Methods, compositions and kits useful for pH gradient cation exchange chromatography

Abstract

The present disclosure relates to methods, compositions and kits useful for the enhanced pH gradient cation exchange chromatography of a variety of analytes. In various aspects, the present disclosure pertains to chromatographic elution buffer solutions that comprise a first buffer salt, a second buffer salt, a third buffer salt, and fourth buffer salt. The first buffer salt may be, for example, a diprotic acid buffer salt, the second buffer salt may be, for example, a divalent buffer salt with two amine groups, the third buffer salt may be, for example, a monovalent buffer salt comprising a single amine group, and the fourth buffer salt may be, for example, a zwitterionic buffer salt. Moreover, the buffer solution has a pH ranging from 3 to 11.

Claims

1. A chromatographic elution buffer solution comprising a plurality of buffer salts, the buffer solution having a first pKa value that ranges from 4.5 to 6.5, a second pKa value that ranges from 6.5 to 7.5, a third pKa value that ranges from 7.5 to 8.5, a fourth pKa value that ranges from 8.5 to 9.5, and a fifth pKa value that ranges from 9.5 to 11.

2. The chromatographic elution buffer solution of claim 1, wherein the buffer solution has a pH ranging from 3 to 11.

3. Chromatographic elution buffer solution of claim 1, wherein the first pKa value is provided by a first buffer salt that is a diprotic acid, (b) wherein the second pKa value and the fourth pKa value are provided by a second buffer salt that is a divalent buffer salt with two amine groups, (c) wherein the third pKa is provided by a third buffer salt that is a monovalent buffer salt comprising a single amine group, and (d) wherein the fifth pKa value is provided by a fourth buffer salt that is a zwitterionic buffer salt.

4. The chromatographic elution buffer solution of claim 3, comprising a single zwitterionic buffer salt.

5. The chromatographic elution buffer of claim 3, wherein the diprotic acid buffer salt is succinic acid.

6. The chromatographic elution buffer of claim 3, wherein the divalent buffer salt is BIS-TRIS propane.

7. The chromatographic elution buffer of claim 3, wherein the monovalent buffer salt is selected from the group consisting of triethanolamine and TRIS.

8. The chromatographic elution buffer of claim 3, wherein the zwitterionic buffer salt is selected from the group consisting of zwitterionic buffer salt comprising a sulfonate group and an amine group and a zwitterionic buffer salt comprising a carboxyl group and an amine group.

9. The chromatographic elution buffer of claim 3, wherein the zwitterionic buffer salt is selected from the group consisting of CAPSO, CAPS and β-alanine.

10. A chromatographic elution buffer kit comprising (a) a first buffer solution comprising a chromatographic elution buffer solution comprising a plurality of buffer salts, the buffer solution having a first pKa value that ranges from 4.5 to 6.5, a second pKa value that ranges from 6.5 to 7.5, a third pKa value that ranges from 7.5 to 8.5, a fourth pKa value that ranges from 8.5 to 9.5, and a fifth pKa value that ranges from 9.5 to 11, wherein the buffer solution has a pH ranging from 3 to 7 or has a pH ranging from 3 to 7 upon dilution with water in a ratio ranging from 1:2 to 1:20 and (b) a second buffer solution comprising a chromatographic elution buffer solution comprising a plurality of buffer salts, the buffer solution having a first pKa value that ranges from 4.5 to 6.5, a second pKa value that ranges from 6.5 to 7.5, a third pKa value that ranges from 7.5 to 8.5, a fourth pKa value that ranges from 8.5 to 9.5, and a fifth pKa value that ranges from 9.5 to 11, wherein the second buffer solution has a pH ranging from 9 to 11 or has a pH ranging from 9 to 11 upon dilution with water in a ratio ranging from 1:2 to 1:20, wherein the second buffer solution comprises a non-buffer salt.

11. The chromatographic elution buffer kit of claim 10, wherein each of the first, second, third and fourth buffer salts is present in the first buffer solution at a concentration ranging from 2 to 20 millimolar and/or wherein each of the first, second, third and fourth buffer salts is present in the second buffer solution at a concentration ranging from 2 to 20 millimolar.

12. The chromatographic elution buffer kit of claim 10, wherein the non-buffer salt comprises (a) a cation selected from the group consisting of alkali metal cations, alkaline earth metal cations, transition metal cations, ammonium cations and (b) an anion selected from the group consisting of halide anions, nitrate anions, sulfate anions, phosphate anions, carbonate anions, chlorate anions, thiocyanate anions and perchlorate anions.

13. The chromatographic elution buffer kit of claim 12, wherein the concentration of the non-buffer salt ranges from 1 to 100 millimolar.

14. The chromatographic elution buffer kit of claim 10, wherein a concentration of each of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a concentration of each of the first, second, third and fourth buffer salts in the second buffer solution.

15. The chromatographic elution buffer kit of claim 10, wherein a total concentration of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a total concentration of the first, second, third and fourth buffer salts in the second buffer solution.

16. The chromatographic elution buffer kit of claim 10, wherein a plot of pH versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

17. The chromatographic elution buffer kit of claim 10, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

18. The chromatographic elution buffer kit of claim 10, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution does not exhibit a negative slope.

19. The chromatographic elution buffer kit of claim 10, wherein the first buffer solution has a conductivity ranging from 0.5 millisiemens (mS) to 3 mS and wherein the second buffer solution has a conductivity ranging from 3 mS to 100 mS.

20. The chromatographic elution buffer kit of claim 10, further comprising an ion-exchange chromatography material.

21. The chromatographic elution buffer kit of claim 20, comprising a separation device comprising a housing comprising an inlet and an outlet, wherein the ion-exchange chromatography material is housed in the housing.

22. The chromatographic elution buffer kit of claim 20, wherein the ion-exchange chromatography material is a cation exchange chromatography material.

23. The chromatographic elution buffer kit of claim 22, wherein the cation exchange chromatography material comprises carboxylate groups.

24. The chromatographic elution buffer kit of claim 22, wherein the cation exchange chromatography material comprises sulfonate groups.

25. A method for analyzing a sample comprising a plurality of analytes, the method comprising: loading the sample onto an ion-exchange chromatography material thereby binding the plurality of analytes to the ion-exchange chromatography material and eluting the plurality of analytes from the ion-exchange chromatography material with a mobile phase comprising a chromatographic elution buffer solution comprising a plurality of buffer salts, the buffer solution having a first pKa value that ranges from 4.5 to 6.5, a second pKa value that ranges from 6.5 to 7.5, a third pKa value that ranges from 7.5 to 8.5, a fourth pKa value that ranges from 8.5 to 9.5, and a fifth pKa value that ranges from 9.5 to 11, thereby separating at least some of the plurality of analytes.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1D show exemplary ion exchange chromatograms and analysis of resolving power for charge variant profiling of infliximab (FIG. 1A), trastuzumab (FIG. 1B), adalimumab (FIG. 1C), and NIST mAb reference material 8671 (FIG. 1D). Calculation of effective peak capacity for infliximab (A), a pseudo resolution value using the acidic variant peak and the main peak for trastuzumab (FIG. 1B), and peak-to-valley ratios of the first lysine variant for adalimumab (FIG. 1C) and NIST mAb (FIG. 1C) are illustrated.

(2) FIGS. 2A-2B show UV chromatograms (FIG. 2B) resulting from the use of a binary pH gradient buffer system (FIG. 2A) with a 3 μm non-porous sulfonated cation exchange stationary phase in a 4.6×50 mm column dimension. Charge variant separations are obtained for panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671.

(3) FIGS. 3A-3C show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of histidine and Gly-Gly (FIG. 3A), citric acid, glycine, and Gly-Gly (FIG. 3B), or MES, glycine, and Gly-Gly (FIG. 3C).

(4) FIGS. 4A-4B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, ACES, 2-Amino-2-methyl-1,3-propanediol (AMPD), TEA, and CAPS (FIG. 4A) or succinic acid, BIS-TRIS propane, TEA, and CAPS (FIG. 4B).

(5) FIGS. 5A-5B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and AMP (FIG. 5A) or succinic acid, BIS-TRIS propane, TEA, and CAPS (FIG. 5B). FIG. 5C shows conductivity traces observed using buffers composed of succinic acid, BIS-TRIS propane, TEA, and AMP.

(6) FIGS. 6A-6B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and CAPS (FIG. 6A), or succinic acid, BIS-TRIS propane, TEA, and CAPSO (FIG. 6).

(7) FIGS. 7A-7B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, EPPS, and CAPSO (FIG. 7A) or (B) succinic acid, BIS-TRIS propane, TEA, and CAPSO (FIG. 7B).

