Methods for detection and quantification of surfactants

Abstract

Disclosed herein are materials and related methods for detecting and quantifying surfactants in solutions containing interfering species, such as proteins.

Description

Attorney Docket No. JPI6076WOPCT1 METHODS FOR DETECTION AND QUANTIFICATION OF SURFACTANTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application US 63 / 697,716, “METHODS FOR DETECTION AND QUANTIFICATION OF SURFACTANTS”, filed 23 Sep 2024, which is included herein by reference. FIELD

[0002] Disclosed herein are methods and related materials for detecting and quantifying surfactants in solutions containing interfering species, such as proteins. BACKGROUND

[0003] Non-ionic surfactants such as polysorbate 20 / 80 (PS20 / PS80), poloxamer, or BRIJ™ are commonly used in protein and gene therapeutic drug formulations to increase stability of the active pharmaceutical ingredient against surface and shear stress. Therefore, it is important to monitor surfactant concentration in drug product at release and throughout a product’s shelf-life. To do so, it is essential to have a robust method to determine the surfactant concentration. The relatively high protein content in biopharmaceutical products formulations poses a challenge to the quantitative surfactant methods.

[0004] Hewitt and Zhang (Journal of Chromatography A, 1215: 156-160, 2008) developed a mixed-mode (anion exchange-hydrophobic) high-performance liquid chromatography (MAX- ELSD) method to quantify polysorbate in mAb formulations. The protein is positively charged at low pH by adding 2% formic acid in the mobile phase. As a result, the mAb protein is repelled by the (positively charged) anion exchange material in the column while the neutral polysorbate molecules are retained by the hydrophobic portion of the column. A step gradient of isopropyl alcohol is used to elute polysorbate, which is detected with a non-volatile particle detector. This HPLC based method is considered superior to other colorimetric and fluorescence-based methods as it allows the specific quantification of polysorbate during product shelf-life.

[0005] This method and variations thereof have been validated and successfully implemented in Janssen Biologics to quantify PS20, PS80 and BRIJ™ L23 in drug products. Furthermore, a reversed phase HPLC method using a non-volatile particle detector was developed, implemented, and validated for the quantitation of P188 in drug products. However, challenges in surfactant analysis arise with high protein concentrations, hydrophobic proteins, proteinAttorney Docket No. JPI6076WOPCT1 fragments, and low surfactant concentrations in the drug formulation. High protein concentration, protein degradation products or hydrophobic proteins can lead to interference peaks at the retention time of the surfactant, reducing the methods specificity, accuracy, and precision. The combination with low surfactant concentration eliminates the option for sample dilution, including organic solvent precipitation.

[0006] There remains a need for methods to detect and quantify surfactants in the presence of interfering biomolecules in bioprocessing, including but not limited to proteins, peptides, viral vectors and antibodies. BRIEF SUMMARY

[0007] Accordingly, provided herein is a method for quantifying a nonionic surfactant in a sample comprising the nonionic surfactant and one or more biomolecules, the method comprising the steps of: (a) applying the sample to an HPLC column comprising (i) a hydrophobic stationary phase or (ii) a mixed-mode stationary phase containing a hydrophobic portion; (b) eluting the HPLC column with a first mobile phase to form a first eluent; (c) eluting the HPLC column with a second mobile phase; (d) passing the first eluent through a cation exchange material to form a second eluent; and (e) detecting the second eluent with a suitable detector, thereby quantifying the nonionic surfactant in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] A full understanding of the invention can be gained from the following description of the preferred embodiments when read in conjunction with the accompanying drawings in which:

[0009] FIG.1 depicts chromatographic equipment (a) without and (b) with cationic exchange columns.

[0010] FIG.2 depicts chromatograms for (a) blank injection, (b) injection of polysorbate (PS), and PS-free mAb formulations (c) without CX column, and (d) with CX column. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0011] Accordingly, provided herein is a method for quantifying a nonionic surfactant in aAttorney Docket No. JPI6076WOPCT1 sample comprising the nonionic surfactant and one or more biomolecules, the method comprising the steps of: (a) applying the sample to an HPLC column comprising (i) a hydrophobic stationary phase or (ii) a mixed-mode stationary phase containing a hydrophobic portion; (b) eluting the HPLC column with a first mobile phase to form a first eluent; (c) eluting the HPLC column with a second mobile phase; (d) passing the first eluent through a cation exchange material to form a second eluent; and (e) detecting the second eluent with a suitable detector, thereby quantifying the nonionic surfactant in the sample.

