阅读说明：本技术 生产经蚀刻的无孔颗粒的方法 (Method for producing etched non-porous particles ) 是由 魏大程 于 2021-06-07 设计创作，主要内容包括：本文提供了生产经蚀刻的无孔颗粒的方法。在一些实施例中,所述方法包括用亲水性聚合物涂覆无孔颗粒并且用酸或碱处理所述经涂覆的颗粒。还提供了经蚀刻的无孔颗粒,所述经蚀刻的无孔颗粒能够分离多种分析物,包括生物分子。(Provided herein are methods of producing etched non-porous particles. In some embodiments, the method comprises coating non-porous particles with a hydrophilic polymer and treating the coated particles with an acid or a base. Etched non-porous particles capable of separating a plurality of analytes, including biomolecules, are also provided.)

1. A method of producing etched non-porous particles, the method comprising:

providing a plurality of non-porous particles at least partially coated with at least one of a hydrophilic polymer, an inorganic metal oxide, a hybrid metal oxide, or a magnetic material; and

contacting the plurality of non-porous particles with an acid or a base to produce a plurality of etched non-porous particles.

2. The method of claim 1, wherein the plurality of non-porous particles are made of one of a polymeric material, an inorganic metal oxide, a hybrid metal oxide, or a magnetic material; and wherein when the non-porous particles are made of a polymeric material then the coating is one of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material, and when the non-porous particles are made of one of the inorganic metal oxide, the hybrid metal oxide, or the magnetic material then the coating is the hydrophilic polymer.

3. The method of claim 2, wherein the non-porous particles are made of a polymeric material at least partially coated with at least one of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material, further at least partially coated with the hydrophilic polymer to produce coated non-porous particles.

4. The method of claim 3, wherein the coated non-porous particles are contacted with the acid or base to produce the plurality of etched non-porous particles.

5. The method of claim 4, wherein the plurality of etched non-porous particles are washed to remove the hydrophilic polymer.

6. The method of claim 2, wherein the inorganic metal oxide or hybrid metal oxide or magnetic material with the hydrophilic polymer coating particles is contacted with an acid or a base to produce the plurality of etched non-porous particles.

7. The method of claim 6, wherein the plurality of etched non-porous particles are washed to remove the hydrophilic polymer.

8. The method of claim 1, wherein the hydrophilic polymer comprises at least one of: poly (N-isopropylacrylamide) (PNIPAM), Polyacrylamide (PAM), poly (acrylic acid), polymethacrylate, poly (ethylene glycol), poly (ethylene oxide), poly (2-oxazoline), and Polyethyleneimine (PEI), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), or a combination thereof.

9. A method of producing etched metal oxide particles, the method comprising:

providing a plurality of inorganic or hybrid metal oxide particles;

optionally coating the inorganic or hybrid metal oxide particles with at least one hydrophilic polymer; and

contacting the plurality of inorganic or hybrid metal oxide particles with an acid or base to produce a plurality of etched metal oxide particles.

10. A particle, comprising:

comprising non-porous inorganic metal oxide or hybrid metal oxide particles etched into the surface,

wherein the particles have from about 0.001 to about 0.1cm³Pore volume per gram, from about 2 to about 100m²Surface area per gram, or combinations thereof.

Background

Since the mid 1980 s, non-porous stationary phases have attracted interest due to their ability to rapidly separate proteins in High Performance Liquid Chromatography (HPLC). An advantage of non-porous particles is that there is no intra-particle diffusion resistance due to the lack of any pore structure, which results in rapid protein separation. However, the retention time of the stationary phase using such particles is short and the sample loading capacity is significantly lower when compared to porous particles. Most non-porous particles are also made of silica, which limits their use under high pH conditions. Thus, there is a need for high pH stable non-porous particles with increased loading capacity and longer retention times.

The present disclosure provides a solution to this problem by synthesizing non-porous inorganic/organic particles with etched rough surfaces. Such particles have a much higher surface area than typical non-porous particles and can increase sample loading capacity and peak capacity. The methods described herein can be performed without the need for particle size classification and are easier and shorter than current methods due to the manufacture of porous particles. In addition, the hybrid chemistry of the particles provides improved high pH stability compared to silica.

Disclosure of Invention

In one aspect, provided herein is a method of producing etched non-porous particles. The method includes providing a plurality of non-porous particles at least partially coated with at least one of a hydrophilic polymer, an inorganic metal oxide, a hybrid metal oxide, or a magnetic material; and contacting the plurality of non-porous particles with an acid or a base to produce a plurality of etched non-porous particles.