(8) FIGS. 8A-8B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of MES, BIS-TRIS propane, TEA, and CAPSO (FIG. 8A) or (B) succinic acid, BIS-TRIS propane, TEA, and CAPSO (FIG. 8B). FIG. 8C shows the conductivity traces observed using buffers composed of MES, BIS-TRIS propane, TEA, and CAPSO (solid line), or succinic acid, BIS-TRIS propane, TEA, and CAPSO (dashed line).

(9) FIGS. 9A-9B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and β-alanine (FIG. 9A) or (B) succinic acid, BIS-TRIS propane, TEA, and CAPS (FIG. 9B).

(10) FIGS. 10A-10B show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO and the ionic strengths were mediated with sodium chloride (FIG. 10A) or potassium chloride (FIG. 10B).

(11) FIGS. 11A-11E show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO when the pH values of the A buffers were titrated to 3.5 (FIG. 11A), 4.2 (FIG. 11B), 5.0 (FIG. 11C), 5.2 (FIG. 11D), or 5.5 (FIG. 11E) while keeping the pH of buffer B at 10.2.

(12) FIG. 12A shows composition details for the pH gradient buffers b-h. FIGS. 12B-12H show pH traces obtained using buffer b (FIG. 12B), buffer c (FIG. 12C), buffer d (FIG. 12D), buffer e (FIG. 12E), buffer f (FIG. 12F), buffer g (FIG. 12G), and buffer h (FIG. 12H) (dashed traces) overlaid with buffer a (solid traces).

(13) FIGS. 13A-13H show ion exchange chromatograms of panitumumab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO with the ionic strengths of buffer salts as specified in FIG. 12A. In particular, FIGS. 13A-13H show pH traces obtained using buffer a (FIG. 13A), buffer b (FIG. 13B), buffer c (FIG. 13C), buffer d (FIG. 13D), buffer e (FIG. 13E), buffer f (FIG. 13F), buffer g (FIG. 13G), and buffer h (FIG. 13H).

(14) FIGS. 14A-14B show exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO with the buffer compositions described as buffer h in FIG. 12A (FIG. 14A) and with a gradient of the buffer salt concentrations starting with 10% lower values in mobile phase A (versus mobile phase B) (FIG. 14B).

(15) FIGS. 15A-15C show ion exchange chromatograms of panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671 obtained using buffers comprised of piperazine, imidazole, and Tris (FIG. 15A), Thermo Scientific CX-1 Buffer Kit (FIG. 15B), or succinic acid, BIS-TRIS propane, TEA, and CAPSO (FIG. 15C).

DETAILED DESCRIPTION

(16) As seen from the detailed description of certain beneficial embodiments to follow, the present disclosure provides novel mobile phase compositions for facilitating high-resolution, reproducible pH gradient cation exchange separations of mAbs having pI values ranging from 6 to 10, among other uses. These mobile phase compositions allow the use of a binary mobile phase system and were developed by means of empirical optimization according to experimental results linking mobile phase composition to resolving power. In the Examples to follow, the particular mobile phases developed are comprised of four buffer salts, namely a single diprotic acid, a single divalent buffer salt with two amine groups, a single monovalent buffer salt comprising a single amine group, and a single zwitterionic buffer salt. In specific embodiments, the diprotic acid is succinic acid, the divalent buffer salt is BIS-TRIS propane (1,3-bis(tris(hydroxymethyl)methylamino)propane), the monovalent buffer salt is TEA (triethanolamine), and the zwitterionic buffer salt is CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), or β-alanine (3-aminopropanoic acid). In one mobile phase, the buffer salts are titrated to a low pH. In the other mobile phase, the buffer salts are titrated to a high pH and supplemented with a non-buffer salt. The pH range employed is 5.0 to 11.0, and more ideally 5.0 to 10.2, as optimized to achieve balanced resolution of mAbs exhibiting a wide range of pI values. The concentrations of the buffer salts and the non-buffer salt are optimized to achieve high mAb resolution, a linear pH trace, and an advantageous conductivity trace. The developed method delivers high resolution for a wide range of mAbs and good pH linearity with individual buffer salt concentrations ranging from 2 to 20 millimolar, more ideally from 5 to 15 millimolar, and most ideally from 8 to 11 millimolar. To achieve optimal resolving power, the second mobile phase buffer is provided with a non-buffer salt, such as sodium chloride (NaCl) or potassium chloride (KCl), at a concentration of 1 to 100 millimolar, or more ideally 5 to 60 millimolar. This non-buffer salt is added to fine tune the conductivity trace associated with the pH gradient. As well, in a preferred embodiment, the binary gradient change from low to high pH values can also optionally entail a gradient change in the concentration of the buffer salts, including one as large as 30% but also as small as 5%. That is, the first mobile phase contains buffer salt concentrations that are 30%, or more ideally 10%, lower than the second mobile phase.

(17) To evaluate the resolving power of pH gradient ion exchange chromatography, five mAbs (panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb reference material 8671) were routinely analyzed. An effective peak capacity of infliximab, a pseudo resolution between the acidic variant and the main peak of trastuzumab, and peak-to-valley ratios (p/v) of the first lysine variant of adalimumab and NIST mAb were calculated from UV chromatograms as demonstrated in FIGS. 1A-1D, respectively.

(18) In one exemplary embodiment of present disclosure, the pH gradient buffers are composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO with the composition described in FIG. 2A. When used in combination with a 3 μm non-porous sulfonated cation exchange stationary phase packed into a 4.6×50 mm column dimension, high resolution separations can be achieved for each of the tested mAbs. UV chromatograms corresponding to these analyses are shown in FIG. 2B along with calculated values on peak capacity, resolution, and p/v ratios. Using this method, good resolution could be achieved for each mAb using a rapid 0.23 pH unit/minute gradient.

(19) Buffer salts used in the present Examples are shown the following Table 1:

(20) TABLE-US-00001 TABLE 1 Short Name IUPAC Name pKa1 pKa2 pKa3 ACES 2-(carbamoylmethylamino)ethane- 6.9 n/a n/a sulfonic acid AMP 2-amino-2-methyl-1-propanol 9.7 n/a n/a AMPD 2-amino-2-methyl-1,3-propanediol 8.8 n/a n/a β-alanine 3-Aminopropanoic acid 3.6 10.4  n/a BIS-TRIS 1,3-bis(tris(hydroxymethyl)meth- 6.8 9.1 n/a Propane ylamino)propane CAPS 3-(Cyclohexylamino)-1- 10.4 n/a n/a propanesulfonic acid CAPSO 3-(Cyclohexylamino)-2-hydroxy-1- 9.6 n/a n/a propanesulfonic acid Citric Acid 2-Hydroxypropane-1,2,3- 3.1 4.8 6.4 tricarboxylic acid EPPS 4-(2-hydroxyethyl)-1-piperazine 7.9 n/a n/a propanesulfonic acid Gly Aminoethanoic acid 2.4 8.1 n/a Gly-Gly 2-[(2-Aminoacetyl)amino] 3.1 8.1 n/a acetic acid Histidine 2-Amino-3-(1H-imidazol-4-yl)pro- 1.8 6.1 9.2 panoic acid Imidazole 1,3-diazacyclopenta-2,4-diene 7.0 n/a n/a MES 2-morpholin-4-ylethanesulfonic 6.2 n/a n/a acid Piperazine 1,4-Diazinane 5.7 9.8 n/a Succinic 1,4-butandioic acid 4.1 5.5 n/a Acid TEA triethanolamine 7.8 n/a n/a Tris 2-Amino-2-(hydroxymethyl)pro- 8.1 n/a n/a pane-1,3-diol

(21) Various unexpected results have been observed which have led to the present disclosure. In this regard, the five above-mentioned mAbs were used to evaluate three different comparative pH gradient mobile phase compositions. Chromatograms resulting from the use of buffers based on two zwitterionic buffer salts bearing both carboxyl and amine groups (histidine and Gly-Gly) are shown in FIG. 3A. Noteworthy resolution was observed for panitumumab and the acidic variant of trastuzumab. However, the basic variant of trastuzumab was not well resolved. In addition, only one major peak was observed from infliximab, and resolution for the basic variants of adalimumab and NIST mAb was unsatisfactory. This buffer system was observed to yield a near linear conductivity trace, but its pH trace was seen to be comparatively non-linear, presumably to the detriment of its resolving power. To test another buffer system, trivalent citric acid was combined with glycine and Gly-Gly, and in turn more linear pH and conductivity traces were obtained. High resolution separations were achieved for panitumumab, adalimumab, and NIST mAb, and reasonably good resolution was even seen for the separation of infliximab (FIG. 3A). The basic variant of trastuzumab, however, was not resolved. In yet another buffer composition, a zwitterionic buffer salt bearing both sulfonate and amine groups (MES) was tested in combination with glycine and Gly-Gly. Despite improving the separations of panitumumab, infliximab, and adalimumab and showing reasonably linear pH and conductivity traces, this buffer system did not yield satisfactory separation of the basic variants of trastuzumab and NIST mAb (FIG. 3C). In sum, the above examples demonstrate that mAb separations should be carefully evaluated during the development of pH gradient ion exchange methods and that the utility of various buffer salts cannot be assumed.

