biomolecule; and o a relative surface aromatic nature of the target biomolecule;• preparing a polymerization precursor mixture:o comprising:■ an aliphatic proportion of aliphatic hydrophobic monomers;■ an aromatic proportion of aromatic hydrophobic monomers;■ a hydrophilic proportion of neutral hydrophilic monomers; and■ at least one of a positive proportion of positively charged monomers and a negative proportion of negatively charged monomers; o exhibiting:■ the hydrophobic-hydrophilic ratio of the aliphatic proportion and the aromatic proportion to the hydrophilic proportion; and■ the aliphatic-aromatic ratio of the aliphatic proportion to the aromatic proportion; and• producing the chromatographic media by: o polymerizing the polymerization precursor mixture to yield copolymer chains comprising the aliphatic hydrophobic monomers, the aromatic hydrophobic monomers, the neutral hydrophilic monomers, the positive charged monomers, and the negative charged monomers; and o grafting the copolymer chains to a substrate material.

7. The method of Claim 6:• further comprising selecting a charge ratio based on: o a target pH for chromatographic purification of the target biomolecule; o a negative surface charge area of the target biomolecule at the target pH; o a negative surface charge intensity of the target biomolecule at the target pH; o a positive surface charge area of the target biomolecule at the target pH; and o a positive surface charge intensity of the target biomolecule at the target pH; and• wherein preparing the polymerization precursor mixture comprises preparing the polymerization precursor mixture exhibiting the charge ratio of the positive proportion to the negative proportion.

8. A method for targeted chromatographic purification of a target biomolecule comprising:• obtaining chromatographic media targeted to the target biomolecule comprising copolymer chains grafted to a substrate material, the copolymer chains comprising:o an aliphatic proportion of aliphatic hydrophobic monomers; o an aromatic proportion of aromatic hydrophobic monomers; o a hydrophilic proportion of neutral hydrophilic monomers; and o at least one of a positive proportion of positively charged monomers and a negative proportion of negatively charged monomers;• preparing a loading buffer as an interpolation between a canonical hydrophobic interaction chromatography loading buffer and a canonical ion-exchange chromatography loading buffer based on the aliphatic proportion, the aromatic proportion, and at least one of the positive proportion and the negative proportion;• preparing a loading solution comprising the loading buffer and a quantity of the target biomolecule;• preparing an elution buffer as an interpolation between a canonical hydrophobic interaction chromatography elution buffer and a canonical ion-exchange chromatography elution buffer based on the aliphatic proportion, the aromatic proportion, and at least one of the positive proportion and the negative proportion;• exposing the loading solution to the chromatographic media, causing selective binding of the quantity of the target biomolecule to the chromatographic media;• exposing the quantity of the target biomolecule bound to the chromatographic media to the elution buffer, causing elution of the quantity of the target biomolecule from the chromatographic media, yielding an elution solution; and• extracting the quantity of the target biomolecule from the elution solution.

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