An online monitoring method for the apparent stability of biological products in chromatographic processes.

Abstract

This disclosure provides an online monitoring method for the apparent stability of biological products in chromatographic processes. Specifically, this disclosure provides an online monitoring method for the apparent stability of biological products in chromatographic processes, and its application in identifying risk factors and / or risk ranges that are detrimental to the apparent stability of biological products in chromatographic processes, as well as its application in screening the types and / or concentrations of buffer systems and / or protective agents.

Description

Technical Field

[0001] This disclosure relates to the field of biopharmaceutical purification, specifically to an online monitoring method for the apparent stability of biopharmaceuticals in chromatographic processes. Background Technology

[0002] With the continuous development of the biopharmaceutical industry, an increasing number of novel molecules are being designed. Some of these novel molecules, such as engineered antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins, exhibit poor stability and are sensitive to numerous factors, including buffer pH, the type and strength of ions in the buffer system, and shear force. They readily form soluble oligomers or polymers, and can further form sub-visible and visible particles, resulting in a milky or turbid appearance of the chromatographic sample, causing significant challenges to the purification process. Identifying risk factors detrimental to sample stability and avoiding risk zones during chromatography is therefore crucial. Furthermore, adding protective agents to the buffer system, such as amino acids, inorganic salts, nonionic surfactants, and non-reducing sugars, can effectively improve the unstable state of molecules during chromatography and inhibit particulate formation.

[0003] Currently, various techniques can be used to evaluate the appearance of samples. Microflow imaging (MFI) systems are commonly used for analyzing samples with low turbidity. They integrate digital microscopy and microfluidic image processing technologies to perform image analysis on the sample, generating data on particle number, size, transparency, and morphology. Turbidity measurement methods are commonly used for analyzing samples with high turbidity. Their built-in light source excites a stable single-wavelength beam; as the beam passes through the sample, it is reflected or scattered by particles, and the reflection and scattering losses can be converted into turbidity data. Visual inspection is the simplest evaluation method; by comparing the appearance of different samples under the same conditions, the degree of turbidity can be directly assessed.

[0004] The methods described above for evaluating sample appearance have certain limitations. MFI cannot detect highly turbid samples, turbidity measurement methods cannot obtain particle size information of the sample, and visual inspection methods are heavily dependent on testing conditions and the experience of the personnel. More importantly, all of the above methods are offline detection methods and cannot monitor changes in sample appearance during chromatography in real time. Summary of the Invention

[0005] This disclosure provides an online monitoring method for the apparent stability of biological products in chromatographic processes, comprising the following steps:

[0006] (a) Perform chromatography on the biological product using a purification instrument with a built-in ultraviolet-visible light sensor;

[0007] (b) Select at least two wavelengths within the wavelength range of 300nm-800nm ​​as effective wavelengths;

[0008] (c) During the chromatography operation, plot the chromatography time or chromatography volume as the independent variable and the absorbance signal value collected at the effective wavelength as the dependent variable, and characterize the apparent stability.

[0009] In some implementations, incident light of different wavelengths is passed through the sample, and the intensity loss can be measured to obtain information about the turbidity of the sample (i.e., apparent stability).

[0010] In some implementations, step (b) includes the following steps:

[0011] (b-1) Use a spectrophotometer to perform ultraviolet-visible wavelength scanning on water and solvent to determine the characteristic absorption wavelength range of the solvent, denoted as λ1;

[0012] (b-2) Select a wavelength range of 300 to 800 nm, and subtract the portion that overlaps with the characteristic absorption wavelength range λ1 of the solvent described in step (b-1) as a candidate wavelength range, denoted as λ2;

[0013] (b-3) Take the biological product and perform gradient dilution with the solvent to obtain N samples, where N≥3;

[0014] (b-4) Select any wavelength within the candidate wavelength range λ2 described in step (b-2) and perform an effectiveness test as follows: Use a turbidity meter to measure the turbidity of the N samples obtained in step (b-3), and record the turbidity values ​​as x1 to x2 respectively. n Use a spectrophotometer to perform a full-wavelength scan on the N samples obtained in step (b-3), and record the absorbance signal values ​​at the selected monitoring points, denoted as y1 to y2 respectively. n Linear fitting was performed with x as the independent variable and y as the dependent variable, and the fitting coefficient R0 was obtained. 2 If z ≥ 0.9, the selected detection wavelength is considered to be an effective wavelength.