(22) Next, alternatives to the preferred buffer composition were explored, and the significance of choosing a divalent buffer salt with two amine groups, specifically, BIS-Tris propane, was substantiated. In contrast to the previous three comparative buffer compositions, a combination of a diprotic acid, specifically, succinic acid, a divalent buffer salt with two amine groups, specifically BIS-TRIS propane, a monovalent buffer salt having an amine group, specifically, TEA, and a zwitterionic buffer salt, specifically, CAPS resulted in notably balanced resolution for infliximab, trastuzumab, adalimumab and NIST mAb (FIG. 4B). Replacing the divalent buffer salt having two amine groups (BIS-TRIS propane), with two buffer salts of comparable pKa values (ACES and AMPD) resulted in similar resolution for panitumumab, infliximab, adalimumab, and the acidic variant of trastuzumab (FIG. 4A). However, resolution on the basic variants of trastuzumab and NIST mAb were better with the buffer containing divalent buffer salt. ACES is a zwitterionic monovalent buffer salt with a sulfonate group and an amine group, and AMPD is a monovalent buffer salt with an amine group. The pH gradient buffer containing a negatively charged sulfonate group from the zwitterionic buffer salt ACES showed lower conductivities at low to medium pH range and have slightly higher conductivities at high pH range compared to the buffer composed of BIS-TRIS propane. Regardless, no benefit to resolution was observed for mAbs with lower to median pI values, and the resolution for higher pI mAbs was better with the buffers composed of BIS-TRIS propane.

(23) In the buffer composed of succinic acid, BIS-TRIS propane, TEA, and CAPS, replacing the zwitterionic CAPS with monovalent AMP resulted in similar resolution for panitumumab, infliximab, trastuzumab, and adalimumab (FIGS. 5A-5B). Although the p/v ratios for the basic variant of NIST mAb were nearly identical using the two buffer compositions, the peak profile obtained with the buffer containing AMP was distorted versus established data sets. The pH traces of the buffers composed of CAPS and AMP were highly similar. However, a concave curve in the conductivity trace was observed at a retention time of 14 minutes when using the buffer composed of AMP, while the conductivity trace remained relatively steady in the same range with the buffer containing CAPS. Without wishing to be bound by theory of operation, the sulfonate group in CAPS is believed to impart a desired linearity to the conductivity trace and, as a consequence, yield reliable peak profiles for mAbs with higher pI values. In this way, buffers comprised of CAPS, but not AMP, are deemed to be beneficial for this method.

(24) When CAPS is replaced by another zwitterionic monovalent buffer salt having a slightly lower pKa value (CAPSO, 9.6 versus 10.4), similar resolution values for panitumumab and infliximab were observed (FIG. 6A-6B). Moreover, the resolution metrics for trastuzumab, adalimumab, and NIST mAb were slightly improved. Since a large portion of mAbs have pI values in the range of 6 to 10, the use of CAPSO may be preferred in some instances. However, in the case of analyzing a mAb with an inordinately high pI, CAPS may be applied to ensure a higher final pH value can be achieved with the described binary gradient. So, while the use of CAPSO is a preferred embodiment in some instances, it could, in other instances, be advantageous to use a buffer containing CAPS.

(25) The replacement of TEA with an alternative buffer salt was also explored. In a buffer composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO, the monovalent buffer salt TEA was replaced with a zwitterionic monovalent buffer salt having a comparable pKa (EPPS) and slightly better resolution was observed for NIST mAb with EPPS. However, the separations of panitumumab, infliximab, trastuzumab, and adalimumab were found to suffer (FIGS. 7A-7B). In addition, a more pronounced baseline shift was observed in the UV chromatograms. Although similar pH traces were observed with the two buffer systems, the conductivity of the buffer system containing EPPS was much higher than the buffer system containing TEA, even though their concentration of non-buffer salt was identical. It appears, as a result, that the buffer system based on TEA gives more balanced capabilities for mAb charge variant profiling. Accordingly, in various beneficial embodiments, TEA is used.

(26) In a similar fashion, the changeability of the diprotic acid buffer salt succinic was investigated. In a buffer composed of succinic acid, BIS-Tris propane, TEA, and CAPSO, the diprotic acid buffer salt succinic acid was replaced with a zwitterionic monovalent buffer salt having comparable pKa (MES). However, no benefit to resolution was observed for infliximab, trastuzumab, and adalimumab, despite a reduction in conductivity at medium to high pH ranges (FIGS. 8A-8B). Moreover, the low pI mAb, panitumumab, was poorly retained with the alternative MES buffer. Furthermore, resolution for NIST mAb was noticeably worse. Thus, a buffer containing the diprotic acid buffer salt succinic acid, rather than the zwitterionic monovalent buffer salt MES, is preferred.

(27) Given the above results, it has been determined that a buffer system based on succinic acid, BIS-Tris propane, TEA, and CAPSO affords a highly capable mAb charge profiling method (FIG. 2B). Nevertheless, it should be recognized that several alternative buffer systems could also be effectively used. For example, CAPSO can be successfully replaced with either CAPS or β-alanine. In both cases, reasonably high resolution can be achieved for panitumumab, infliximab, trastuzumab, adalimumab and NIST mAb (FIGS. 9A-9B). In addition, with either buffer system, the pH and conductivity traces were found to be relatively linear. In this way, other zwitterionic buffer salts with a carboxyl group, such as amino acids or short peptides are expected to be suitable for use in combination with succinic acid, BIS-Tris propane, and TEA. In another alternative embodiment, the non-buffer salt sodium chloride can be replaced with potassium chloride without compromising resolution capabilities (FIGS. 10A-10B). In certain beneficial embodiments, the concentration of the non-buffer salt in the second mobile phase buffer is 1 to 100 millimolar, and more ideally 5 to 60 millimolar.

(28) Based on exploration of pH gradient buffer compositions, the pH range of buffers composed of succinic acid, BIS-Tris propane, TEA, and CAPSO was optimized by varying the pH of the first mobile phase buffer (buffer A) from 3.5 to 5.5 while keeping the pH of the second mobile phase buffer (buffer B) at 10.2. The peak capacities of infliximab and the p/v ratios of the basic variant of NIST mAb were found to improve slightly as the pH of buffer A increased from 3.5 to 5.5. Meanwhile, the resolution of trastuzumab and adalimumab remained relatively constant (FIGS. 11A-11E). Most notably, the retention of panitumumab, a mAb with a pI of 6.7, was poor when the pH of buffer A was titrated to 5.5 (FIG. 11E). Lowering the pH of buffer A to 5.2 improved the retention of panitumumab, but the peak profile was somewhat distorted comparing to established results (FIG. 11D). Hence, in a certain beneficial embodiments, a buffer system composed of succinic acid, BIS-TRIS propane, TEA, and CAPSO is prepared so as to have one mobile phase titrated to a pH of 5.0 and the other to 10.2.

