CN111018947B - Method for removing HCP residues in yeast cells - Google Patents
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Abstract
The invention provides a method for removing HCP residues in yeast cells, belonging to the technical field of biological engineering. The invention carries out cation chromatography, hydrophobic chromatography, CHT chromatography and cation chromatography on the yeast cell sap, and then uses an HCP detection kit to detect HCP residues. The invention removes HCP residues in yeast, especially protein expressed outside yeast cells, by optimizing process routes and parameters, can solve the problem that HCP is not easy to remove due to the similar chemical properties of HCP and target protein, such as isoelectric point, molecular weight and other factors, and develops a solution to the problem so as to reduce the residual concentration to be within 100ppm (ng/mg) and achieve related standards. The invention solves the problems of more HCP residues and difficult removal commonly existing in the prior fermentation liquor, and has obvious effect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for removing HCP residues in yeast cells.
Background
HCPs are host cell residual proteins, which are key quality attributes (CQAs) of biological products, directly affecting the safety, efficacy and stability of biological products. Therefore, throughout the production of biologies, it is desirable to remove or control the HCP to as low a level as possible.
Currently, sandwich ELISA test kits are gold standards for detecting HCP residues. However, this method is highly dependent on the coverage of the total host cell protein by the anti-HCP antibody in the kit. If the antibody coverage used in the ELISA assay is insufficient, some residual HCPs may be missed in the final product, which in turn may cause problems with immune responses and safety of other drugs in the patient to whom the drug is administered.
We also see that events leading to project set aside may occur because HCP remaining in the injection causes adverse reactions in patients. The united states pharmacopeia and european pharmacopeia have successively placed new requirements on HCP antibody coverage assays in 2016 and 2017.
Most of the HCPs have isoelectric points between 3 and 6, and thus can be removed by relevant processes and parameters, but currently, no specific removal of HCP residues in biological products is available, and most of the existing reports mainly aim at the removal of HCP residues in animal cells, such as the removal of HCP residues in CHO cells, mainly aim at the removal of HCP residues by affinity methods, and no specific aim at the removal of residues in yeast cells, such as the removal of HCP residues in pichia pastoris. HCP residues are ubiquitous in fermentation broth, especially intracellular, but most of them are slowly removed as the downstream process proceeds, but some are not, thus requiring special process routes and parameters for removal.
The invention mainly removes HCP residues in pichia pastoris, especially extracellular expressed protein through a process route and parameters, can solve the problem that the HCP and the target protein are not easy to remove due to the similar chemical properties of the HCP and the target protein, such as isoelectric point, molecular weight and other factors at present although the step of cell disruption is not carried out.
Disclosure of Invention
The invention aims to provide a method for removing HCP residues in yeast cells, which can well remove the HCP residues in the yeast cells to reach relevant standards.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for removing HCP residues in yeast cells, comprising:
(1) Carrying out cation exchange chromatography on the yeast cell sap, eluting, and collecting an elution peak to obtain an elution sample solution;
(2) Carrying out hydrophobic chromatography and elution on the elution sample liquid obtained in the step (1), and collecting an elution peak to obtain an elution sample liquid;
(3) Performing CHT chromatography on the elution sample liquid obtained in the step (2), eluting, and collecting an elution peak to obtain an elution sample liquid;
(4) And (4) carrying out cation chromatography on the elution sample liquid obtained in the step (3), eluting, and collecting an elution peak.
And (5) detecting HCP (HCP) of the elution sample solution obtained in the step (4): and (3) detecting HCP residues by using a pichia pastoris HCP detection kit F640, wherein the concentration of the residues is within 100ppm (ng/mg), namely the residues meet the relevant standard.
Further, the method specifically comprises the following steps:
(1) Carrying out cation chromatography on yeast cell sap, firstly balancing a chromatographic column by using a balance buffer solution, continuously washing 2 column volumes by using the balance buffer solution in a sample loading stage, eluting at the flow rate of 2.0mL/min by using 2.8-3.6%, 8.2-11.8%, 23.8-27.3%, 47.9-51.5% and 100% gradient elution buffer solution respectively, collecting an elution peak, and detecting at the wavelength of 280nm to obtain an elution sample solution;
the balance buffer solution: 52mM sodium acetate-acetic acid, pH4.2;
the elution buffer: 52mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
the main function of the step is to capture target protein and remove HCP for the first time, the principle is that according to the difference of charged properties of the protein, the cation chromatography protein is positively charged and can be adsorbed by the filler, and the negatively charged protein can flow through, so that non-target protein and part of HCP can be removed.