[0012] In some embodiments, the HPLC column further comprises (iii) an anion exchange material.

[0013] In some embodiments, step (b) is initiated after completion of step (a). In some embodiments, step (a) is initiated and completed during performance of step (b).

[0014] In some embodiments, step (c) is initiated after completion of step (b). In some embodiments, step (c) is initiated immediately after completion of step (b).

[0015] In some embodiments, step (d) is concurrent with step (c). In some embodiments, step (d) is initiated before initiation of step (c). In some embodiments, step (d) is initiated after initiation of step (c).

[0016] In some embodiments, step (e) is concurrent with step (d). In some embodiments, step (e) is initiated after initiation of step (d).

[0017] In some embodiments, the method comprises the steps of: (b) eluting the HPLC column with a first mobile phase, with the flow directed to a waste; and (c) eluting the HPLC column with a second mobile phase, with the flow directed to a cation exchange material.

[0018] In some embodiments, the method further comprises the steps of: prior to initiation of step (b), configuring the HPLC column to deliver the eluent to a waste; and prior to initiation of step (d), configuring the HPLC column to deliver the eluent to a cation exchange material.Attorney Docket No. JPI6076WOPCT1

[0019] In some embodiments, the method comprises the step of: (b) eluting the HPLC column with a first mobile phase comprising a gradient of water and a first water miscible organic solvent.

[0020] In some embodiments, the method comprises the step of: (c) eluting the HPLC column with a second mobile phase comprising a gradient of water and a second water miscible organic solvent.

[0021] In some embodiments: the first mobile phase comprises a solvent chosen from water and a first water miscible organic solvent, or a mixture or gradient thereof; and the second mobile phase comprises a solvent chosen from water and a second water miscible organic solvent, or a mixture or gradient thereof; wherein the first water miscible organic solvent and the second water miscible organic solvent are the same or different.

[0022] In some embodiments, the first water miscible organic solvent and the second water miscible organic solvent are the same. In some embodiments, the first water miscible organic solvent and the second water miscible organic solvent are different.

[0023] In some embodiments, the first mobile phase comprises a mixture or gradient of water and a first water miscible organic solvent. In some embodiments, the second mobile phase comprises a mixture or gradient of water and a second water miscible organic solvent.

[0024] In some embodiments, the first mobile phase comprises a gradient of water and a first water miscible organic solvent. In some embodiments, the second mobile phase comprises a gradient of water and a second water miscible organic solvent.

[0025] In some embodiments, the first water miscible organic solvent is chosen from an alcohol, an ester, a ketone, and acetonitrile. In some embodiments, the first water miscible organic solvent is chosen from 2-propanol, ethyl acetate, acetone, and acetonitrile. In some embodiments, the first water miscible organic solvent is chosen from an alcohol and acetonitrile. In some embodiments, the first water miscible organic solvent is 2-propanol.

[0026] In some embodiments, the second water miscible organic solvent is chosen from an alcohol, an ester, a ketone, and acetonitrile. In some embodiments, the second water miscible organic solvent is chosen from 2-propanol, ethyl acetate, acetone, and acetonitrile. In someAttorney Docket No. JPI6076WOPCT1 embodiments, the second water miscible organic solvent is chosen from an alcohol and acetonitrile. In some embodiments, the first water miscible organic solvent is 2-propanol.

[0027] In some embodiments, the first mobile phase comprises an acid. In some embodiments, the acid is an organic acid. In some embodiments, the organic acid is chosen from formic acid, acetic acid, and trifluoroacetic acid. In some embodiments, the concentration of acid in first mobile phase is between 0.1% and 5% (w / v), inclusive. In some embodiments, the concentration of acid in first mobile phase is 2%.

[0028] In some embodiments, the second mobile phase is identical to the first mobile phase.

[0029] In some embodiments, both the first mobile phase and the second mobile phase comprise water and an water miscible organic solvent. In some embodiments, the water miscible organic solvent is chosen from an alcohol and acetonitrile. In some embodiments, the water miscible organic solvent is 2-propanol or a mixture of acetonitrile and 2-propanol.