[0015] (b-5) Repeat step (b-4) to obtain the at least two effective wavelengths.

[0016] In some implementations, in step (b-3), N is an integer from 3 to 20, such as an integer from 5 to 10.

[0017] In some implementations, in step (b-4), x n and y n The value of the subscript n corresponds to that of N.

[0018] In some implementation schemes, z≥0.95, z≥0.96, z≥0.97, z≥0.98, or z≥0.99.

[0019] In some implementations, one effective wavelength is ≤450nm and the other effective wavelength is ≥500nm; for example, one effective wavelength is ≤400nm and the other effective wavelength is ≥550nm.

[0020] In some implementations, one effective wavelength is about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, or about 350 nm, and another effective wavelength is about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, or about 620 nm.

[0021] In some implementations, the effective wavelengths are approximately 340 nm and approximately 600 nm.

[0022] In some embodiments, the bioproduct is selected from peptides or proteins.

[0023] In some embodiments, the bioproduct is selected from any one of monoclonal antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins.

[0024] In some embodiments, the biological product is a monoclonal antibody having a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2.

[0025] In some embodiments, the chromatography is selected from one or more of affinity chromatography, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, and complex mode chromatography.

[0026] In some embodiments, the solvent is selected from any one of an acetate buffer system, a phosphate buffer system, a citrate buffer system, a Tris buffer system, and a disodium hydrogen phosphate-citric acid buffer system.

[0027] In some embodiments, the buffer system further comprises one or more of amino acids, inorganic salts, nonionic surfactants, non-reducing sugars, and alcohols.

[0028] In some embodiments, the amino acid is selected from one or more of arginine, arginine hydrochloride, histidine, histidine hydrochloride, and glycine;

[0029] The inorganic salt is selected from one or more of sodium chloride, sodium acetate, sodium sulfate, and potassium chloride;

[0030] The nonionic surfactant is selected from one or more of Tween 20, Tween 80 and poloxamer;

[0031] The non-reducing sugar is selected from sucrose, fucose, or mixtures thereof; and / or

[0032] The alcohols are selected from ethanol, isopropanol, polyethylene glycol, or mixtures thereof.

[0033] In some embodiments, the ratio of the absorbance signal values ​​of the solvent to that of water at the effective wavelength is greater than 3, for example greater than 4, greater than 5, or greater than 6.

[0034] In some implementations, the ultraviolet-visible wavelength scanning is selected from: ultraviolet-visible full-wavelength scanning, ultraviolet-visible partial-band scanning, and ultraviolet-visible single-wavelength or multi-wavelength scanning.

[0035] In some implementations, the purification instrument has a built-in ultraviolet-visible light sensor. For example, purification instruments include, but are not limited to, AKTA purifier, AKTA pure, AKTA avant, and AKTA pilot.

[0036] In some embodiments, the purification instrument further includes one or more sensors selected from the following: pH sensor, conductivity sensor, temperature sensor, concentration sensor, and pressure sensor.

[0037] In some embodiments, the turbidimeter includes an optical system consisting of a stable excitation source, a scattered light detector, and a transmitted light detector.

[0038] In some implementations, the turbidimeter includes, but is not limited to, HACH 2100Q or HACH TU5200.

[0039] In some implementations, step (c) includes the following steps:

[0040] (c-1) During the chromatography operation, while acquiring absorbance signal values ​​at the effective wavelength, one or more parameters among pH, conductivity, temperature, pressure, and concentration are acquired simultaneously.