In another aspect, provided herein is a method of producing etched metal oxide particles. The method comprises providing a plurality of inorganic or hybrid metal oxide particles, optionally coating the inorganic or hybrid metal oxide particles with at least one hydrophilic polymer, and contacting the plurality of inorganic or hybrid metal oxide particles with an acid or a base to produce a plurality of etched metal oxide particles.

In a further aspect, a non-porous inorganic metal oxide or hybrid metal oxide particle is provided. The particles include an etched surface and have from about 0.001 to about 0.1cm³Pore volume per gram, from about 2 to about 100m²Surface area per gram, or combinations thereof.

Advantageously, in some embodiments, the method provides etched particles having a longer retention time than non-porous particles, similar retention time compared to wide-pore, surface-porous particles and comparable retention time to fully porous, wide-pore particles. Advantageously, in some embodiments, the method provides etched particles that are not susceptible to limited diffusion of large biomolecules, such as proteins and antibody fragments.

Additional features and advantages of various embodiments will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description herein.

Drawings

The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application. FIGS. 1A-1B show chromatograms for protein separation using a non-porous etched silica core column according to one embodiment (FIG. 1A) and a commercially available porous particle column (Agilent advanced Bio RP-mAb, FIG. 1B). Chromatographic conditions are as follows: the temperature is 60 ℃; the flow rate is 0.3 mL/min; the injection volume was 2.0 μ L; gradient 20% -56% B over 9 min; the MW range of the samples was 12-240 kDa. Samples tested: ribonuclease A (13.5kDa), cytochrome C (12.3kDa), holotransferrin (76.5kDa), alpha-lactalbumin (14.2kDa), catalase (240kDa) and carbonic anhydrase (30 kDa). In some embodiments, for the gradient, mobile phase a is 0.1% TFA in water and mobile phase B is 0.08% TFA in acetonitrile. The chromatogram is under a gradient of flow B from 20% to 56% in 9 min.

Fig. 2A-2B show the particle size distributions for example batch 1 (fig. 2A) and example batch 2 (fig. 2B). In one embodiment, batch 1 has an average particle size of 1.6 μm and d₉₀/d₁₀Etched particles of 1.17. In one embodiment, batch 2 has an average particle size of 2.7 μm and d₉₀/d₁₀Etched particles of 1.14.

Figures 3A-3B show chromatograms of protein separations from a non-porous silica core column (figure 3A) and an etched hybrid core column according to one embodiment (figure 3B). Samples tested: ribonuclease A (13.5kDa), cytochrome C (12.3kDa), holotransferrin (76.5kDa), alpha-lactalbumin (14.2kDa), catalase (240kDa) and carbonic anhydrase (30 kDa). For protein separation, the etched hybrid particles (3A) showed better separation (narrower peak width and higher peak capacity) and longer retention time than the non-porous particles (3B).

Fig. 4A-4B show chromatograms of peptide separation from a non-porous silica core column (fig. 4A) and an etched hybrid core column (fig. 4B) according to one embodiment. Chromatographic conditions are as follows: the temperature is 60 ℃; the flow rate is 0.3 mL/min; the injection volume was 2.0 μ L; gradient 15% -65% B over 12.5 min; the MW range of the samples was 0.7-2.8kDa (bradykinin fragment 1-7, 756.85; bradykinin, 1060.21; angiotensin II (human), 1045.53; neurotensin 1672.92; angiotensin I (human), 1296.48; porcine renin substrate, 1759.01, [ Ace-F-3, -2H-1] angiotensinogen (1-14), 2231.61; Ser/Thr protein phosphatase (15-31), 1952.39, [ F14] Ser/Thr protein phosphatase (15-31), 2099.00, melittin (bee venom), 2846.46. all molecular weights are in kDa).

Fig. 5A-5B are SEM (scanning electron microscopy) images of particles before (fig. 5A) and after (fig. 5B) etching, showing the formation of a rough surface after etching.

Fig. 6 shows that the etched particle loaded column has a comparable back pressure to the porous particle filled column. Plotting Back pressure (bar) against 1/Dp²Graph of change, wherein Dp is mean particle size. All particles were packed in the same way under high pressure and at 70/30H₂The test was carried out as mobile phase in O/acetonitrile. In one embodiment, the etched particles have a particle strength equal to or greater than a comparable size of the porous particles.

