阅读说明：本技术 色谱方法、在色谱方法中测定至少一种化合物的浓度的方法及获得至少一种色谱方法参数的方法 (Chromatographic method, method for determining the concentration of at least one compound in a chromatographic method and method for obtaining at least one chromatographic parameter ) 是由 简·施韦伦巴赫 沃克马尔·汤姆 多米尼克·斯坦 于 2020-05-08 设计创作，主要内容包括：本发明涉及色谱方法、在色谱方法中测定至少一种化合物的浓度的方法及获得至少一种色谱方法参数的方法。(The present invention relates to a chromatographic method, a method for determining the concentration of at least one compound in a chromatographic method and a method for obtaining at least one chromatographic method parameter.)

1. A method of determining the concentration of at least one compound in a chromatographic method, the method comprising the steps of:

(ia) selecting the at least one compound;

(ib) selecting a stationary phase;

(ic) selecting a mobile phase;

(id) selecting a chromatography device having a chromatography bed comprising said stationary phase and said mobile phase;

(ie) selecting a chromatographic temperature;

wherein at least one of the composition of the mobile phase and the chromatographic temperature is varied;

(iia) obtaining an adsorption isotherm of the at least one compound on the stationary phase for the varying composition of the mobile phase and/or the varying chromatographic temperature;

(iib) determining at least the flow velocity v of the mobile phase in the chromatography bed and the bulk porosity epsilon of the chromatography bed for the varying composition of the mobile phase and/or the varying chromatography temperature_b；

(iii) Based on the adsorption isotherm, the flow velocity v and the bulk porosity epsilon_bCalculating the concentration c (z, t) of the at least one compound in the mobile phase at a predetermined position z of the chromatography device and at a predetermined time t.

2. The method of claim 1, wherein the first and second light sources are selected from the group consisting of,

wherein the stationary phase is reversibly swellable.

3. The method according to claim 1 or 2,

wherein in step (iib) the axial dispersion coefficient D of the at least one compound in the chromatography bed is also determined for the varying composition of the mobile phase and/or for the varying chromatography temperature_axAnd in step (iii) the concentration c (z, t) is calculated based also on the axial dispersion coefficient D_ax。

4. The method of any one of claims 1 to 3,

wherein in step (iii), c (z, t) is calculated based on the following equation

Wherein Z is a compound comprising

And

wherein

q (z, t) represents the binding capacity of the at least one compound through the stationary phase.

5. The method of claim 4, wherein the first and second light sources are selected from the group consisting of,

wherein said sum Z further comprises an item

6. The method according to any one of the preceding claims,

wherein the mobile phase is an aqueous medium.

7. The method according to any one of the preceding claims,

wherein a gel is formed on at least a portion of a surface of the stationary phase when the mobile phase is in contact with the stationary phase.

8. The method according to any one of the preceding claims,

wherein at least a portion of a surface of the stationary phase is comprised of a polymer bound to a surface of a stationary phase support structure.

9. The method according to any one of the preceding claims,

wherein the at least one compound comprises a protein and/or a drug.

10. A method of obtaining at least one chromatography method parameter selected from the group consisting of a stationary phase, a mobile phase and a chromatography device, said method comprising the steps of:

(I) performing the method according to any of the preceding claims n times, wherein n is an integer of 2 or more, wherein the n times of performing differ from each other with respect to at least one of steps (ib) to (id); and

(II) selecting at least one stationary phase, at least one mobile phase and/or at least one chromatographic device based on the results of step (I).

11. The method of claim 10, wherein the first and second light sources are selected from the group consisting of,

wherein step (I) is performed such that in each of said n executions at least two different compounds are selected in step (ia) and in each of said n executions said at least two different compounds are the same.

12. A chromatographic process comprising

A method of determining the concentration of at least one compound in a chromatographic method according to any one of claims 1 to 9, and

(iv) performing chromatography.

13. The chromatographic method according to claim 12,

wherein step (iii) comprises calculating the concentration c of the compound in the mobile phase at the outlet of the chromatography device at several time points t_out(t) and step (iv) comprises collecting c therein_out(t)>0.0mmol/L of the mobile phase at time t.

14. A chromatographic process comprising

A method of obtaining at least one chromatography parameter according to claim 10 or 11, and

(III) a step of performing chromatography based on the at least one chromatography parameter selected in step (II).

Examples

Example 1: determination of porosity data based on Process conditions (step (iib))

The determination of reversible swelling behavior can be easily performed using inverse size exclusion chromatography (iesc) while changing the desired process conditions. Here, specific examples are given.

The stationary phase isAnd (4) S membrane adsorbent. As an adsorbent for the membrane, it is preferred,s has no internal porosity (. epsilon.)_b＝ε_T). It shows a significant reversible swelling behavior resulting from its charged hydrogel surface modification. The chromatographic apparatus was 3mL (V)_b3mL) having a bed height of 8mmNano。

Dead volume V of chromatography apparatus_DeadDetermined by using 5 μ L injection of 0.25g/L dextran (Mw 2000kDa, determined by size exclusion chromatography) and 0.319 ± 0.03mL by RI detector in case of a no-spectrum device. Peak maxima and first momentum analysis were used to determine dead volume, respectively. The dead volume of the chromatography apparatus was 1 mL.