(29) In another study, the concentrations of the components in the preferred buffer system were optimized. Buffer composition a in FIG. 12A was taken as a starting point to optimize for linear pH traces across a gradient between pH 5.0 and 10.2. Although this buffer composition is effective in providing a linear pH trace at relatively shallow gradients, it was found that an experimental pH trace obtained with a rapid gradient of 0.46 pH unit/minute showed deviations from a theoretical, linear trace (FIG. 12B). In an attempt to mitigate kinetic effects, the present inventors also empirically evaluated how the linearity of the pH trace was affected by changes to the concentrations of each individual buffer salts. Through this work, it was seen that increasing the concentrations of BIS-Tris propane or CAPSO gave slight improvements to pH linearity. Conversely, changing the concentrations of TEA did not appear to have a significant effect (FIG. 12B-12G). In turn, the present inventors were successful in determining an optimal ratio of succinic acid:BIS-TRIS propane:TEA:CAPSO (FIG. 12H). By evaluating effects on mAb resolution, an effective concentration range for each individual buffer salt was determined. Chromatograms obtained with various buffers (a-h) are shown in FIGS. 13A-13H. Varying the concentration of BIS-Tris propane by ±25% from 8.3 mM did not affect the resolution of infliximab or trastuzumab, but it did appear to cause slight fluctuations in the resolution of the basic variants of adalimumab and NIST mAb (FIGS. 13A-13C). Similarly, varying the concentration of CAPSO by ±33% from 6.0 mM and the concentration of TEA by ±22% from 9.0 mM only resulted in slight fluctuations of the resolution for the five tested mAbs (FIGS. 13D-13G). Ultimately, the separations observed for infliximab, trastuzumab, adalimumab and NIST mAb with buffer h were comparable to the results achieved with buffers a-g (FIG. 13H). However, it was noticed that the resolution of panitumumab with buffer h was compromised if compared to buffers a-g, likely due to its relatively high ionic strength. In order to improve the resolution of a low pI mAb, the concentrations of the four buffer salts in mobile phase A were each decreased by 10% (FIGS. 14A-14B). In doing this, the resolution of panitumumab visibly improved, while the resolution of the other mAbs remained unchanged (FIG. 14B). In a preferred embodiment, the pH gradient buffer system of this disclosure is comprised of individual buffer salts (for example succinic acid, BIS-TRIS propane, TEA, and CAPSO) at concentrations ranging from 2 to 20 mM. As well, in a preferred embodiment, the binary gradient corresponding to the lowest and highest pH values can also optionally include a gradient change in the concentration of the buffer salts, including a gradient change as large as 30% but also as small as 5%.

(30) The chromatographic capabilities of the developed pH gradient buffer system are exemplary and this can be visualized in a comparative example. To this end, a study was performed to compare the buffer system of this disclosure to that prepared by a commercially-available product from Thermo Scientific (CX-1 Buffer Kit) (Fekete, S.; Beck, A.; Fekete, J.; Guillarme, D., “Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach.” Journal of pharmaceutical and biomedical analysis 2015, 102, 282-9) and a previously published buffer system (see Zhang et al., supra). Direct comparisons of mAb charge variant separations using these three salt-mediated pH gradient cation exchange chromatography methods were performed using a 3 μm non-porous sulfonated cation exchange stationary phase and gradients of matching slope (0.23 pH unit/minute). Implementation of the buffer system described by Zhang et al. (composed of piperazine, imidazole, and Tris) failed to provide robust separations of medium to high pI mAbs, although the low pI mAb, panitumumab, was well resolved (FIG. 15A). The inadequacy of this method in the present case originates from the fact that it was designed for use with a carboxymethyl weak cation exchange stationary phase and not a sulfonated strong cation exchange stationary phase. In contrast, the CX-1 buffer kit was designed to be used with either type of ion exchanger. Versus the buffer system described herein, similar resolution for infliximab and trastuzumab could be achieved with the mobile phases prepared with a Thermo CX-1 Buffer Kit (FIGS. 15B and 15C). However, through this comparison, enhanced resolution for high pI mAbs, e.g. adalimumab and NIST mAb, was achieved with the compositions of the present disclosure. In summary, the buffer system of this disclosure has been found to provide the most balanced resolving power for mAb charge variant profiling. Additionally, it has been found that it is particularly advantageous to employ the inventive compositions with a 3 μm non-porous sulfonated strong cation exchange stationary phase. That is, the mobile phase buffer system describe here in would be ideal in pairing with the cation exchange stationary phases described in U.S. Provisional Patent Application No. 62/635,699, filed Feb. 27, 2018 and entitled “Polymer Particles with a Gradient Composition and Methods of Production Thereof” which is hereby incorporated by reference. Additionally, this mobile phase buffer system could be advantageously paired with other cation exchange columns, including but not limited to Thermo Scientific MAb Pac SCX, Thermo Scientific Pro Pac SCX, Thermo Scientific Pro Pac WCX, and YMC BioPro SP-F.

(31) As seen from the above, advantages of the present disclosure include the ability to perform charge variant profiling of mAb-based therapeutics with enhanced peak resolution and reproducibility.

(32) Although optimized to achieve high resolution for mAb charge variants, the methods, compositions and kits described herein can be used to separate other analytes, including other types of biomolecules, particular examples of which include peptides, other proteins including naturally occurring non-mAb proteins, fusion proteins and antibody drug conjugates (ADCs), among others.

(33) Further aspects of the present disclosure will now be described in the following enumerated paragraphs.

(34) Aspect A1. A chromatographic elution buffer solution comprising (a) a first buffer salt comprising a first pKa value, (b) a second buffer salt comprising a second pKa value, (c) a third buffer salt comprising a third pKa value, and (d) a fourth buffer salt comprising a fourth pKa value, wherein the first pKa value is less than the second pKa value, wherein the second pKa value is less than the third pKa value, and wherein the third pKa value is less than the fourth pKa value, and wherein the buffer solution has a pH ranging from 3 to 11.

(35) Aspect A2. The chromatographic elution buffer of Aspect A1, wherein the first pKa value differs from the second pKa value by a first amount ranging from 0.2-2.0, wherein the second pKa value differs from the third pKa value by a second amount ranging from 0.2-2.0, and wherein the third pKa value differs from the fourth pKa value by a third amount ranging from 0.2-2.0.

(36) Aspect A3. The chromatographic elution buffer of Aspect A1, wherein the first pKa value ranges from 3 to 5, wherein the second pKa value ranges from 5 to 7, wherein the third pKa value ranges from 7 to 9, and wherein the fourth pKa value ranges from 9 to 11.

(37) Aspect A4. A chromatographic elution buffer solution of any of Aspects A1-A3, wherein the first buffer salt is a diprotic acid, (b) wherein the second buffer salt is a divalent buffer salt with two amine groups, (c) wherein the third buffer salt is a monovalent buffer salt comprising a single amine group, and (d) wherein the fourth buffer salt is a zwitterionic buffer salt.

(38) Aspect A5. The chromatographic elution buffer of Aspect A4, wherein the diprotic acid buffer salt is succinic acid.

(39) Aspect A6. The chromatographic elution buffer of any of Aspects A4-A5, wherein the divalent buffer salt is BIS-TRIS propane.

(40) Aspect A7. The chromatographic elution buffer of any of Aspects A4-A6, wherein the monovalent buffer salt is selected from triethanolamine and TRIS.

(41) Aspect A8. The chromatographic elution buffer of any of Aspects A4-A7, wherein the zwitterionic buffer salt is selected from zwitterionic buffer salt comprising a sulfonate group and an amine group and a zwitterionic buffer salt comprising a carboxyl group and an amine group.

(42) Aspect A9. The chromatographic elution buffer of any of Aspects A4-A8, wherein the zwitterionic buffer salt is selected from CAPSO, CAPS and β-alanine.

(43) Aspect A10. A chromatographic elution buffer kit comprising (a) a first buffer solution comprising a chromatographic elution buffer in accordance with any of Aspects A1-9, wherein the buffer solution has a pH ranging from 3 to 7 or has a pH ranging from 3 to 7 upon dilution with water in a ratio ranging from 1:2 to 1:20 and (b) a second buffer solution comprising a chromatographic elution buffer in accordance with any of Aspects A1-9 and further comprising a non-buffer salt, wherein the second buffer solution has a pH ranging from 9 to 11 or has a pH ranging from 9 to 11 upon dilution with water in a ratio ranging from 1:2 to 1:20.

(44) Aspect A11. The chromatographic elution buffer kit of Aspect A10, wherein each of the first, second, third and fourth buffer salts is present in the first buffer solution at a concentration ranging from 2 to 20 millimolar and/or wherein each of the first, second, third and fourth buffer salts is present in the second buffer solution at a concentration ranging from 2 to 20 millimolar.

(45) Aspect A12. The chromatographic elution buffer kit of any of Aspects A10-A11, wherein the non-buffer salt comprises (a) a cation selected from alkali metal cations, alkaline earth metal cations, transition metal cations, ammonium cations and (b) an anion selected from selected from halide anions, nitrate anions, sulfate anions, phosphate anions, carbonate anions, chlorate anions, thiocyanate anions and perchlorate anions

(46) Aspect A13. The chromatographic elution buffer kit of Aspect A12, wherein the concentration of the non-buffer salt ranges from 1 to 100 millimolar.

(47) Aspect A14. The chromatographic elution buffer kit of any of Aspects A10-A13, wherein a concentration of each of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a concentration of each of the first, second, third and fourth buffer salts in the second buffer solution.