FIGS. 7A-7D show chromatograms for protein separation from (FIG. 7A) non-porous silica core columns, (FIG. 7B) columns containing etched hybrid core particles according to one embodiment, (FIG. 7C) Agilent advanced Bio RP-mAb columns, and (FIG. 7D) Zorbax RRHD 300 columns. Etched hybrid core particles with thin rough surfaces have longer retention times than non-porous cores and exhibit similar retention times compared to wide-pore surface porous particles and only slightly lower retention times than fully porous wide-pore particles.

Fig. 8 illustrates one embodiment of etched particles described herein. In one embodiment, a is a hollow or solid core of inorganic or inorganic/organo-metallic oxide or polymer or magnetic material, B is a second solid layer of inorganic or inorganic/organo-metallic oxide or polymer or magnetic material, and C is a thin etched layer of inorganic metal oxide or inorganic/organo-metallic oxide.

Fig. 9A-9C show a comparison of SEM images of a non-porous silica core (fig. 9A), an etched hybrid silica core according to one embodiment (fig. 9B), and porous silica particles (advanced bio RP-mAb, fig. 9C).

10A-10B show TEM (transmission electron microscopy) edge images of a non-porous silica core (FIG. 10A) and an etched hybrid core (FIG. 10B) according to some embodiments.

Fig. 11A-11B show TEM images of the edges of the core-shell particles (fig. 11A) and the etched core particles (fig. 11B). The particles in fig. 11A have thick porous layers, but are not readily accessible to biomolecules (e.g., proteins, antibody fragments, intact mabs, etc.) due to small pore sizes that result in limited diffusion. In contrast, the etched surface of the particles in fig. 11B has no problem of limited diffusion.

Detailed Description

Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. Although the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

Values expressed in range format are to be interpreted in a flexible manner throughout the document to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Likewise, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y, or about Z".

In this document, the terms "a", "an" or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The statement "at least one of a and B" or "at least one of a or B" has the same meaning as "A, B, or a and B". Also, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid in reading documents and should not be construed as limiting; information related to the section title may appear within or outside of that particular section. All publications, patents, and patent documents mentioned in this document are incorporated by reference herein in their entirety as if individually incorporated by reference.

In the methods described herein, the acts may be performed in any order, unless time or order of operation is explicitly recited. Further, specified actions can be taken concurrently unless explicitly stated in the claim language that they are taken separately. For example, the claimed act of doing X and the claimed act of doing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed process.

Definition of

The term "about" as used herein may allow for a degree of variation in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or stated range limit, and includes the exact stated value or range.

The term "substantially" as used herein refers to a majority or majority, such as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free" as used herein may mean no or negligible amounts of such materials such that the amount of material present does not affect the material properties of a composition comprising the materials such that the composition is from about 0 wt% to about 5 wt%, or from about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less of the materials. The term "substantially free" can mean having an insignificant amount, such that the composition is from about 0 wt% to about 5 wt%, or from about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt% of the material.

The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond is present between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to, inter alia vinyl, -CH-C-CCH₂、-CH＝CH(CH₃)、-CH＝C(CH₃)₂、-C(CH₃)＝CH₂、-C(CH₃)＝CH(CH₃)、-C(CH₂CH₃)＝CH₂Cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.

The term "alkynyl" as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons, or in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to-C.ident.CH, -C.ident.C (CH), among others₃)、-C≡C(CH₂CH₃)、-CH₂C≡CH、-CH₂C≡C(CH₃) and-CH₂C≡C(CH₂CH₃)。

The term "aryl" as used herein refers to a cyclic aromatic hydrocarbon group containing no heteroatoms in the ring. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptenylene, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylene, pyrenyl, naphthoenyl, and,Mesityl, biphenylene, anthracenyl and naphthyl. In some embodiments, the aryl group is atThe ring portion of the group contains from about 6 to about 14 carbons. Aryl groups, as defined herein, may be unsubstituted or substituted. Representative substituted aryl groups may be mono-substituted or substituted more than once, such as, but not limited to, phenyl substituted at any one or more of the 2, 3, 4, 5, or 6 positions of the phenyl ring or naphthyl substituted at any one or more of the 2 to 8 positions thereof.