[0030] In some embodiments, both the first mobile phase and the second mobile phase comprise an acid. In some embodiments, the acid is an organic acid. In some embodiments, the organic acid is chosen from formic acid, acetic acid, and trifluoroacetic acid. In some embodiments, the concentration of acid in first mobile phase is between 0.1% and 5% (w / v), inclusive. In some embodiments, the concentration of acid in first mobile phase is 2%.

[0031] In some embodiments, the HPLC column comprises an anion exchange material. In some embodiments, the anion exchange material comprises quaternary ammonium moieties.

[0032] In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is chosen from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0033] In some embodiments, the nonionic surfactant is a glycol polymer or copolymer. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is chosen from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.

[0034] In some embodiments, the nonionic surfactant is an ether of a PEG and a fatty alcohol. In some embodiments, the nonionic surfactant is a BRIJ™. In some embodiments, the nonionic surfactant is chosen from BRIJ™-35, BRIJ™-58, BRIJ™-93, and BRIJ™-L23. In some embodiments, the nonionic surfactant is chosen from BRIJ™ CS20, BRIJ™ C2, BRIJ™ C10, BRIJ™ C20, BRIJ™ IC20, BRIJ™ IC20-70, BRIJ™ L4, BRIJ™ L23, BRIJ™ L23-69, BRIJ™Attorney Docket No. JPI6076WOPCT1 O2, BRIJ™ O3, BRIJ™ O5, BRIJ™ O10, BRIJ™ O20, BRIJ™ S2, BRIJ™ S10, BRIJ™ S20, BRIJ™ S100, and BRIJ™ S721.

[0035] In some embodiments, the nonionic surfactant is PEG. In some embodiments, the average Mnfor PEG is chosen from 250, 300, 350, 400, 500, 600, 700, 1000, 1500, 3400, 3500, 4000, 6000, and 10,000.

[0036] In some embodiments, each of the one or more biomolecules are chosen independently from proteins, viral vectors, and antibodies. In some embodiments, the viral vector is an adeno- associated virus (“AAV”).

[0037] In some embodiments, the one or more biomolecules comprises a protein. In some embodiments, the one or more biomolecules comprises an antibody. In some embodiments, the one or more biomolecules comprises a viral vector. In some embodiments, the one or more biomolecules comprises an AAV. Abbreviations

[0038] AX = anion exchange; BSA = bovine serum albumin; CX = cation exchange; ELSD evaporative light scattering detector / detection; HPLC = high-performance liquid chromatography; mAb= monoclonal antibody; PEG = polyethylene glycol; PS = polysorbate; RP = reverse phase. Definitions

[0039] The term “biomolecule”, as used herein, refers to a molecule having a biological origin. In some embodiments, a biomolecule is a macromolecule. The macromolecule may be chosen from the group consisting of peptides, proteins, carbohydrates, and nucleic acids. In some embodiments, the biomolecule is a noncovalent association of macromolecule moieties. In some embodiments, the biomolecule is an antibody, or a fragment thereof. In some embodiments, the biomolecule is a viral vector.

[0040] In some embodiments, the biomolecule has a molecular weight of 10000 kDa or less, optionally 500 kDa or less, optionally 2000 kDa or less, optionally 1000 kDa or less, optionally 500 kDa or less, optionally 200 kDa or less, optionally 100 kDa or less, optionally 50 kDa or less, optionally 25 kDa or less. In some embodiments, the biomolecule has a molecular weight of 1 kDa or greater, optionally 5 kDa or greater, optionally 10 kDa or greater, optionally 50 kDa or greater, optionally 100 kDa or greater, optionally 200 kDa or greater, optionally 500 kDa orAttorney Docket No. JPI6076WOPCT1 greater, optionally 1000 kDa or greater, optionally 2000 kDa or greater.

[0041] In some embodiments, the biomolecule has a mean size of 2 nm or greater, optionally 5 nm or greater, optionally 10 nm or greater. Method according to the invention

[0042] Provided in FIG.1(a) is a depiction of an apparatus for chromatographic analysis. Mobile phase from bottles 5 is delivered with pump 10 into injector 15. A chromatographic run is initiated by introducing sample 20, containing a mixture of compounds to be identified and quantified, into injector 15. The flow of mobile phase from pump 10 drives the sample through chromatography column 25, which is packed with a stationary phase. The affinity of each compound for the stationary phase determines its relative mobility in column 25. Compounds with low affinity for the stationary phase will pass rapidly down the column, in some cases at rates approaching that of the bulk mobile phase. Compounds with high affinity for the stationary phase will pass slower through the column. The compounds will emerge from column 25 in order of increasing affinity.