[0041] (c-2) Plot the absorbance signal value and parameter value collected in step (c-1) as dependent variables with chromatography time or chromatography volume as independent variables, and evaluate the synchronicity between the change of absorbance signal value and the change of parameter value, in order to determine the influencing factors and / or risk range of the influencing factors on the apparent stability of the bioproduct in the chromatography process.

[0042] In some implementations, step (c) includes the following steps:

[0043] (ci) During the chromatography operation, one or more factors are changed, absorbance signal values ​​at the effective wavelength are acquired, and the correlation between apparent stability and the factors is evaluated.

[0044] In some implementations, the factors include, but are not limited to, one or more of the following: buffer pair type, buffer pair concentration, buffer system pH, buffer system conductivity, chromatography temperature, protective agent type, and protective agent concentration.

[0045] In some embodiments, the protective agent is selected from one or more of amino acids, inorganic salts, nonionic surfactants, non-reducing sugars, and alcohols.

[0046] In some embodiments, the amino acid is selected from one or more of arginine, arginine hydrochloride, histidine, histidine hydrochloride, and glycine;

[0047] The inorganic salt is selected from one or more of sodium chloride, sodium acetate, sodium sulfate, and potassium chloride;

[0048] The nonionic surfactant is selected from one or more of Tween 20, Tween 80 and poloxamer;

[0049] The non-reducing sugar is selected from sucrose, fucose, or mixtures thereof; and / or

[0050] The alcohols are selected from ethanol, isopropanol, polyethylene glycol, or mixtures thereof.

[0051] This disclosure provides the application of any of the above methods in identifying risk factors and / or risk ranges that are detrimental to the apparent stability of biopharmaceuticals during the chromatographic process of biopharmaceuticals.

[0052] In some implementations, the risk factors include, but are not limited to, one or more of the following: buffer pair type, buffer pair concentration, buffer system pH, buffer system conductivity, chromatography temperature, protective agent type, and protective agent concentration.

[0053] This disclosure provides the application of any of the above methods in screening the types and / or concentrations of buffer systems and / or protective agents used in biochromatographic processes.

[0054] This disclosure also provides a method for identifying risk factors and / or risk ranges that are detrimental to the apparent stability of biological products in a biochromatographic process, comprising:

[0055] (a) Perform chromatography on the biological product using a purification instrument with a built-in ultraviolet-visible light sensor;

[0056] (b) Select at least two wavelengths within the wavelength range of 300nm-800nm ​​as effective wavelengths;

[0057] (c) During the chromatography operation, while acquiring absorbance signal values ​​at the effective wavelength, one or more parameters among pH, conductivity, temperature, pressure, and concentration are acquired simultaneously.

[0058] (d) Plot the absorbance signal value and parameter value collected in step (c) as dependent variables with chromatography time or chromatography volume as independent variables, and evaluate the synchronicity between the change in absorbance signal value and the change in parameter value, in order to determine the risk factors and / or risk range of the apparent stability of the bioproduct in the chromatography process.

[0059] This disclosure provides a method for screening the type and / or concentration of buffer systems and / or protective agents used in biochromatographic processes, comprising:

[0060] (a) Perform chromatography on the biological product using a purification instrument with a built-in ultraviolet-visible light sensor;

[0061] (b) Select at least two wavelengths within the wavelength range of 300nm-800nm ​​as effective wavelengths;

[0062] (c) During the chromatography operation, one or more factors are changed, absorbance signal values ​​at the effective wavelength are acquired, and the correlation between apparent stability and the factors is evaluated.

[0063] As an advantageous aspect of this disclosure, by using incident light of different wavelengths to pass through a sample and detecting its intensity loss, information about the sample's turbidity can be obtained. Calculating the signal ratio of incident light at different wavelengths can reveal the particle size distribution information within the sample. The principle is as follows: light, as an electromagnetic wave, undergoes reflection, refraction, and scattering when it encounters particles in a solution. According to the theoretical model, when the particle radius is less than 1 / 10 of the incident light wavelength, lateral scattering mainly occurs; when the particle radius reaches approximately 1 / 4 of the wavelength, strong forward scattering occurs; when the particle radius is greater than the incident light wavelength, while scattering forward, it also emits scattered light with varying intensity at different angles to the sides. Therefore, it can be seen that the intensity loss of the light beam passing through the sample is correlated with the incident wavelength, the number of particles, and the particle radius. When the incident light wavelength is fixed, the intensity loss increases with the number of particles; when the number and size of particles are fixed, shorter wavelength incident light is more prone to intensity loss; and when the number and wavelength of particles are fixed, larger diameter particles are more likely to cause intensity loss.