The term "amine" as used herein refers to a compound having, for example, the formula N (group)₃Wherein each group can be independently H or non-H, such as alkyl, aryl, and the like. Amines include, but are not limited to, R-NH₂Such as alkyl amines, aryl amines, alkyl aryl amines; r₂NH, wherein each R is independently selected, such as dialkylamine, diarylamine, aralkylamine, heterocyclylamine, or the like; and R₃N, wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

The term "amino" as used herein refers to-NH₂、-NHR、-NR₂、-NR₃ ⁺(ii) substituents of the form wherein each R is independently selected, and the respective protonated form, except-NR which cannot be protonated₃ ⁺. Thus, any compound substituted with an amino group can be considered an amine. In this context, an "amino group" within the meaning may be a primary, secondary, tertiary or quaternary amino group. "alkylamino" groups include monoalkylamino, dialkylamino, and trialkylamino groups.

The term "halo", "halogen" or "halide" group as used herein by itself or as part of another substituent means (unless otherwise specified) a fluorine, chlorine, bromine or iodine atom.

The term "hydrocarbon" or "hydrocarbyl group" as used herein refers to a molecule or functional group that comprises carbon and hydrogen atoms. The term may also refer to molecules or functional groups that typically contain both carbon and hydrogen atoms, but in which all hydrogen atoms are replaced by other functional groups.

As herein describedThe term "hydrocarbyl", as used, refers to a functional group derived from a straight, branched, or cyclic hydrocarbon, and may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. The hydrocarbyl group may be represented by (C)_a-C_b) A hydrocarbyl group, wherein a and b are integers and mean having any one of a to b carbon atoms. For example, (C)₁-C₄) Hydrocarbyl means that the hydrocarbyl group may be methyl (C)₁) Ethyl (C)₂) Propyl group (C)₃) Or butyl (C)₄) And (C)₀-C_b) Hydrocarbyl means that in certain embodiments no hydrocarbyl groups are present.

The term "solvent" as used herein refers to a liquid that can dissolve solids, liquids or gases. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids and supercritical fluids.

The term "independently selected" as used herein means that the groups referred to are the same, different or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X¹、X²And X³Independently selected from the group consisting of noble gases "would include the following: wherein for example X¹、X²And X³Are all the same; wherein X¹、X²And X³Are all different; wherein X¹And X²Same but X³Different; and other similar arrangements.

The term "room temperature" as used herein refers to a temperature of about 15 ℃ to 28 ℃.

The term "standard temperature and pressure" as used herein refers to 20 ℃ and 101 kPa.

Method for producing etched non-porous particles

In one embodiment, a method of producing etched non-porous particles includes providing a plurality of non-porous particles at least partially coated with at least one of a hydrophilic polymer, an inorganic metal oxide, a hybrid metal oxide, or a magnetic material; and contacting the plurality of non-porous particles with an acid or a base to produce a plurality of etched non-porous particles.

In some casesIn embodiments, the non-porous particles may have a diameter or average diameter of from about 0.3 μm to about 10 μm. In one embodiment, the non-porous particles may have a diameter or average diameter of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.5, 5.6, 6, 7.6, 7, 8, 6.6, 7.6, 7, 8, 6.6, 7, 8.6, 7, 8, 6, 7.6, 7, 7.6, 8, 6, 7, 8, 6, 7.5.0, 5.9, 5.1, 6, 8, 6, 8.6, 6, 8, 8.6.6, 6, 8, 8.6, 8, 6, 6.6, 7.6, 8.6, 7, 8.6, 8, 8.6, 6.6, 7, 8.6, 7.6, 8.6, 8, 6, 8.7, 8.6.7.7.6, 8.6, 8, or more. The non-porous particles may have a particle size of from about 0.01m²G to about 2m²A surface area per gram, the surface area being the surface area of the non-porous particles prior to exposure to any of the etching methods described herein. In one embodiment, the surface area of the non-porous particles may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0m²(ii) in terms of/g. In some embodiments, D of the non-porous particles₉₀/D₁₀The ratio may be from about 1.00 to about 1.50. In one embodiment, D of said non-porous particles₉₀/D₁₀The ratio may be about 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or about 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50. In some embodiments, the non-porous particles are not monodisperse. In some embodiments, the non-porous particles are monodisperse.

The portion of the coating on the non-porous particles can comprise at least about or equal to about 50% or less, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, 99%, 99.5%, or about 99.9% of the surface area of a given non-porous particle. The amount of surface area coated on the non-porous particles can be an average surface area such that the average coated surface area of the plurality of non-porous particles is at least about or equal to about 50% or less, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, 99%, 99.5%, or about 99.9%. In some embodiments, the coated surface area or average surface area of the non-porous particle or particles is completely (100%) coated.