[0043] Switch valve 30 can be set to direct the flow emerging from the column either to drain 35 or to detector 40. This functionality can be used to divert poorly retained, early eluting compounds in the sample, which elute with or shortly after the void volume, from the detector, thereby increasing detector lifetime.

[0044] Output from detector 40 is fed into data processing hardware 45, from which the composition of sample 20 can be derived.

[0045] In some methods, the stationary phase includes a hydrophobic, reverse-phase material. Highly polar compounds in a high polarity mobile phase (including but not limited to water and alcohols, and mixtures thereof) will pass relatively unhindered through the hydrophobic stationary phase, whereas compounds with intermediate or low polarity will interact with the hydrophobic stationary phase. This interaction, governed by the hydrophobic effect, will slow the passage of relatively nonpolar compounds down in column 25.

[0046] In theory, each compound in such a sample can be identified by the time period required for the compound to pass through the length of column 25, i.e., the compound’s retention time. In practice, different compounds can have similar retention times, and may overlap at the detector, depending on the breadth of the corresponding peaks. Therefore, quantitation, and evenAttorney Docket No. JPI6076WOPCT1 identification, of compounds with similar retention times can be difficult.

[0047] In some methods, column 25 can contain anion exchange material as well as hydrophobic material. Due to electrostatics, this positively charged material will repel cationic analytes from the stationary phase. Cationic analytes will therefore emerge sooner from the column, i.e., have a shorter retention time.

[0048] In certain situations, this method can be modified to separate certain proteins from nonionic compounds, including surfactants, such as polysorbate 80. Depending on its isoelectric point, a protein can be converted to ionic form by adjusting the pH of the mobile phase, and its mobility down a column containing charged materials will likewise depend on mobile phase pH. The nonionic surfactant will be unaffected by changing the pH and, all else being equal, its retention by the stationary phase will be relatively unaffected by changes in mobile phase pH.

[0049] It will be understood that some cationic compounds, despite repulsion by the ionic exchange material of column 25, may still be held up by the stationary phase and cause problems with coelution with the nonionic analyte of interest. This may occur for compounds with low net positive charge, or for compounds with significant hydrophobic attraction to reverse-phase material in column 25.

[0050] Improved separation of interfering cationic proteins from nonionic surfactants can be achieved with the apparatus depicted in FIG.1(b). As with the aforementioned method, sample 20 is applied, through injector 15, to column 25, followed by elution with the mobile phase, provided from bottles 5, optionally comprising an acid to form cations from proteins having basic sites. The hydrophobic surfactants are retained on the column while the cationic proteins pass rapidly down the column. Preferably, these proteins are subsequently shunted from the detector with switch valve 30 into bottle 35.

[0051] The apparatus of FIG.1(b) further comprises cation exchange column 50, downstream from column 25 and switch valve 30. The cation exchange material, having a negative charge, will attract and retain cations, slowing or stopping their passage through column 50. As with the anion exchange material that may be incorporated into column 25, the neutral surfactant will be unaffected by the charged stationary phase of column 50. In contrast, cations which elute slowly from column 25, and which potentially co-elute with the nonionic surfactant, will be held up by the cation exchange material.Attorney Docket No. JPI6076WOPCT1 Example 1. Apparatus and methods.

[0052] In order to demonstrate the usefulness of including cation exchange column 50, the following experiments were conducted.

[0053] As indicated below, the apparatus as depicted in either FIG.1(a) (without cation exchange column 50) or FIG.1(b) (with cation exchange column 50) was used, with a Waters OASIS MAX column (2.1 mm x 20 mm, 30-μm particle size) as column 25. Injection size was chosen based on the nature of the analyte, and was generally 30 μL. The switch valve is initially set to shunt eluent from column 25 to waste, and is generally switched after 3 min to direct eluent to the ELSD detector.

[0054] Columns were eluted with 2% formic acid in water / 2-propanol. Step gradients were utilized to elute nonionic surfactant.