[0064] As another advantageous aspect of this disclosure, by using an ultraviolet-visible light sensor integrated within the purification instrument, one or more effective wavelength response signals are collected when the sample flows through the sensor. This converts the turbidity of the sample into a readable effective wavelength response signal, enabling real-time online monitoring of the apparent stability of biological products in chromatographic processes. This method is simple and convenient, and can also be used to screen stability influencing factors and risk ranges. Furthermore, when two or more effective wavelength signal values ​​are monitored simultaneously, this technology can also obtain information on the particle size distribution of the particles contained in the sample, resulting in more accurate monitoring results. Attached Figure Description

[0065] Figure 1 The UV-Vis scan spectra of water, solvent-1, and solvent-2 are shown.

[0066] Figure 2 The appearance of sample mAb-a and samples diluted 2, 4, 8, and 16 times is shown.

[0067] Figure 3 The results of the monitoring wavelength effectiveness test (optical path 10mm) are displayed;

[0068] Figure 4 The results of the chromatography influence factor screening are shown, including: (a) the collected chromatography signal; and (b) the appearance of the collected samples.

[0069] Figure 5 The results of the screening of chromatographic protectants are shown, including: (a) the signal acquired after chromatography with the addition of 100 mM sodium chloride; (b) the signal acquired after chromatography with the addition of 100 mM arginine hydrochloride; (c) the appearance of the sample collected after chromatography with the addition of 100 mM sodium chloride; and (d) the appearance of the sample collected after chromatography with the addition of 100 mM arginine hydrochloride.

[0070] Figure 6 The results of the screening of chromatographic protectant concentrations are shown, including: (a) the signal acquired after chromatography with the addition of 20 mM arginine hydrochloride; (b) the signal acquired after chromatography with the addition of 50 mM arginine hydrochloride; (c) the signal acquired after chromatography with the addition of 100 mM arginine hydrochloride; (d) the appearance of the eluted sample collected after chromatography with the addition of 20 mM arginine hydrochloride; (e) the appearance of the eluted sample collected after chromatography with the addition of 50 mM arginine hydrochloride; and (f) the appearance of the eluted sample collected after chromatography with the addition of 100 mM arginine hydrochloride. Detailed Implementation

[0071] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] The term "buffer solution" refers to a solution that resists pH changes through the action of its acid-base pairs. A variety of buffer solutions are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., Ed. Calbiochem Corporation (1975), depending on, for example, the desired buffer pH.

[0073] The term "turbidity," also known as turbidity or degree of turbidity, refers to a measure of the decrease in the transparency of a liquid caused by the presence of substances in water that reflect or scatter light. Particles in a liquid scatter and absorb light passing through a sample; turbidity is characterized by the light-scattering properties of these particles, and the measurement result is measured in NTUs (Nephelometric Turbidity Units).

[0074] The term "conductivity" refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, current flows through ion transport, and the higher the amount of ions present in the aqueous solution, the higher the conductivity. The basic unit for measuring conductivity is the Siemens (or ohm), or ohm (mS / cm), and it can be measured using a conductivity meter, such as various models of the Orion conductivity meter. Because electrolytic conductivity is the ability of ions in a solution to carry current, the conductivity of a solution can be changed by altering the concentration of ions within it. For example, the desired conductivity can be achieved by changing the concentration of buffers and / or salts (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution.

[0075] The term "about" refers to a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance of a number or numerical range preceded by the term "about" also includes embodiments of a given number. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially includes" should be assumed to be within an acceptable margin of error for that specific value.