In one embodiment, the non-porous particles may be made of a polymeric material and the coating is at least one of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material. Suitable polymeric materials may include polystyrene, poly (methyl methacrylate), poly (ethylene oxide), polyurethane, poly (vinylbenzylchloride), poly (vinylpyrrolidone), and the like. Suitable polymeric materials may also include copolymers such as acrylonitrile butadiene styrene, poly (styrene acrylic acid), poly (styrene methyl methacrylate), and the like.

The inorganic metal oxide coating is not particularly limited and may be a metal oxide or mixed metal oxide in some embodiments. In some embodiments, the inorganic metal oxide may be ZnO, SiO₂Etc.; or mixed metal oxides, such as M/SiO₂Wherein M may be Au, Ag, Ni, Fe, Co, FeNi, ZnO, CdS, AgI, CdTe, CdSe, CaCO₃And the like. In some embodiments, the hybrid metal oxide coating can be a combination of any of the inorganic metal oxides and organic components described herein. The organic component may contain a plurality of C-H groups. In one embodiment, the organic component of the hybrid metal oxide includes an organosilyl group. Suitable organosilyl groups include one or more R-Si moieties, wherein the R group may include a hydrocarbyl, alkenyl, alkynyl, aryl, hydroxyl, carboxylic acid, ester, ether, or amide group, or combinations thereof, and include linear, branched, and cyclic forms of the foregoing. In one embodiment, the plurality of non-porous particles are surface at least partially coated with at least one organo-siliconAlkyl silicon dioxide (SiO)₂) And (3) granules. The magnetic material may be any suitable ferromagnetic substance (such as Fe)₃O₄) A neodymium-based material, or the like, or a combination of the aforementioned magnetic material and any of the aforementioned inorganic metal oxides and/or mixed metal oxides.

The hydrophilic polymer may be at least one of: poly (N-isopropylacrylamide) (PNIPAM), Polyacrylamide (PAM), poly (acrylic acid), polymethacrylates, poly (ethylene glycol), poly (ethylene oxide), poly (2-oxazoline), and Polyethyleneimine (PEI), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), and the like, or combinations thereof. In one embodiment, the hydrophilic polymer is PVP. In some embodiments, the hydrophilic polymer may have a number average molecular weight or a mass average molecular weight of from about 5kDa to about 200 kDa. In some embodiments, the hydrophilic polymer may have a number average molecular weight or a mass average molecular weight of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or about 200 kDa.

In one embodiment, the non-porous particles are made of a polymeric material at least partially coated with at least one of an inorganic metal oxide, a mixed metal oxide, or a magnetic material, further at least partially coated with the hydrophilic polymer to produce coated non-porous particles. In one embodiment, the plurality of non-porous particles are made of an inorganic metal oxide, a mixed metal oxide, or a magnetic material, and are coated with a hydrophilic polymer.

In one embodiment, coating any of the non-porous particles described herein with a hydrophilic polymer can comprise heating the plurality of non-porous particles in an aqueous medium in the presence of at least one hydrophilic polymer described herein. In one embodiment, the heating may be a temperature from about 30 ℃ to about 95 ℃. In some embodiments, the heating can be at a temperature of about 30 ℃, 35 ℃, 40 ℃,45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, or about 95 ℃. In some embodiments, the heating may be for a period of time of about 0.5, 1, 2, 3, 4, 5, 6, 7, or about 8 hours, or from about 1 hour to about 8 hours.

Etching of

In one embodiment, any of the non-porous particles described herein coated with a hydrophilic polymer can be contacted with an acid or a base to produce a plurality of etched non-porous particles. In one embodiment, a plurality of non-porous particles made of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material and having a hydrophilic polymer coating can be contacted with an acid or a base to produce a plurality of etched non-porous particles. In some embodiments, the contacting with the acid or base may be for a period of time from about 2 hours to about 24 hours. In one embodiment, the contacting with the acid or base may be for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours. In one embodiment, the plurality of etched non-porous particles are washed to remove the hydrophilic polymer. Washing may be performed, for example, by reslurrying the etched non-porous particles about one to ten times in water and methanol. Contacting may include, for example, stirring a suspension or slurry of non-porous particles coated with a hydrophilic polymer as described herein with an aqueous solution of an acid or base. The base may be any suitable base that generates hydroxide ions in aqueous solution and brings the pH above 7. In one embodiment, the base may be an alkali metal hydroxide, ammonium hydroxide, tetramethylammonium hydroxide, and the like, or combinations thereof. Suitable alkali metal hydroxides include LiOH, NaOH, KOH, RbOH, and CsOH. In one embodiment, the acid may be ammonium fluoride, hydrofluoric acid, or a combination thereof. In some embodiments, the acid may be a suitable fluoride salt, such as NaF, KF, and the like. In one embodiment, the acid produces a water soluble fluoride anion (F) when in solution^-) In this case, the etching may be performed at room temperature. The acid or base concentration used in the etching may be, for example, about 1.0M to about 5.0M, or about 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 32, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, or about 5.0M.