[0055] Table 1 provides details of the cation exchange columns tested with this study. Table 1. Cation Exchange Columns Ion Exchange Column Dimensions Part no. OASIS MCX, 2.1 mm 2.1 x 20 mm 186002046 OASIS MCX, 4.6 mm 4.6 x 20 mm 186002049 SPHERISORB® SCX 4.6 x 30 mm PSS839471 MABPACK™ SCX 2.0 x 5.0 mm 075749 PROTEOMIX® SCX, 10 mm 4.0 x 10.0 mm 401NP10-4001 PROTEOMIX® SCX, 50 mm 4.6 x 50.0 mm 401NP10-4605 Example 2. Chromatograms.

[0056] Chromatograms from selected runs are depicted in FIG.2.

[0057] FIG.2(a) depicts a blank injection using the apparatus of FIG.1(b), using column 401NP10-4605. As expected, no peaks are seen in the detector.

[0058] FIG.2(b) depicts injection of a polysorbate standard using the apparatus of FIG.1(b), using column 401NP10-4605.

[0059] FIG.2(c) depicts a chromatogram for a mAb formulation, without polysorbate, using the apparatus of FIG.1(a), i.e., without cation exchange column 50. Ideally this chromatogram would appear similar to the blank depicted in FIG.2(a), due to efficient fast elution of the mAb,Attorney Docket No. JPI6076WOPCT1 due to the anion exchange material, during the initial shunt phase of operation. The appearance of a peak at 6.4 minutes in this chromatogram suggests that, under these conditions, mAb would interfere with polysorbate quantitation, reducing the specificity, accuracy and precision of this method. The additional detector signal at 7-9 minutes indicates the elution of additional material from this formulation.

[0060] FIG.2(d) depicts a chromatogram for the same mAb formulation, without polysorbate, using the apparatus of FIG.1(b), i.e., with cation exchange column 50. It will be observed that this chromatogram is similar in appearance to a blank injection, presumably due to the affinity of positively charged species for the cation exchange material of column 50. This material does not enter the detector over the period of observation, indicating the reduced interference with detection and quantitation of surfactants under these conditions. Example 3. Scoring of cation exchange columns.

[0061] The cation exchange columns of Table 1 were evaluated for their performance in reducing the likelihood and degree of interference with surfactant. Performance of the columns with a PS standard are reported in Table 2. Table 2. Performance of cation exchange columns with a PS standard. Second column Platecount tailing factor peak width at retention Change in 50% time chromatographic profile None 5410 1.31 0.14 4.367 NA OASIS MCX 5303 1.5 0.148 4.5 no 2.1 mm OASIS MCX 5109 1.56 0.152 4.625 no 4.6 mm SPHERISORB® NA to much NA to much NA to much 4.701 Additional peak at SCX interference of interference of interference of 3.5 min and peak at peak at 3.5 peak at 3.5 peak at 3.5 PS retention time minutes minutes minutes PROTEOMIX® 6275 1.38 0.131 4.423 Additional peaks at SCX, 10 mm 3.5 min and 6 minAttorney Docket No. JPI6076WOPCT1 PROTEOMIX® 6450 1.34 0.138 4.705 no SCX, 50 mm

[0062] The columns were tested with 65 μL aliquots of the following analytes:

[0063] (a) BSA (50 mg / mL);

[0064] (b) Stelara (90 mg / mL);

[0065] (c) KLK2xCD3 (100 mg / mL)

[0066] Height of the analyte at the PS peak, in LSU, are reported in Table 3. Table 3. Analysis of analytes Second column Analyte 50 mg / mL BSA 90 mg / mL Stelara 100 mg / mL KLK2xCD3 none 16 LSU 15-20 LSU 11 LSU OASIS MCX <1 LSU for 8 injections 5 LSU (only 1 injection 7 LSU (only 1 injection 2.1 mm performed) performed) OASIS MCX <1 LSU for 45 3.5 LSU (only 1 <1 LSU for 4 injections, 6 4.6 mm injections (increasing injection performed) LSU at the 6th injections ( peak after 45th height increased with every injection) injection after the first 4) SPHERISORB® <2 LSU for 40+ <2 LSU for 4 injections <2 LSU (only 1 injection SCX injections (experiment (only 4 injections performed) stopped before column performed) was at capacity) PROTEOMIX® <1 LSU for 40 9 LSU (only 1 injection not performed SCX, 10 mm injections (increasing performed) peak after 40th injection) PROTEOMIX® <5 LSU for 235 4 LSU (only 1 injection 4 LSU , for 60+ injections SCX, 50 mm injections (experiment performed) (experiment was stopped was stopped before the before the column was at column was at capacity) capacity)Attorney Docket No. JPI6076WOPCT1