[0076] Example

[0077] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0078] Example 1: Selection of Effective Wavelength

[0079] UV-Vis wavelength scans were performed on water, solvent-1 (20 mM phosphate, pH 7.4), and solvent-2 (50 mM acetate, pH 5.0) using a spectrophotometer. The results are as follows: Figure 1 As shown in the scan spectrum, the characteristic absorption wavelengths of Solvent-1 and Solvent-2 are both less than 300 nm, and there is no overlap with the wavelength range of 300–800 nm.

[0080] Take the monoclonal antibody mAb-a (heavy chain sequence as shown in SEQ ID NO: 1, light chain sequence as shown in SEQ ID NO: 2) from the chromatography sample, and dilute it 2-fold, 4-fold, 8-fold, and 16-fold with solvent-1, respectively, to obtain a total of 5 samples, which are labeled as No. 1 to No. 5. The appearance of the samples is as follows. Figure 2 As shown.

[0081] The turbidity of samples 1 to 5 was measured using a turbidimeter (HACH 2100Q), and the values ​​were 524 NTU, 306 NTU, 78.7 NTU, 22.9 NTU, and 8.96 NTU, respectively.

[0082] The signal values ​​(optical path 10 mm) at 340 nm for samples 1-5 were measured and were 2.84, 1.61, 0.63, 0.24, and 0.08, respectively. The signal values ​​(optical path 10 mm) at 600 nm for samples 1-5 were measured and were 1.39, 0.92, 0.38, 0.17, and 0.10, respectively.

[0083] The signal values ​​and turbidity at a wavelength of 340 nm were fitted using samples 1 to 5, and the correlation coefficient R was obtained. 2 The correlation coefficient R is 0.9951. The signal values ​​and turbidity at 600 nm were fitted using samples 1–5, and the correlation coefficient R0 was [value missing]. 2 It is 0.9931. For example... Figure 3 As shown, 340nm and 600nm were selected as effective monitoring wavelengths.

[0084] mAb-a:

[0085] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDDYMHWVRQAPGQGLEWMGWI

[0086] DPENGDTEYASKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLYGNFYYFDY

[0087] WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS

[0088] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR

[0089] VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE

[0090] VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV

[0091] SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV

[0092] EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL

[0093] HNHYTQKSLSLSLGK

[0094] SEQ ID NO:1EIVLTQSPATLSLSPGERATLSCSASSSINYMHWYQQKPGQAPKRWIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSFSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0095] SEQ ID NO:2

[0096] Example 2, Screening of Influencing Factors and Risk Intervals of Sample Turbidity in Chromatography Process

[0097] Clean the Mabselect Prism A chromatography column with 0.5M sodium hydroxide solution for 3–5 column volumes. Wash with 0.02M phosphate + 1M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Load the cell culture clarification buffer of monoclonal antibody mAb-a onto the chromatography column. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.02M phosphate, pH 7.4 solution for 3–5 column volumes. Elute the antibody with 0.05M acetate, pH 3.6 solution. Collect signal values ​​at 280 nm, 340 nm, and 600 nm, as well as pH, conductivity, temperature, and pressure. Wash the chromatography column with 0.1M acetic acid solution for 3 to 5 column volumes.

[0098] like Figure 4 As shown in (a) and (b), conductivity, temperature, and pressure remained stable during elution and showed no significant correlation with the signal values ​​at 340 nm, 600 nm, and 280 nm. Significant correlations were observed between the signal values ​​at 340 nm and 600 nm and pH: the signal values ​​at 340 nm and 600 nm increased as pH decreased from 7.4 to 6.0, and decreased as pH decreased from 6.0 to 3.6. This indicates that pH significantly affects the appearance of the sample during chromatography, and the pH range of 5.0–6.5 is considered a risk region.