(48) Aspect A15. The chromatographic elution buffer kit of any of Aspects A10-A13, wherein a total concentration of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a total concentration of the first, second, third and fourth buffer salts in the second buffer solution.

(49) Aspect A16. The chromatographic elution buffer kit of any of Aspects A10-A14, wherein a plot of pH versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

(50) Aspect A17. The chromatographic elution buffer kit of any of Aspects A10-A16, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

(51) Aspect A18. The chromatographic elution buffer kit of any of Aspects A10-A14, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution does not exhibit a negative slope.

(52) Aspect A19. The chromatographic elution buffer kit of any of Aspects A10-A17, wherein the first buffer solution has a conductivity ranging from 0.5 millisiemins (mS) to 3 mS and wherein the second buffer solution has a conductivity ranging from 3 mS to 100 mS.

(53) Aspect A20. The chromatographic elution buffer kit of any of Aspects A10-A19, further comprising an ion-exchange chromatography material.

(54) Aspect A21. The chromatographic elution buffer kit of Aspect A20, comprising a separation device comprising a housing comprising an inlet and an outlet that is configured to accept and hold the ion-exchange chromatography material.

(55) Aspect A22. The chromatographic elution buffer kit of any of Aspects A20-A21, wherein the ion-exchange chromatography material is a cation exchange chromatography material.

(56) Aspect A23. The chromatographic elution buffer kit of Aspect A22, wherein the cation exchange chromatography material comprises carboxylate groups.

(57) Aspect A24. The chromatographic elution buffer kit of Aspect A22, wherein the cation exchange chromatography material comprises sulfonate groups.

(58) Aspect A25. A method for analyzing a sample comprising a plurality of analytes, the method comprising: loading the sample onto an ion-exchange chromatography material thereby binding the plurality of analytes to the ion-exchange chromatography material and eluting the plurality of analytes from the ion-exchange chromatography material with a mobile phase comprising a chromatographic elution buffer in accordance with any of Aspects A1-9, thereby separating at least some of the plurality of analytes.

(59) Aspect A26. The method of Aspect A25, wherein eluting the plurality of analytes from the ion-exchange chromatography material with the mobile phase comprises a course of elution in which a pH of the mobile phase is altered over time, in which an ionic strength of the mobile phase is altered over time, or both.

(60) Aspect A27. The method of Aspect A25, wherein eluting the plurality of analytes from the ion-exchange chromatography material with the mobile phase comprises a course of elution in which a pH of the mobile phase is increased over time, in which an ionic strength of the mobile phase is increased over time, or both.

(61) Aspect A28. The method of any of Aspects A26-A27, wherein the pH of the mobile phase is increased during the course of elution.

(62) Aspect A29. The method of Aspect A28, wherein the pH of the mobile phase increases from 5.0 to 11.0 during the course of elution.

(63) Aspect A30. The method of any of Aspects A28-A29, wherein there is a linear increase in the pH of the mobile phase during the course of elution.

(64) Aspect A31. The method of any of Aspects A26-A30, wherein the ionic strength of the mobile phase is increased during the course of elution.

(65) Aspect A32. The method of any of Aspects A26-A30, wherein there is a linear increase in the ionic strength of the mobile phase during the course of elution.

(66) Aspect A33. The method of any of Aspects A26-A32, wherein a conductivity of the mobile phase increases during the course of elution.

(67) Aspect A34. The method of any of Aspects A26-A32, wherein there is a linear increase in the conductivity during the course of elution.

(68) Aspect A35. The method of any of Aspects A33-A34, wherein the conductivity of the mobile phase increases between 1.5-fold and 10-fold during the course of elution.

(69) Aspect A36. The method of any of Aspects A33-A35, wherein the conductivity of the mobile phase increases from a first value between 0.5 mS and 3 mS to a second value between 3 mS and 100 mS during the course of elution.

(70) Aspect A37. The method of any of Aspects A26-A36, wherein a concentration of each of the first, second, third and fourth buffer salts in the mobile phase increases between 10% and 40% during the course of elution.

(71) Aspect A38. The method of any of Aspects A26-A36, wherein a total concentration of all of the first, second, third and fourth buffer salts in the mobile phase increases between 10% to 45% during the course of elution.

(72) Aspect A39. The method of any of Aspects A26-A38, wherein an automated system is used to mix two or more solutions to form the mobile phase.

(73) Aspect A40. The method of any of Aspects A26-A38, wherein an automated system is used to mix three or more solutions to form the mobile phase.

(74) Aspect A41. The method of any of Aspects A26-A40, wherein during at least a portion of the course of elution, the mobile phase comprises a non-buffer salt.

(75) Aspect A42. The method of Aspect A41, wherein the course of elution comprises a period during which a concentration of the non-buffer salt increases over time.

(76) Aspect A43. The method of any of Aspects A41-A42, wherein during the course of elution the mobile phase is formed from a buffer mixture that comprises (a) a first buffer solution comprising the first, second, third and fourth buffer salts, the first buffer solution having a pH ranging from 3 to 7 and (b) a second buffer solution comprising the first, second, third and fourth buffer salts and further comprising the non-buffer salt, the second buffer solution having a pH ranging from 9 to 11.

(77) Aspect A44. The method of Aspect A35, wherein the buffer mixture comprises a binary mixture of the first buffer solution and the second buffer solution.

(78) Aspect A45. The method of any of Aspects A43-A44, wherein during the course of elution a first volume percent of the first buffer solution in the buffer mixture is decreased over time while at the same time increasing a second volume percent of the second buffer solution over time in a concentration gradient separation

(79) Aspect A46. The method of Aspect A45, wherein the first volume percent in the buffer mixture decreases linearly over time and the second volume percent in the buffer mixture increases linearly during the course of elution.

(80) Aspect A47. The method of any of Aspects A25-A46, further comprising detecting the plurality of analytes.

(81) Aspect A48. The method of any of Aspects A25-A47, wherein the plurality of analytes comprises a plurality of biomolecules.

(82) Aspect A49. The method of any of Aspects A25-A47, wherein the plurality of analytes comprises a plurality of proteins.

(83) Aspect A50. The method of any of Aspects A25-A47, wherein the plurality of analytes comprises a plurality of mAb species having pI values ranging from 6 to 10.

(84) Aspect B1. A chromatographic elution buffer solution comprising a plurality of buffer salts, the buffer solution having a first pKa value that ranges from 4.5 to 6.5, a second pKa value that ranges from 6.5 to 7.5, a third pKa value that ranges from 7.5 to 8.5, a fourth pKa value that ranges from 8.5 to 9.5, and a fifth pKa value that ranges from 9.5 to 11.

(85) Aspect B2. The chromatographic elution buffer solution of Aspect B1, wherein the buffer solution has a pH ranging from 3 to 11.

(86) Aspect B3. The chromatographic elution buffer solution of any of Aspects B1-B2, wherein the first pKa value is provided by a first buffer salt that is a diprotic acid, (b) wherein the second pKa value and the fourth pKa value are provided by a second buffer salt that is a divalent buffer salt with two amine groups, (c) wherein the third pKa is provided by a third buffer salt that is a monovalent buffer salt comprising a single amine group, and (d) wherein the fifth pKa value is provided by a forth buffer salt that is a zwitterionic buffer salt.

(87) Aspect B4. The chromatographic elution buffer solution of Aspect B3, comprising a single zwitterionic buffer salt.

(88) Aspect B5. The chromatographic elution buffer of any of Aspects B3-B4, wherein the diprotic acid buffer salt is succinic acid.

(89) Aspect B6. The chromatographic elution buffer of any of Aspects B3-B5, wherein the divalent buffer salt is BIS-TRIS propane.

(90) Aspect B7. The chromatographic elution buffer of any of Aspects B3-B6, wherein the monovalent buffer salt is selected from triethanolamine and TRIS.

(91) Aspect B8. The chromatographic elution buffer of any of Aspects B3-B7, wherein the zwitterionic buffer salt is selected from zwitterionic buffer salt comprising a sulfonate group and an amine group and a zwitterionic buffer salt comprising a carboxyl group and an amine group.

(92) Aspect B9. The chromatographic elution buffer of any of Aspects B3-B8, wherein the zwitterionic buffer salt is selected from CAPSO, CAPS and βalanine.