For the iSEC experiment, the buffer used was 10mM potassium phosphate buffer (KPi), pH 7. The salt concentration (NaCl) is 0.01 to 0.8M. Membrane sorbent (MA) 15 membrane volumes were equilibrated with the desired salt concentration before loading 50. mu.L of injection 0.5g/L dextran 2000 kDa. The resulting peak response was recorded using an IR detector.

The porosity epsilon is determined based on the following equation.

The porosity values obtained depend on the salt concentration used, as shown in fig. 2.

The data sets were fitted using boltzmann functions, as shown below.

The fitting parameters were determined as follows:

parameter(s)	Value of
		A1	-54.022
A2	0.759
		x0	-0.865
dx	0.125

Example 2: determination of porosity number based on process conditionsAccording to (step (iib))

In the case of chromatographic media having external and internal porosity, both values can be determined using reverse volume size exclusion chromatography as described above. Chromatographic media with internal porosity exhibit different achievable volume fractions of the chromatographic bed depending on the tracer molecule size. Fractogel EMD SO₃ ^-An example of (M) is shown in FIG. 3.

Porosity epsilon of stationary phase_spThe total porosity ε may be used_TAnd external porosity ε_b(void fraction).

Total porosity epsilon_TIs accessible by the tracer molecules which fully reach the internal porosity. In this example, acetone was used. Completely excluded tracer molecules allow the determination of the bulk porosity ε_b. In this example, dextran with a molecular weight Mw of 2000kDa was used. Using the above equation, the resulting ε may be used_TAnd ε_bTo calculate the stationary phase porosity epsilon depending on the salt concentration_sp. Obtained ε including Boltzmann fitting_sp(stationary phase porosity) and ε_bThe values of (external porosity) and the corresponding parameters are shown in fig. 4 and 5.

Porosity epsilon of stationary phase_sp：

Bulk porosity ε_b：

Parameter(s)	Value of
		A1	-72.89
A2	0.539
		x0	-2.548
dx	0.31

When using the boltzmann function, the porosity value ∈_bAnd ε_spAn excellent fit was obtained.

Example 3: obtaining equilibrium adsorption data (step (iia))

In example 3, Bovine Serum Albumin (BSA) was obtainedEquilibrium adsorption data on Q.

For theQ (average pore diameter of 3 μm and ligand density of) For example, Bovine Serum Albumin (BSA) of 0.1 to 5g/L is used in the treatment of diseases based on the protein such as Antibodies 2018,7(1), 13; https:// doi.org/10.3390/anti 7010013, "Evaluation of Continuous Membrane Chromatography concept with Enhanced Process SimulinatDetermination of equilibrium adsorption data for the batch experiments shown in on Approach ", Zobel, Stein, Strube. The buffer used was 20mM TRIS HCl buffer, pH7, NaCl concentration 0 to 0.3M NaCl. The pH was adjusted using HCl or NaOH. Mixing a circular shape with a diameter of 20cm and a height of 0.024-0.028cmThe Q Membrane Adsorbent (MA) samples were equilibrated in 20mM TRIS HCl buffer with respective pH and salt concentration for 30 minutes. The volume of buffer was 200 times the volume of MA. After 30 minutes equilibration time, the MA was wiped with paper and transferred to a 12-well plate chamber. BSA was dissolved in TRIS HCl buffer corresponding to the experimental pH and salt concentration. The concentration of the BSA feed solution was measured by UV/Vis spectroscopy at 280nm and 4mL was added to the MA in a 12-well plate. After a residence time of at least 8 hours, the supernatant concentration was measured, and the MA was again wiped with paper and transferred to a new well plate. Subsequently, MA was eluted with 4mL of 20mM TRIS HCl and 1M NaCl for at least 4 hours. The supernatant concentration was measured after 4 hours of elution time. The above process is schematically depicted in fig. 6.

The data set obtained for the three different salt concentrations is shown in fig. 7.

Example 3 a: obtaining equilibrium adsorption data for several compounds

The method of example 3 is also feasible for multi-component analysis. A prominent example is the simultaneous determination of equilibrium adsorption data for monoclonal antibodies (mabs) and their aggregates and contaminants. Batch experiments can be performed in the same way, but the supernatant and the eluate must be analyzed in a way that allows to distinguish all components (mAb monomers, aggregates and other contaminants). This can be achieved, for example, by size exclusion chromatography.

The resulting peaks of the obtained size exclusion chromatograms (see figure 8 of the present example) can be evaluated using appropriate calibration to determine the concentration of the component of interest during the batch experiment shown in figure 6. This gives equilibrium adsorption data. For a given salt concentration, an example is shown in fig. 9.

Practice ofExample 3 b: obtaining equilibrium adsorption data for varying salt concentration and varying pH

In example 3 above, only the salt concentration was changed. Other influencing factors such as pH can also be varied in the same way to obtain a multidimensional adsorption data map. FIGS. 10 and 11 show that monoclonal antibody IgG and its dimer are dependent on pH and salt concentrationEquilibrium adsorption data on Q.