In some embodiments, etching of the non-porous particles increases the pore volume of the particles from about 8 to about 20 times compared to their pore volume prior to etching. In one embodiment, etching of the non-porous particle increases the pore volume of the particle by at least about or about 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, 10, 10.2, 10.4, 10.6, 10.8, 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2, 13.4, 13.6, 13.8, 14, 14.2, 14.4, 14.6, 14.8, 15, 15.2, 15.4, 15.6, 15.8, 16, 16.2, 16.4, 16.6, 16.8, 17, 17.2, 17.4, 17.6, 17.8, 18, 18.2, 18.4, 18.6, 18.8, 19.19, 19.6, 19.0, or about 0 times its pore volume prior to etching.

In one embodiment, a method of producing etched metal oxide particles includes providing a plurality of inorganic or hybrid metal oxide particles; coating the inorganic or hybrid metal oxide particles with at least one hydrophilic polymer; and contacting the plurality of inorganic or hybrid metal oxide particles with an acid or base to produce a plurality of etched metal oxide particles.

Etched non-porous particles

In one embodiment, non-porous inorganic metal oxide or hybrid metal oxide particles having an etched surface are provided. The etched non-porous inorganic metal oxide particles or etched hybrid metal oxide particles can be comprised of any of the respective materials described herein.

In one embodiment, the etched non-porous particles can have from about 0.001 to about 0.1cm³Pore volume per gram, from about 2 to about 100m²Surface area per gram, or combinations thereof. In some embodiments, the etched non-porous particles may have a pore volume of about 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024, 0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044, 0.046, 0.048, 0.05, 0.052, 0.054, 0.056, 0.058, 0.06, 0.062, 0.064, 0.066, 0.068, 0.07, 0.072, 0.074, 0.076, 0.08, 0.082, 0.084, 0.086, 0.088, 0.084, 0.092, 0.091, 0.092, 0.098, 0.0925 g/cm.In some embodiments, the etched non-porous particles can have about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or about 100m²Surface area in g.

In one embodiment, the non-porous inorganic or hybrid metal oxide particle comprises a core comprising a hollow core, an inorganic metal oxide core, a mixed metal oxide core, a polymer core, or a magnetic core. The inorganic metal oxide core, hybrid metal oxide core, polymeric core, or magnetic core can be comprised of any of the corresponding materials described herein or a combination of these materials. In one embodiment, the non-porous particles have the structure shown in fig. 8. In some embodiments, the non-porous inorganic or hybrid metal oxide particles can have a washable hydrophilic polymer layer. The hydrophilic polymer layer may be comprised of any of the hydrophilic polymers mentioned herein or a combination of these hydrophilic polymers. In one embodiment, the non-porous inorganic metal oxide or hybrid metal oxide particles can be silica particles that are at least partially coated with at least one hydrophilic group, hydrophobic group, or a combination thereof. In some embodiments, the hydrophilic group can be a moiety comprising at least one of a nitrogen-containing heterocycle, an amide, a carbamate, a carboxylic acid, a carboxylate, a methyl ether, a cyano, an amine, an ammonium, a sulfonamide, a sulfonate, a urea, a thiourea, a hydroxyl, a thiol, PEG, a zwitterionic group, and a sulfonic acid. In some embodiments, the core and/or shell may comprise various geometric shapes, such as spherical, ovoid, oblate spheroid, elliptical, and the like.

Examples

Various embodiments of the present application may be better understood by reference to the following examples, which are provided by way of illustration. The scope of the present application is not limited to the examples given herein.

Chromatography method

All chromatographic tests described in the figure use mobile phase a of 0.1% TFA (trifluoroacetic acid)/water and mobile phase B of 0.08% TFA/acetonitrile. Analytes were detected using Agilent 1290Infinity LC with an 8nm bandwidth using UV absorbance at 220nm at 60 ℃.