(93) Aspect B10. A chromatographic elution buffer kit comprising (a) a first buffer solution comprising a chromatographic elution buffer in accordance with any of Aspects B1-B9, wherein the buffer solution has a pH ranging from 3 to 7 or has a pH ranging from 3 to 7 upon dilution with water in a ratio ranging from 1:2 to 1:20 and (b) a second buffer solution comprising a chromatographic elution buffer in accordance with any of Aspects B1-B9 and further comprising a non-buffer salt, wherein the second buffer solution has a pH ranging from 9 to 11 or has a pH ranging from 9 to 11 upon dilution with water in a ratio ranging from 1:2 to 1:20.

(94) Aspect B11. The chromatographic elution buffer kit of Aspect B10, wherein each of the first, second, third and fourth buffer salts is present in the first buffer solution at a concentration ranging from 2 to 20 millimolar and/or wherein each of the first, second, third and fourth buffer salts is present in the second buffer solution at a concentration ranging from 2 to 20 millimolar.

(95) Aspect B12. The chromatographic elution buffer kit of any of Aspects B10-B11, wherein the non-buffer salt comprises (a) a cation selected from alkali metal cations, alkaline earth metal cations, transition metal cations, ammonium cations and (b) an anion selected from selected from halide anions, nitrate anions, sulfate anions, phosphate anions, carbonate anions, chlorate anions, thiocyanate anions and perchlorate anions

(96) Aspect B13. The chromatographic elution buffer kit of Aspect B12, wherein the concentration of the non-buffer salt ranges from 1 to 100 millimolar.

(97) Aspect B14. The chromatographic elution buffer kit of any of Aspects B10-B13, wherein a concentration of each of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a concentration of each of the first, second, third and fourth buffer salts in the second buffer solution.

(98) Aspect B15. The chromatographic elution buffer kit of any of Aspects B10-B13, wherein a total concentration of the first, second, third and fourth buffer salts in the first buffer solution is from 10% to 30% lower than a total concentration of the first, second, third and fourth buffer salts in the second buffer solution.

(99) Aspect B16. The chromatographic elution buffer kit of any of Aspects B10-B14, wherein a plot of pH versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

(100) Aspect B17. The chromatographic elution buffer kit of any of Aspects B10-B16, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution is linear.

(101) Aspect B18. The chromatographic elution buffer kit of any of Aspects B10-B14, wherein a plot of conductivity versus volume percent of the first buffer solution relative to a total volume for a binary mixture of the first buffer solution and the second buffer solution does not exhibit a negative slope.

(102) Aspect B19. The chromatographic elution buffer kit of any of Aspects B10-B17, wherein the first buffer solution has a conductivity ranging from 0.5 millisiemins (mS) to 3 mS and wherein the second buffer solution has a conductivity ranging from 3 mS to 100 mS.

(103) Aspect B20. The chromatographic elution buffer kit of any of Aspects B10-B19, further comprising an ion-exchange chromatography material.

(104) Aspect B21. The chromatographic elution buffer kit of Aspect B20, comprising a separation device comprising a housing comprising an inlet and an outlet that is configured to accept and hold the ion-exchange chromatography material.

(105) Aspect B22. The chromatographic elution buffer kit of any of Aspects B20-B21, wherein the ion-exchange chromatography material is a cation exchange chromatography material.

(106) Aspect B23. The chromatographic elution buffer kit of Aspect B22, wherein the cation exchange chromatography material comprises carboxylate groups.

(107) Aspect B24. The chromatographic elution buffer kit of Aspect B22, wherein the cation exchange chromatography material comprises sulfonate groups.

(108) Aspect B25. A method for analyzing a sample comprising a plurality of analytes, the method comprising: loading the sample onto an ion-exchange chromatography material thereby binding the plurality of analytes to the ion-exchange chromatography material and eluting the plurality of analytes from the ion-exchange chromatography material with a mobile phase comprising a chromatographic elution buffer in accordance with any of Aspects B1-9, thereby separating at least some of the plurality of analytes.

(109) Aspect B26. The method of Aspect B25, wherein eluting the plurality of analytes from the ion-exchange chromatography material with the mobile phase comprises a course of elution in which a pH of the mobile phase is altered over time, in which an ionic strength of the mobile phase is altered over time, or both.

(110) Aspect B27. The method of Aspect B25, wherein eluting the plurality of analytes from the ion-exchange chromatography material with the mobile phase comprises a course of elution in which a pH of the mobile phase is increased over time, in which an ionic strength of the mobile phase is increased over time, or both.

(111) Aspect B28. The method of any of Aspects B26-B27, wherein the pH of the mobile phase is increased during the course of elution.

(112) Aspect B29. The method of Aspect B28, wherein the pH of the mobile phase increases from 5.0 to 11.0 during the course of elution.

(113) Aspect B30. The method of any of Aspects B28-B29, wherein there is a linear increase in the pH of the mobile phase during the course of elution.

(114) Aspect B31. The method of any of Aspects B26-B30, wherein the ionic strength of the mobile phase is increased during the course of elution.

(115) Aspect B32. The method of any of Aspects B26-B30, wherein there is a linear increase in the ionic strength of the mobile phase during the course of elution.

(116) Aspect B33. The method of any of Aspects B26-B32, wherein a conductivity of the mobile phase increases during the course of elution.

(117) Aspect B34. The method of any of Aspects B26-B32, wherein there is a linear increase in the conductivity during the course of elution.

(118) Aspect B35. The method of any of Aspects B33-B34, wherein the conductivity of the mobile phase increases between 1.5-fold and 10-fold during the course of elution.

(119) Aspect B36. The method of any of Aspects B33-B35, wherein the conductivity of the mobile phase increases from a first value between 0.5 mS and 3 mS to a second value between 3 mS and 100 mS during the course of elution.

(120) Aspect B37. The method of any of Aspects B26-B36, wherein a concentration of each of the first, second, third and fourth buffer salts in the mobile phase increases between 10% and 40% during the course of elution.

(121) Aspect B38. The method of any of Aspects B26-B36, wherein a total concentration of all of the first, second, third and fourth buffer salts in the mobile phase increases between 10% to 45% during the course of elution.

(122) Aspect B39. The method of any of Aspects B26-B38, wherein an automated system is used to mix two or more solutions to form the mobile phase.

(123) Aspect B40. The method of any of Aspects B26-B38, wherein an automated system is used to mix three or more solutions to form the mobile phase.

(124) Aspect B41. The method of any of Aspects B26-B40, wherein during at least a portion of the course of elution, the mobile phase comprises a non-buffer salt.

(125) Aspect B42. The method of Aspect B41, wherein the course of elution comprises a period during which a concentration of the non-buffer salt increases over time.

(126) Aspect B43. The method of any of Aspects B41-B42, wherein during the course of elution the mobile phase is formed from a buffer mixture that comprises (a) a first buffer solution comprising the first, second, third and fourth buffer salts, the first buffer solution having a pH ranging from 3 to 7 and (b) a second buffer solution comprising the first, second, third and fourth buffer salts and further comprising the non-buffer salt, the second buffer solution having a pH ranging from 9 to 11.

(127) Aspect B44. The method of Aspect B35, wherein the buffer mixture comprises a binary mixture of the first buffer solution and the second buffer solution.

(128) Aspect B45. The method of any of Aspects B43-B44, wherein during the course of elution a first volume percent of the first buffer solution in the buffer mixture is decreased over time while at the same time increasing a second volume percent of the second buffer solution over time in a concentration gradient separation

(129) Aspect B46. The method of Aspect B45, wherein the first volume percent in the buffer mixture decreases linearly over time and the second volume percent in the buffer mixture increases linearly during the course of elution.

(130) Aspect B47. The method of any of Aspects B25-B46, further comprising detecting the plurality of analytes.

(131) Aspect B48. The method of any of Aspects B25-B47, wherein the plurality of analytes comprises a plurality of biomolecules.

(132) Aspect B49. The method of any of Aspects B25-B47, wherein the plurality of analytes comprises a plurality of proteins.

(133) Aspect B50. The method of any of Aspects B25-B47, wherein the plurality of analytes comprises a plurality of mAb species having pI values ranging from 6 to 10.

(134) Further aspects of the present disclosure are detailed in the Examples to follow.