(2) Performing hydrophobic chromatography on the elution sample liquid obtained in the step (1), firstly balancing a chromatographic column by using an equilibrium buffer solution, in a sample loading stage, continuously washing 2 column volumes by using the equilibrium buffer solution, performing linear elution by using 0-100% of elution buffer solution, eluting at the flow rate of 2.0mL/min, collecting an elution peak, and detecting at the wavelength of 280nm to obtain an elution sample liquid;
equilibration buffer: 21mM NaAc-HAc,1M (NH) 4 ) 2 SO 4 pH5.5；
Elution buffer: 21mM NaAc-HAc, pH5.5;
the method mainly comprises the steps of loading high salt, changing exposed groups of protein by using the high salt, exposing hydrophobic groups of the protein, binding to hydrophobic filler, eluting by reducing the salt concentration in the later period, exposing hydrophilic groups of the protein after the salt concentration is reduced, hiding the hydrophobic groups into the protein, and enabling the protein to flow along with the polarity change of a solution according to a polarity compatibility principle, so that HCP can be further removed.
(3) Performing CHT chromatography on the elution sample liquid obtained in the step (2), firstly balancing a chromatographic column by using an equilibrium buffer solution, in a sample loading stage, continuously washing 2 column volumes by using the equilibrium buffer solution, eluting by using the elution equilibrium solution at the flow rate of 2.0mL/min, collecting flow-through peaks, and detecting at the wavelength of 280nm to obtain an elution sample liquid;
further, the equilibration buffer: 20mM PB buffer, pH7.2;
further, the elution equilibrium solution: 400mM PB buffer, 1.0M sodium chloride, pH7.2;
the step adopts the principle of combining metal chelation and cation exchange for separation, the main function is metal chelation, proteins with similar properties can be separated, certain proteins cannot be carried on the filler when being loaded, but target proteins flow through in the flow-through stage, HCP is hung on the filler, and therefore HCP can be removed.
(4) Carrying out cation exchange chromatography on the elution sample liquid obtained in the step (3), firstly balancing a chromatographic column by using a balance buffer solution, continuously washing 2 column volumes by using the balance buffer solution in a sample loading stage, eluting at the flow rate of 2.0mL/min, respectively at the flow rate of 2.1-4.8%, at the flow rate of 5.9-8.6%, at the flow rate of 9.3-12.7%, at the flow rate of 23.5-27.6% and at the flow rate of 100% of the elution buffer solution, collecting elution peaks, and detecting the wavelength of 280nm;
further, the equilibration buffer: 50mM sodium acetate-acetic acid, pH4.2;
further, the elution equilibrium solution: 50mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2.
Further, the steps (1) to (4) adopt an AKTA pure 150 chromatography system, and all the chromatography columns are BXP16/30.
Further, the packing material of the chromatographic column in the step (1): any one of NanoSP 30L, nuvia and HR-S;
further, the packing material of the chromatographic column in the step (2): phenyl bestarose FF (HS);
further, the packing material of the chromatographic column in the step (3): CHT;
further, the packing material of the chromatographic column in the step (4): SP Bestrose HP.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the invention can remove HCP (with similar property to target protein) remained in extracellular secretion protein in yeast, and the removal can reach related standards through detection, thereby being beneficial to the development of related biological processes and the progress of the whole biological industry.
Drawings
FIG. 1 is a chromatogram obtained by elution in step (1) according to a preferred embodiment of the present invention;
FIG. 2 is a chromatogram obtained by elution in step (2) in the example of FIG. 1 according to the present invention;
FIG. 3 is a chromatogram obtained by elution in step (3) in the example of FIG. 1 according to the present invention;
FIG. 4 is a chromatogram obtained by elution in step (4) in the example of FIG. 1 according to the present invention;
reference numerals are as follows: 1. a protein peak; 2. eluting a conductivity peak; 3. peak ratio of eluent.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for removing HCP residues in yeast cells comprises the following steps:
(1) Performing cation exchange chromatography on pichia pastoris cell sap, adopting an AKTA pure 150 chromatography system, firstly balancing a chromatography column by using an equilibrium buffer solution, continuously washing 2 column volumes by using the equilibrium buffer solution in a loading stage, collecting elution peaks, and determining the protein yield, wherein the chromatography process conditions are as follows:
a chromatographic column: BXP16/30;
gradient: 2.8-3.6%, 8.2-11.8%, 23.8-27.3%, 47.9-51.5%, 100%;
flow rate: 2.0mL/min;
detection wavelength: 280nm;
the chromatographic column is filled with: any one of NanoSP 30L, nuvia and HR-S;
the balance buffer solution: 52mM sodium acetate-acetic acid, pH4.2;
the elution buffer: 52mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
the main function of the step is to capture target protein and remove HCP for the first time, the principle is that according to the difference of charged properties of the protein, the cation chromatography protein is positively charged and can be adsorbed by the filler, and the negatively charged protein can flow through, so that non-target protein and part of HCP can be removed.