The main advantage of non-porous particles due to the lack of any pore structure is the absence of intra-particle diffusion resistance, which leads to rapid protein separation. However, the retention time of the stationary phase using such particles is short and the sample loading capacity is significantly lower when compared to porous particles (fig. 1A-1B).

Example 1: method for producing etched non-porous particles

The following procedure can be used to make etched non-porous hybrid silica core particles, and can be applied to other non-porous particles described herein.

1. The following were combined in a 500mL flask: 40g of non-porous hybrid silica core particles, 400mL of water, and 20g of polyvinylpyrrolidone (PVP). For example, hybrid silica core particles can be made according to the procedure in U.S.9,284,456.

2. The aqueous mixture of particles and PVP was heated at 92 ℃ for 3 hours.

3. After heating, the mixture was centrifuged and the particles were washed twice with water. After this step, the particles were coated with PVP.

4. The PVP coated particles from step 3 were redispersed in 400mL of water to form a slurry.

5. A solution of 32g KOH in 200mL of water was added to the particle slurry.

6. The slurry formed in step 5 was stirred at room temperature for 4 hours.

7. The slurry was centrifuged and the particles were washed twice with water.

8. The washed particles were filtered and dried at 110-120 ℃ overnight.

Three different etching conditions were applied using various bases, various PVP molecular weights and various reaction temperatures (table 1).

Table 1: selected etching reaction conditions

TMAOH ═ tetramethylammonium hydroxide; RT ═ room temperature

Surprisingly, in one embodiment, the etching methods described herein can have 0.004cm after etching³Pore volume per gram of non-porous particles converted to a pore volume of 0.040-0.047cm³Per gram of particles. Very narrow particle size distribution, where D₉₀/D₁₀Less than 1.20 and without any size grading (fig. 2A-2B). Advantageously, the processing time is much shorter than the time required to make other porous particles that require at least twelve weeks to complete.

When the same protein test was run on the etched hybrid nuclei column, a narrower peak width and higher peak capacity were observed compared to the peak width and peak capacity on the solid nuclei column (fig. 3A-3B). In one embodiment, the performance of the etched particles is optimized for this type of separation with the best commercial columns (Agilent Advance Bio RP-mAb) and (Agilent ZORBAX RRHD)) Matched (fig. 7B-7D), while also exhibiting superior pH stability, for example, compared to particles such as those described in u.s.9,284,456 and u.s.6,686, 035. The same conclusions can be drawn from the peptide separations in FIGS. 4A-4B.

SEM images of the particles before and after etching clearly show the formation of rough surfaces during etching (fig. 5A-5B, 9A-9B, 10A-10B). TEM images of the particles before and after etching clearly show that shallow rough surfaces are formed during etching (fig. 10A-10B) and that the thickness of the roughened surface is much thinner than commercial surface porous particles (fig. 11A-11B) which result in greatly reduced diffusion paths for efficient separation of biomolecules.

The etched particle loaded column showed comparable back pressure compared to the porous particle filled column (fig. 6). This indicates that the etched particles according to some embodiments have better or compatible mechanical strength than the commercial particles. In one embodiment, the methods described herein may be applied to produce other etched surface core particles having various physical combinations for different purposes. For example, the hollow core may reduce particle density and may reduce the impact on particles or other inorganic/organo-metallic oxides impacting the outlet frit during packaging for better pH stability and/or application (fig. 8).

Example 2: commercial process for producing etched non-porous particles

As but one example, table 2 lists four stages in the manufacture of finished, non-porous etched particles suitable for commercial applications.

Table 2: etched non-porous particles can be produced in one week, which is significantly shorter than porous particles.

In stage 1, the desired non-porous particles are synthesized, purified and dried according to one embodiment. The synthesis of the non-porous particles may be performed as described herein or by another suitable method known in the art.

In stage 2, according to one embodiment, the non-porous particles are coated with a hydrophilic polymer according to the method described herein.

According to one example, in stage 3, the coated non-porous particles are etched, filtered and dried according to the methods described herein. The surface area of the dried etched particles is determined and the particles are classified based on silane calculations. The surface silanol density of the porous silica was estimated to be 8OH/nm². Thus, the amount of silane added can be calculated based on a 1:1 OH to silane ratio.