[0099] Example 3: Screening of Chromatography Protectants

[0100] Clean the Mabselect Prism A chromatography column with 0.5M sodium hydroxide solution for 3–5 column volumes. Wash with 0.02M phosphate + 1M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Load the clarified cell culture buffer of monoclonal antibody mAb-a onto the chromatography column. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.02M phosphate, pH 7.4 solution for 3–5 column volumes. Antibodies were eluted using 0.05M acetate + 100mM sodium chloride, pH 3.6 and 0.05M acetate + 100mM arginine hydrochloride, pH 3.6 solutions, respectively. Signal values ​​at 280 nm, 340 nm, and 600 nm, as well as pH, conductivity, temperature, and pressure, were collected. The column was washed with 0.1M acetic acid solution for 3–5 column volumes.

[0101] The inhibition effects of different additives (sodium chloride and arginine hydrochloride) on signal values ​​at 340 nm and 600 nm were statistically analyzed, and the results are shown in Table 1. It can be seen that, under the same conditions, arginine hydrochloride has a better inhibition effect than sodium chloride, and the proportion of large-diameter particles in the eluted sample is lower.

[0102] Table 1 Comparison of the protective efficacy of sodium chloride and arginine hydrochloride

[0103]

[0104] Example 4: Screening of Protective Agent Concentration

[0105] Clean the Mabselect Prism A chromatography column with 0.5M sodium hydroxide solution for 3–5 column volumes. Wash with 0.02M phosphate + 1M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Load the clarified cell culture buffer of monoclonal antibody mAb-a onto the chromatography column. Wash with 0.025M Tris-HCl, 0.15M sodium chloride, pH 7.4 solution for 3–5 column volumes. Wash with 0.02M phosphate, pH 7.4 solution for 3–5 column volumes. Antibodies were eluted using solutions of 0.05M acetate + 20mM arginine hydrochloride, pH 3.6, 0.05M acetate + 50mM arginine hydrochloride, pH 3.6, and 0.05M acetate + 100mM arginine hydrochloride, pH 3.6, respectively. Signal values ​​at 280 nm, 340 nm, and 600 nm, as well as pH, conductivity, temperature, and pressure, were collected. The column was washed with 0.1M acetic acid solution for 3–5 column volumes.

[0106] The suppression effects of different concentrations of arginine hydrochloride on signal values ​​at 340 nm and 600 nm were statistically analyzed, and the results are shown in Table 2. It can be seen that within the range of 20–100 mM, with increasing arginine hydrochloride concentration, the signal values ​​at both 340 nm and 600 nm continuously decreased, while the ratio of the 340 nm signal value to the 600 nm signal value continuously increased. This indicates that within the range of 20–100 mM, with increasing arginine hydrochloride concentration, the appearance of the eluted sample improved, and the proportion of large-diameter particles in the eluted sample continuously decreased.

[0107] Table 2 Comparison of protective efficacy of arginine hydrochloride at different concentrations

[0108]

[0109] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

1. A method for online monitoring of apparent stability of a biological product in a chromatography process, comprising the following steps: (a) performing a chromatography operation on the biological product using a purification instrument with built-in UV-Vis light sensor; (b) selecting at least two wavelengths as effective wavelengths in the range of 300 nm-800 nm; (c) during the chromatography operation, plotting and characterizing the apparent stability with absorbance signal values acquired at the effective wavelengths as dependent variables and chromatography time or chromatography volume as independent variables, wherein step (b) comprises the following steps: (b-1) performing UV-Vis wavelength scanning on water, solvent using a spectrophotometer to determine the solvent characteristic absorption wavelength range, denoted as λ1; (b-2) selecting a wavelength range of 300-800 nm, excluding the overlapping part of the solvent characteristic absorption wavelength range λ1 as candidate wavelength range, denoted as λ2; (b-3) taking the biological product, performing gradient dilution using the solvent to obtain N samples, N≥3; (b-4) selecting a wavelength in the candidate wavelength range λ2 as an effective wavelength; (b-5) repeating step (b-4) to obtain the at least two effective wavelengths.

2. The method of claim 1, wherein in step (b-4), z≥0.

95.

3. The method of claim 1, wherein in step (b-4), z≥0.

98.