[0067] Both mAb and BSA were studied, and scores for both reduction of potential interference and for protein capacity were assigned. In addition, the profile of PS surfactant was characterized. Results are provided in Table 4. Table 4. Performance of cation exchange columns. Cation Exchange Interference Protein Capacity Column reduction score score PS profile OASIS MCX, 2.1 mm 4 / 5 1 / 5 Same as without OASIS MCX, 4.6 mm 4 / 5 3 / 5 Same as without SPHERISORB SCX 5 / 5 5 / 5 Peak observed upon operating switch valve and at PS retention time > 1LSU MABPACK™ SCX (a) (a) (a) PROTEOMIX® SCX, 4 / 5 3 / 5 Peak observed upon operating switch 10 mm valve that decreases for each injection. Peak observed on reversion to aqueous PROTEOMIX® SCX, 4 / 5 5 / 5 Same as without 50 mm (a) Could not be tested, backpressure too high for system.

[0068] While the methods and manufactures have described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

Attorney Docket No. JPI6076WOPCT1 CLAIMS What is claimed is:

1. A method for quantifying a nonionic surfactant in a sample comprising the nonionic surfactant and one or more biomolecules, the method comprising the steps of: (a) applying the sample to an HPLC column comprising (i) a hydrophobic stationary phase or (ii) a mixed-mode stationary phase containing a hydrophobic portion; (b) eluting the HPLC column with a first mobile phase to form a first eluent; (c) eluting the HPLC column with a second mobile phase; (d) passing the first eluent through a cation exchange material to form a second eluent; and (e) detecting the second eluent with a suitable detector, thereby quantifying the nonionic surfactant in the sample.

2. The method of claim 1, wherein the HPLC column further comprises (iii) an anion exchange material.

3. The method of claim 1, wherein: the first mobile phase comprises a solvent chosen from water and a first water miscible organic solvent, or a mixture thereof; the second mobile phase comprises a solvent chosen from water and a second water miscible organic solvent, or a mixture thereof; and the first water miscible organic solvent and the second water miscible organic solvent are the same or different.

4. The method of claim 1, comprising the step of: (b) eluting the HPLC column with a first mobile phase comprising a gradient of water and a first water miscible organic solvent to form a first eluent.

5. The method of claim 1, comprising the step of: (c) eluting the HPLC column with a second mobile phase comprising a gradient of water and a second water miscible organic solvent to form a first eluent.Attorney Docket No. JPI6076WOPCT1 6. The method of claim either one of claims 4 and 5, wherein the gradient of step (c) consists of increasing the relative amount of the second water miscible organic solvent.

7. The method of any one of claims 3 – 6, wherein the first water miscible organic solvent and the second water miscible organic solvent are independently chosen from an alcohol and acetonitrile, or a combination of both.

8. The method of claim 7, wherein the alcohol is 2-propanol.

9. The method of claim 1, wherein the first mobile phase further comprises an acid.

10. The method of claim 1, wherein the second mobile phase further comprises an acid.

11. The method of either one of claims 9 and 10, wherein the concentration of acid is 2% (v / v).

12. The method of any one of claims 9 – 11, wherein the acid is an organic acid.

13. The method of claim 12, wherein the organic acid is formic acid.

14. The method of claim 1, wherein the eluent from the HPLC column is discarded prior to initiation of step (d).

15. The method of claim 1, wherein the nonionic surfactant is a polysorbate or a poloxamer.

16. The method of claim 15, wherein the polysorbate is chosen from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

17. The method of claim 15, wherein the poloxamer is chosen from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.

18. The method of claim 1, wherein the nonionic surfactant is an ether of a polyethylene glycol [PEG] and a fatty alcohol.

19. The method of claim 1, wherein the nonionic surfactant is a BRIJ™.

20. The method of claim 1, wherein the one or more biomolecules comprises a protein.

21. The method of claim 1, wherein the one or more biomolecules comprises an antibody.Attorney Docket No. JPI6076WOPCT1 22. The method of claim 1, wherein the one or more biomolecules comprises a virus.

23. The method of claim 1, wherein the one or more biomolecules comprises an adeno- associated virus [AAV].

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