In stage 4, the etched non-porous particles are bonded/functionalized with silanes of a desired length, according to one embodiment. For example, surface modification can be achieved using trialkoxy (octadecyl) silane (C18 functionalized), trialkoxy (octyl) silane (C8 functionalized), or a combination of C8 and C18 silylating agents according to art-recognized methods. Capping may be used to react any free silanol (Si-OH) groups on the surface of the etched non-porous particles by reacting the particles with a capping agent, such as trimethylsilyl chloride or another suitable agent known in the art.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the application. Thus, it should be understood that although particular embodiments and optional features are described herein, modification and variation of the compositions, methods, and concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of embodiments of this application.

In summary, the present invention includes the following clauses.

1. A method of producing etched non-porous particles, the method comprising:

providing a plurality of non-porous particles at least partially coated with at least one of a hydrophilic polymer, an inorganic metal oxide, a hybrid metal oxide, or a magnetic material; and

contacting the plurality of non-porous particles with an acid or a base to produce a plurality of etched non-porous particles.

2. The method of clause 1, wherein the plurality of non-porous particles are made from a polymeric material and the coating is at least one of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material.

3. The method of clause 2, wherein the polymeric material is at least partially coated with at least one of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material, further at least partially coated with the hydrophilic polymer to produce coated non-porous particles.

4. The method of clause 3, wherein the coated non-porous particles are contacted with the acid or base to produce the plurality of etched non-porous particles.

5. The method of clause 4, wherein the plurality of etched non-porous particles are washed to remove the hydrophilic polymer.

6. The method of clause 1, wherein the plurality of non-porous particles are made of an inorganic metal oxide, a hybrid metal oxide, or a magnetic material, coated with the hydrophilic polymer.

7. The method of clause 6, wherein the inorganic metal oxide or hybrid metal oxide or magnetic material with the hydrophilic polymeric coating particles is contacted with an acid or base to produce the plurality of etched non-porous particles.

8. The method of clause 7, wherein the plurality of etched non-porous particles are washed to remove the hydrophilic polymer.

9. The method of clause 1, wherein the plurality of non-porous particles are silica particles having surfaces at least partially coated with at least one organosilyl group.

10. The method of clause 1, wherein the hydrophilic polymer comprises at least one of: poly (N-isopropylacrylamide) (PNIPAM), Polyacrylamide (PAM), poly (acrylic acid), polymethacrylate, poly (ethylene glycol), poly (ethylene oxide), poly (2-oxazoline), and Polyethyleneimine (PEI), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), or a combination thereof.

11. The method of clause 1, wherein the non-porous particles are contacted with the hydrophilic polymer by heating the non-porous particles and the hydrophilic polymer in an aqueous medium.

12. The method of clause 11, wherein the heating is at a temperature of from about 30 ℃ to about 95 ℃.

13. The method of clause 11, wherein the contacting is for a period of time from about 2 hours to about 24 hours.

14. The method of clause 1, wherein the base comprises at least one of: an alkali metal hydroxide, ammonium hydroxide, tetramethylammonium hydroxide, or a combination thereof, and the acid comprises at least one of: ammonium fluoride, hydrofluoric acid, or a combination thereof.

15. A method of producing etched metal oxide particles, the method comprising:

providing a plurality of inorganic or hybrid metal oxide particles;

optionally coating the inorganic or hybrid metal oxide particles with at least one hydrophilic polymer; and

contacting the plurality of inorganic or hybrid metal oxide particles with an acid or base to produce a plurality of etched metal oxide particles.

16. A particle, comprising:

comprising non-porous inorganic metal oxide or hybrid metal oxide particles etched into the surface,

wherein the particles have from about 0.001 to about 0.1cm³Pore volume per gram, from about 2 to about 100m²Surface area per gram, or combinations thereof.

17. The particle of clause 16, wherein the non-porous inorganic metal oxide or hybrid metal oxide particle comprises a core comprising a hollow core, an inorganic metal oxide core, a hybrid metal oxide core, a polymeric core, or a magnetic core.

18. The particle of clause 16, wherein the non-porous inorganic metal oxide or hybrid metal oxide particle comprises a washable, hydrophilic polymer layer.

19. The particle of clause 18, wherein the non-porous inorganic metal oxide or hybrid metal oxide particles comprise silica particles at least partially coated with at least one hydrophilic group, hydrophobic group, or a combination thereof.

20. The particle of clause 16, wherein the non-porous inorganic metal oxide or hybrid metal oxide particle comprises a diameter of from about 0.3 μ ι η to about 10 μ ι η.