Example 4: fitting the data set to SMA isotherms (step (iia))

The data set obtained in example 3 was used to calculate the protein characteristic charge v_iAnd the equilibrium constant k, in addition, at three different salt concentrations (c)_S0.05, 0.15, 0.25M) using computer-aided least squares regression with spatial factor σ_iThe SMA adsorption isotherms were fitted to obtain the necessary adsorption model parameters in the studied salt concentration region. The ion capacity lambda is 0.97 mol/L: (Q)。

Adsorption constant: k is 7.55

Spatial factor: sigma_i＝46.04

Characteristic charge: v. of_i＝2.72

Salt concentration: c. C₁＝0.05M

Example 5: determination of hydrodynamic behavior and axial Dispersion coefficient

Selection of proteins from GE HealthcareExplorer as chromatographic apparatus (step (id)).

The parameter V is determined as follows_SYS、V_ST、V_DPFAndpulsed injection of acetone (2 vol% in reverse osmosis water) or mAb (4 mg/mL in potassium phosphate (KPi) buffer, 10mM, 20mM NaCl, pH 6) was performed in the absence of chromatography media. Experiments were performed using different volumetric flow rates and buffer conditions. The resulting peak signals were evaluated by the moment method and regressed using a least squares fitting procedure to obtain the desired values. The results of this procedure are given in fig. 12 and 13.

Example 6 determination of kinetic and diffusion coefficients

Use of hydrogel modified membrane adsorbents according to a focused pore modelMathematical correlation of S determines the effective membrane diffusion coefficient D_eff：

Bulk diffusion coefficients were calculated using the einstein-stokes equation. Specifically, intravenous immunoglobulin (IVIG, human γ -Globulin, SeraCare; r ═ 5.2nm) was dissolved in a sodium phosphate buffer (20mM, pH ═ 7) having a viscosity η of 1.05mPa · s at a temperature of 298K.

The porosity values were determined using reverse volume size exclusion chromatography (iesc). Briefly, the bed was equilibrated with 50 Column Volumes (CV) of the desired buffer (sodium phosphate buffer (20mM, pH 7)) and then a solution (100 μ L) containing amylopectin molecules with narrow molecular weight distribution (2mg/mL) was injected. The average molecular weight, which is directly related to the average hydrodynamic radius of the applied amylopectin samples, varied for each injection, covering a wide range (Mn 320-. The elution curve was recorded and analyzed by an RI detector.

The effective diffusion coefficient was calculated using the following correlation.

Internal porosity ε was determined by iSEC as described above_pThe value of (c).

For the membrane adsorbent, the following values were calculated from the corresponding target compounds NaCl, acetone and monoclonal antibody IgG:

example 7: prediction of protein purification Process (step (iii))

The set of model parameters obtained in example 1 and example 5 was used to predict the hydrodynamic behavior of the acetone tracer pulse signal. The adsorption isotherm of acetone on the membrane adsorbent used was found to be 0 (acetone does not bind to the stationary phase). Liquid Chromatography (LC) systems and Membrane Adsorbents (MAs) were simulated by Equilibrium Dispersion Modeling (EDM). Furthermore, the chromatographic apparatus is considered to be a combination of ST, DPF and chromatographic column as described above and as shown in the following equation (see fig. 17).

Wherein

V_SYS＝V_ST+V_DPF

c(t＝0,z)＝0

The overall LC system had a tube length of 2943mm, a volume of 1.3mL and a porosity of 1. The entire MA device is represented by a 5.73mm bed height, a 3.5mL bed volume, and a porosity of 76-71% (0.76-0.71). The MA used was in 10mM KPi buffer at pH7And S. The tracer experiment was performed with 2mL injection volumes of KPi buffer containing 5% acetone and 0 or 0.8M additional sodium chloride at 4 mL/min. In fig. 14, the salt concentration simulation results and experimental data are lower. At low salt concentrations, similar results were obtained with the conventional and improved model methods. At high salt concentrations, the process according to the invention gives much better results than processes which do not take into account the reversible swelling of the stationary phase (see fig. 15). Fig. 14 and 15 show the significant effect of different porosity values on the fluid dynamic behavior.

The following table shows a comparison of the conventional simulation method with the method according to the invention, which corresponds to the full width at half maximum FWHM and the centre of the peak. The method of the present invention gives smaller deviations from the experimental values than the conventional method.

	FWHM	Center of a ship
			Deviation/% of conventional simulation from experiment	2.2	10.6
Deviation/% of simulation and experiment of the invention	2.0	2.6

Example 8: fitting the data set to Langmuir isotherms (step (iia))

A data set of equilibrium adsorption data obtained for IVIG on hydrogel grafted chromatographic membranes was fitted to Langmuir isotherms, taking into account the different NaCl concentrations. The results are shown in fig. 16(a) to 16 (c).

As can be seen from fig. 16(a), the Langmuir fitting approximates the isotherm adsorption data with high accuracy.