Example 1. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, BIS-TRIS Propane, TEA, and CAPSO

(135) FIGS. 2 and 15C present chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(136) TABLE-US-00002 TABLE 2 Buffer Compositions: mM Succinic acid Bis-Tris Propane TEA CAPSO NaCl pH Buffer 9.5 9.9 8.1 9.9 0 5.03 A Buffer 10.5 11.0 9.0 11.0 40.0 10.27 B

(137) TABLE-US-00003 TABLE 3 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 4.6 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/ml Flow Rate: 0.72 mL/min Column 30° C. Temperature: Injection Volume: 5 μL Detection: 280 nm

(138) TABLE-US-00004 TABLE 4 Gradient Table: Time(min) Flow Rate(mL/min) % A % B Curve Initial 0.720 100 0 Initial 1.00 0.720 100 0 6 23.60 0.720 0 100 6 26.60 0.720 0 100 6 27.60 0.720 100 0 6 40.00 0.720 100 0 6

(139) FIG. 8B present chromatograms obtained with the buffer compositions and LC conditions listed above, and gradient table listed below:

(140) TABLE-US-00005 TABLE 5 Gradient Table: Time(min) Flow Rate(mL/min) % A % B Curve Initial 0.720 100 0 Initial 1.00 0.720 100 0 6 12.30 0.720 0 100 6 15.30 0.720 0 100 6 16.30 0.720 100 0 6 20.00 0.720 100 0 6

(141) FIGS. 6B, 7B, 10A, and 11A present chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below, and FIG. 10B presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below except sodium chloride in buffer C was replaced with potassium chloride:

(142) TABLE-US-00006 TABLE 6 Buffer Compositions: succinic Bis-Tris mM acid Propane TEA CAPSO NaCl pH Buffer A 7.5 7.5 7.5 10.5 0 3.50 Buffer B 7.5 9.0 7.5 7.5 0 10.20 Buffer C 0 0 0 0 500 not adjusted

(143) TABLE-US-00007 TABLE 7 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column 30° C. Temperature: Injection Volume: 1 μL Detection: 280 nm

(144) TABLE-US-00008 TABLE 8 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 15.60 0.150 0 92 8 6 18.60 0.150 0 92 8 6 19.60 0.150 100 0 0 6 25.00 0.150 100 0 0 6

(145) FIG. 11B presents chromatograms obtained with the buffer compositions listed for FIG. 11A except the pH of buffer A was titrated to 4.22, and the gradient table listed below:

(146) TABLE-US-00009 TABLE 9 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 14.00 0.150 0 92 8 6 17.00 0.150 0 92 8 6 18.00 0.150 100 0 0 6 25.00 0.150 100 0 0 6

(147) FIG. 11C presents chromatograms obtained with the buffer compositions listed for FIG. 11A except the pH of buffer A was titrated to 5.00, and the gradient table listed below:

(148) TABLE-US-00010 TABLE 10 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 12.30 0.150 0 92 8 6 15.30 0.150 0 92 8 6 16.30 0.150 100 0 0 6 20.00 0.150 100 0 0 6

(149) FIG. 11D presents chromatograms obtained with the buffer compositions listed for FIG. 11A except the pH of buffer A was titrated to 5.22, and the gradient table listed below:

(150) TABLE-US-00011 TABLE 11 Gradient Table: Time(min) Flow Rate(mLmin) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 11.80 0.150 0 92 8 6 14.80 0.150 0 92 8 6 15.80 0.150 100 0 0 6 20.00 0.150 100 0 0 6

(151) FIG. 11E presents chromatograms obtained with the buffer compositions listed for FIG. 11A except the pH of buffer A was titrated to 5.49, and the gradient table listed below:

(152) TABLE-US-00012 TABLE 12 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 11.20 0.150 0 92 8 6 14.20 0.150 0 92 8 6 15.20 0.150 100 0 0 6 20.00 0.150 100 0 0 6

(153) FIG. 13A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(154) TABLE-US-00013 TABLE 13 Buffer Compositions: succinic Bis-Tris mM acid Propane TEA CAPSO NaCl pH Buffer A 10.5 8.3 9.0 6.0 0 4.99 Buffer B 10.5 8.3 9.0 6.0 0 10.21 Buffer C 0 0 0 0 500 not adjusted

(155) TABLE-US-00014 TABLE 14 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column 30° C. Temperature: Injection Volume: 1 μL Detection: 280 nm

(156) TABLE-US-00015 TABLE 15 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 12.30 0.150 0 92 8 6 15.30 0.150 0 92 8 6 16.30 0.150 100 0 0 6 25.00 0.150 100 0 0 6

(157) FIG. 13B presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(158) TABLE-US-00016 TABLE 16 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 6.0 9.0 6.0 0 5.00 Buffer B 10.5 6.0 9.0 6.0 0 10.21 Buffer C 0 0 0 0 500 not adjusted

(159) FIG. 13C presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(160) TABLE-US-00017 TABLE 17 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 10.5 9.0 6.0 0 5.00 Buffer B 10.5 10.5 9.0 6.0 0 10.20 Buffer C 0 0 0 0 500 not adjusted

(161) FIG. 13D presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(162) TABLE-US-00018 TABLE 18 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 8.3 9.0 4.0 0 4.99 Buffer B 10.5 8.3 9.0 4.0 0 10.22 Buffer C 0 0 0 0 500 not adjusted

(163) FIG. 13E presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(164) TABLE-US-00019 TABLE 19 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 8.3 9.0 8.0 0 4.99 Buffer B 10.5 8.3 9.0 8.0 0 10.20 Buffer C 0 0 0 0 500 not adjusted

(165) FIG. 13F presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(166) TABLE-US-00020 TABLE 20 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 8.3 11.0 6.0 0 5.00 Buffer B 10.5 8.3 11.0 6.0 0 10.20 Buffer C 0 0 0 0 500 not adjusted

(167) FIG. 13G presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 13A, and the buffer compositions listed below:

(168) TABLE-US-00021 TABLE 21 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 8.3 7.0 6.0 0 5.00 Buffer B 10.5 8.3 7.0 6.0 0 10.21 Buffer C 0 0 0 0 500 not adjusted

(169) FIGS. 13H and 14A present chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(170) TABLE-US-00022 TABLE 22 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPSO NaCl pH Buffer A 10.5 11.0 9.0 11.0 0 5.00 Buffer B 10.5 11.0 9.0 11.0 0 10.20 Buffer C 0 0 0 0 500 not adjusted Buffer D Milli-Q water

(171) TABLE-US-00023 TABLE 23A LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column 30° C. Temperature: Injection Volume: 1 μL Detection: 280 nm

(172) TABLE-US-00024 TABLE 23B Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 100 0 0 0 Initial 1.00 0.150 100 0 0 0 6 12.30 0.150 0 92 8 0 6 15.30 0.150 0 92 8 0 6 16.30 0.150 100 0 0 0 6 25.00 0.150 100 0 0 0 6

(173) FIG. 14B presents chromatograms obtained with the LC conditions and gradient table listed for FIG. 14A, and the buffer compositions listed below:

(174) TABLE-US-00025 TABLE 24 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 90 0 0 10 Initial 1.00 0.150 90 0 0 10 6 12.30 0.150 0 92 8 0 6 15.30 0.150 0 92 8 0 6 16.30 0.150 90 0 0 10 6 25.00 0.150 90 0 0 10 6

Example 2. Ion Exchange Chromatography with Buffers Composed of Histidine and Gly-Gly

(175) FIG. 3A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(176) TABLE-US-00026 TABLE 25 Buffer Compositions: mM Histidine Gly-Gly NaCl pH Buffer A 20.0 11.5 0 5.39 Buffer B 20.0 11.5 0 10.42 Buffer C 0 0 500 not adjusted

(177) TABLE-US-00027 TABLE 26 Gradient Table: Time(min) Flow Rate(mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial 1.00 0.150 100 0 0 6 11.80 0.150 0 92 8 6 14.80 0.150 0 92 8 6 15.80 0.150 100 0 0 6 20.00 0.150 100 0 0 6

Example 3. Ion Exchange Chromatography with Buffers Composed of Glycine, Citric Acid, and Gly-Gly

(178) FIG. 3B presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(179) TABLE-US-00028 TABLE 27 Buffer Compositions: mM Glycine Citric acid Gly-Gly NaCl pH Buffer A 10.0 10.0 11.0 0 5.20 Buffer B 10.0 10.0 11.0 0 10.41

(180) TABLE-US-00029 TABLE 28 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column 30° C. Temperature: Injection Volume: 1 μL Detection: 280 nm

(181) TABLE-US-00030 TABLE 29 Gradient Table: Time(min) Flow Rate(mL/min) % A % B Curve Initial 0.150 100 0 Initial 1.00 0.150 100 0 6 12.30 0.150 0 100 6 15.30 0.150 0 100 6 16.30 0.150 100 0 6 20.00 0.150 100 0 6

Example 4. Ion Exchange Chromatography with Buffers Composed of Glycine, MES, and Gly-Gly