4. The method of claim 1, wherein the biological product is selected from any one of a monoclonal antibody, a bispecific antibody, a multispecific antibody, and a fusion protein.

5. The method of claim 1, wherein the biological product is a monoclonal antibody having a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO:

2.

6. The method of claim 1, wherein the chromatography is selected from one or more of affinity chromatography, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, and complex mode chromatography.

7. The method of claim 1, wherein the ratio of absorbance signal values of the solvent to water at the effective wavelengths is greater than 3.

8. The method of claim 1, wherein the ratio of absorbance signal values of the solvent to water at the effective wavelengths is greater than 5. (b-4) optionally one wavelength in the candidate wavelength range λ2 described in step (b-2), the effectiveness test is carried out as follows: turbidity of the N samples obtained in step (b-3) is determined using a turbidimeter, and the turbidity determination values are respectively recorded as x1 to xN n ; absorbance signal values of the N samples obtained in step (b-3) are recorded using a spectrophotometer, and the absorbance signal values are respectively recorded as y1 to yN n ; linear fitting is carried out with x as the independent variable and y as the dependent variable, and the fitting coefficient R 2 ≥z is regarded as the selected detection wavelength being an effective wavelength, and z≥0.9; 9. The method of claim 1, wherein the solvent is selected from any one of acetate buffer system, phosphate buffer system, citrate buffer system, Tris buffer system, and disodium hydrogen phosphate-citric acid buffer system.

10. The method of claim 1, wherein the UV-Vis wavelength scanning is selected from UV-Vis full wavelength scanning, UV-Vis partial waveband scanning, UV-Vis single wavelength or multiple wavelength scanning.

11. The method of claim 1, wherein the purification instrument is selected from any one of AKTA purifier, AKTA pure, AKTA avant, and AKTA pilot.

12. The method of claim 1, wherein the turbidimeter comprises an optical system consisting of a stable excitation light source, a scattered light detector, and a transmitted light detector. ​ ​ ​ ​ ​ ​ ​ ​ 13. The method of claim 1, wherein step (c) comprises the following steps: (c-1) during the chromatographic operation, synchronously collecting one or more parameter values of pH, conductivity, temperature, pressure, concentration while collecting absorbance signal values at the effective wavelength; (c-2) plotting the absorbance signal values and the parameter values collected in step (c-1) as a function of chromatographic time or chromatographic volume, respectively, to assess the synchronicity of the changes in the absorbance signal values and the changes in the parameter values, to determine the impact factor and / or the risk interval of the apparent stability of the biological product in the chromatographic process.

14. The method of claim 1, wherein step (c) comprises the following steps: (c-i) during the chromatographic operation, changing one or more factors, collecting the absorbance signal values at the effective wavelength, and assessing the correlation between the apparent stability and the factors; the factors are selected from one or more of the following: buffer pair species, buffer pair concentration, buffer system pH, buffer system conductivity, chromatographic temperature, protective agent species, and protective agent concentration.

15. The method of claim 14, wherein the protective agent is selected from one or more of the following: amino acid, inorganic salt, non-ionic surfactant, non-reducing sugar, and alcohol.

16. The method of claim 15, wherein: the amino acid is selected from one or more of the following: arginine, arginine hydrochloride, histidine, histidine hydrochloride, and glycine; the inorganic salt is selected from one or more of the following: sodium chloride, sodium acetate, sodium sulfate, and potassium chloride; the non-ionic surfactant is selected from one or more of the following: Tween 20, Tween 80, and poloxamer; the non-reducing sugar is selected from sucrose, fucose, or a mixture thereof; and / or the alcohol is selected from ethanol, isopropanol, polyethylene glycol, or a mixture thereof.

17. Use of the method of any one of claims 1-16 in identifying risk factors and / or risk intervals that are detrimental to the apparent stability of a biological product in a chromatographic process of the biological product.

18. Use of the method of any one of claims 1-16 in screening buffer systems and / or protective agent species and / or concentrations for use in a chromatographic process of a biological product.

Images