(182) FIG. 3C presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(183) TABLE-US-00031 TABLE 30 Buffer Compositions: mM Glycine MES Gly-Gly NaCl pH Buffer A 10.0 12.0 11.0 0 5.45 Buffer B 10.0 12.0 11.0 0 10.41 Buffer C 0 0 0 500 not adjusted

(184) TABLE-US-00032 TABLE 31 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation exchange Phase: stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column 30° C. Temperature: Injection Volume: 1 μL Detection: 280 nm

(185) TABLE-US-00033 TABLE 32 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial  1.00 0.150 100 0 0 6 11.80 0.150 0 98 2 6 14.80 0.150 0 98 2 6 15.80 0.150 100 0 0 6 20.00 0.150 100 0 0 6

Example 5. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, ACES, 2-Amino-2-methyl-1,3-propanediol (AMPD), TEA, and CAPS

(186) FIG. 4A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(187) TABLE-US-00034 TABLE 33 Buffer Compositions: mM Succinic Acid ACES AMPD TEA CAPS NaCl pH Buffer A 5.5 4.5 5.5 3.0 4.5 0 3.49 Buffer B 5.5 4.5 5.5 3.0 4.5 0 11.00 Buffer C 0 0 0 0 0 500 not adjusted

(188) TABLE-US-00035 TABLE 34 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column Temperature: 30° C. Injection Volume: 1 μL Detection: 280 nm

(189) TABLE-US-00036 TABLE 35 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial  1.00 0.150 100 0 0 6 17.30 0.150 0 92 8 6 20.30 0.150 0 92 8 6 21.30 0.150 100 0 0 6 30.00 0.150 100 0 0 6

Example 6. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, BIS-TRIS Propane, TEA, and CAPS

(190) FIGS. 4B and 6A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(191) TABLE-US-00037 TABLE 36 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA CAPS NaCl pH Buffer A 10.0 10.0 10.0 14.0 0 3.50 Buffer B 10.0 12.0 10.0 10.0 0 11.01 Buffer C 0 0 0 0 500 not adjusted Buffer D 18.2 MΩ purified water

(192) TABLE-US-00038 TABLE 37 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column Temperature: 30° C. Injection Volume: 1 μL Detection: 280 nm

(193) TABLE-US-00039 TABLE 38 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 75 0 0 25 Initial 1.00 0.150 75 0 0 25 6 17.30 0.150 0 68 7 25 6 20.30 0.150 0 68 7 25 6 21.30 0.150 75 0 0 25 6 30.00 0.150 75 0 0 25 6

(194) FIGS. 5B and 9B present chromatograms obtained with the buffer compositions and LC conditions listed for FIG. 4B, and the gradient table listed below:

(195) TABLE-US-00040 TABLE 39 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 75 0 0 25 Initial 1.00 0.150 75 0 0 25 6 11.00 0.150 0 67 8 25 6 14.00 0.150 0 67 8 25 6 15.00 0.150 75 0 0 25 6 20.00 0.150 75 0 0 25 6

Example 7. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, BIS-TRIS propane, TEA, and AMP

(196) FIG. 5A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(197) TABLE-US-00041 TABLE 40 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA AMP NaCl pH Buffer A 13.0 10.0 10.0 10.0 0 3.53 Buffer B 13.0 10.0 10.0 10.0 0 11.01 Buffer C 0 0 0 0 500 not adjusted Buffer D 18.2 MΩ purified water

(198) TABLE-US-00042 TABLE 41 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column Temperature: 30° C. Injection Volume: 1 μL Detection: 280 nm

(199) TABLE-US-00043 TABLE 42 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 75 0 0 25 Initial 1.00 0.150 75 0 0 25 6 11.00 0.150 0 69 6 25 6 14.00 0.150 0 69 6 25 6 15.00 0.150 75 0 0 25 6 20.00 0.150 75 0 0 25 6

Example 8. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, BIS-TRIS propane, EPPS, and CAPSO

(200) FIG. 7A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(201) TABLE-US-00044 TABLE 43 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane EPPS CAPSO NaCl pH Buffer A 14.0 12.0 10.0 6.0 0 4.00 Buffer B 14.0 12.0 10.0 6.0 0 10.21 Buffer C 0 0 0 0 500 not adjusted

(202) TABLE-US-00045 TABLE 44 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column Temperature: 30° C. Injection Volume: 1 μL Detection: 280 nm

(203) TABLE-US-00046 TABLE 45 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C Curve Initial 0.150 100 0 0 Initial  1.00 0.150 100 0 0 6 14.50 0.150 0 92 8 6 17.50 0.150 0 92 8 6 18.50 0.150 100 0 0 6 25.00 0.150 100 0 0 6

Example 9. Ion Exchange Chromatography with Buffers Composed of MES, BIS-TRIS Propane, TEA, and CAPSO

(204) FIG. 8A present chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(205) TABLE-US-00047 TABLE 46 Buffer Compositions: BIS-TRIS mM MES Propane TEA CAPSO NaCl pH Buffer A 9.0 9.9 8.1 9.9 0 5.59 Buffer B 10.0 11.0 9.0 11.0 40.0 10.22

(206) TABLE-US-00048 TABLE 47 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 4.6 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.72 mL/min Column Temperature: 30° C. Injection Volume: 5 μL Detection: 280 nm

(207) TABLE-US-00049 TABLE 48 Gradient Table: Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.720 100 0 Initial  1.00 0.720 100 0 6 11.00 0.720 0 100 6 14.00 0.720 0 100 6 15.00 0.720 100 0 6 20.00 0.720 100 0 6

Example 10. Ion Exchange Chromatography with Buffers Composed of Succinic Acid, BIS-TRIS Propane, TEA, and β-alanine

(208) FIG. 9A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(209) TABLE-US-00050 TABLE 49 Buffer Compositions: Succinic BIS-TRIS mM Acid Propane TEA β-alanine NaCl pH Buffer 9.0 11.0 10.0 9.0 0 3.51 A Buffer 9.0 11.0 10.0 9.0 0 11.01 B Buffer 0 0 0 0 500 not adjusted C Buffer 18.2 MΩ purified water D

(210) TABLE-US-00051 TABLE 50 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 2.1 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.15 mL/min Column Temperature: 30° C. Injection Volume: 1 μL Detection: 280 nm

(211) TABLE-US-00052 TABLE 51 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C % D Curve Initial 0.150 75 0 0 25 Initial 1.00 0.150 75 0 0 25 6 11.00 0.150 0 67 8 25 6 14.00 0.150 0 67 8 25 6 15.00 0.150 75 0 0 25 6 20.00 0.150 75 0 0 25 6

Example 11. Ion Exchange Chromatography with Buffers Composed of Piperazine, Imidazole, and TRIS

(212) FIG. 15A presents chromatograms obtained with the buffer compositions, LC conditions, and gradient table listed below:

(213) TABLE-US-00053 TABLE 52 Buffer Compositions: mM Piperazine Imidazole TRIS NaCl pH Buffer A 4.0 4.0 4.0 0  5.05 Buffer B 4.0 4.0 4.0 0 10.76 Buffer C 0 0 0 500 not adjusted

(214) TABLE-US-00054 TABLE 53 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 4.6 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.72 mL/min Column Temperature: 30° C. Injection Volume: 5 μL Detection: 280 nm

(215) TABLE-US-00055 TABLE 54 Gradient Table: Time (min) Flow Rate (mL/min) % A % B % C Curve Initial 0.720 100 0 0 Initial  1.00 0.720 100 0 0 6 26.20 0.720 0 96.8 3.2 6 29.20 0.720 0 96.8 3.2 6 30.20 0.720 100 0 0 6 45.00 0.720 100 0 0 6

Example 12. Ion Exchange Chromatography with Thermo CX-1 pH Gradient Buffers

(216) FIG. 15B presents chromatograms obtained with the LC conditions and gradient table listed below:

(217) TABLE-US-00056 TABLE 55 LC Conditions System ACQUITY UPLC H-Class Bio Column Stationary 3 μm non-porous sulfonated strong cation Phase: exchange stationary phase Column Dimension 4.6 × 50 mm Sample: Dilute panitumumab, infliximab, trastuzumab, adalimumab, and NIST mAb with 18.2 MΩ purified water to concentrations of 5 mg/mL Flow Rate: 0.72 mL/min Column Temperature: 30° C. Injection Volume: 5 μL Detection: 280 nm

(218) TABLE-US-00057 TABLE 56 Gradient Table: Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.720 100 0 Initial  1.00 0.720 100 0 6 21.00 0.720 0 100 6 24.00 0.720 0 100 6 25.00 0.720 100 0 6 30.00 0.720 100 0 6