(2) Carrying out hydrophobic chromatography on the elution sample liquid obtained in the step (1):
the instrument comprises the following steps: AKTA pure 150 chromatography system;
a chromatographic column: BXP16/30;
filling: phenyl bestarose FF (HS);
and (3) an equilibrium buffer: 21mM NaAc-HAc,1M (NH) 4 ) 2 SO 4 pH5.5；
Elution buffer: 21mM NaAc-HAc, pH5.5;
firstly, balancing the chromatographic column by using an equilibrium buffer solution, and continuously washing 2 column volumes by using the equilibrium buffer solution in a loading stage;
linear elution: 0-100% elution buffer;
flow rate: 2.0mL/min;
and (3) detection: a wavelength of 280nm;
and collecting an elution peak, and determining the protein yield.
The method mainly comprises the steps of loading high salt, changing exposed groups of protein by using the high salt, exposing hydrophobic groups of the protein, binding to hydrophobic filler, eluting by reducing the salt concentration in the later period, exposing hydrophilic groups of the protein after the salt concentration is reduced, hiding the hydrophobic groups into the protein, and enabling the protein to flow along with the polarity change of a solution according to a polarity compatibility principle, so that HCP can be further removed.
(3) Performing CHT chromatography on the elution sample liquid obtained in the step (2), and firstly, balancing a chromatographic column by using an equilibrium buffer solution, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: AKTA pure 150 chromatography system;
filling: CHT (CHT);
a chromatographic column: BXP16/30;
and (3) an equilibrium buffer: 20mM PB buffer, pH7.2;
eluting the equilibrium solution: 400mM PB buffer, 1.0M sodium chloride, pH7.2;
the parameters and requirements of the loading stage are as follows:
flow rate: 2.0mL/min;
and (3) detection: a wavelength of 280nm;
collecting: flow through the peak;
the step adopts the principle of combining metal chelation and cation exchange for separation, the filler can chelate metal calcium ions in protein, the protein with similar properties can be separated, certain protein cannot be carried on the filler when being loaded, but target protein flows off in the flow-through stage, HCP is hung on the filler, and therefore HCP can be removed.
(4) Carrying out cation exchange chromatography on the elution sample liquid obtained in the step (3), firstly balancing a chromatographic column by using a balance buffer solution, in a sample loading stage, continuously washing 2 column volumes by using the balance buffer solution, eluting at the flow rate of 2.0mL/min, eluting at the flow rate of 5.9-8.6%, eluting at the flow rate of 9.3-12.7%, eluting at the flow rate of 23.5-27.6% and eluting at the flow rate of 100% by using the balance buffer solution, collecting an elution peak, and detecting at the wavelength of 280nm to obtain an elution sample liquid;
the instrument comprises the following steps: AKTA pure 150 chromatography system;
the chromatographic column is BXP16/30;
filling: SP Bestrose HP;
and (3) an equilibrium buffer: 50mM sodium acetate-acetic acid, pH4.2;
eluting the equilibrium solution: 50mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
this step is similar to step (1), and the HCP is removed again by cation exchange chromatography;
(5) And (3) detecting HCP (HCP) of the elution sample liquid obtained in the step (4): and (3) detecting HCP residues by using a pichia pastoris HCP detection kit F640, wherein the concentration of the residues is within 100ppm (ng/mg), namely the residues meet the relevant standard.
Example 1
A method for removing HCP residues in yeast cells (HCP residues in each step are detected by using a pichia pastoris HCP detection kit F640) comprises the following steps:
(1) Performing cation chromatography on pichia pastoris cell fluid (the initial protein concentration is 2.5 (mg/mL)), the initial HCP residual quantity is 5000ppm (ng/mg)), using an AKTA pure 150 chromatographic system, firstly balancing a chromatographic column by using an equilibrium buffer solution, continuously washing 2 column volumes by using the equilibrium buffer solution in a loading stage, collecting elution peaks, and determining the protein yield, wherein the chromatographic process conditions are as follows:
and (3) chromatographic column: BXP16/30;
gradient: 2.8%,8.9%,26.3%,50.5%,100%;
flow rate: 2.0mL/min;
detection wavelength: 280nm;
the chromatography column is filled with: any one of NanoSP 30L, nuvia and HR-S;
the balance buffer solution: 52mM sodium acetate-acetic acid, pH4.2;
the elution buffer: 52mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
the sampled protein concentration was 1.6 (mg/mL), the protein yield was 64%, and the residual amount of HCP was 2000ppm (ng/mg);
the main function of the step is to capture target protein and remove HCP for the first time, the principle is that the cation chromatography protein is positively charged and can be adsorbed by the filler according to different charged properties of the protein, and the negatively charged protein can flow through to remove non-target protein and part of HCP.
Referring to FIG. 1, since the target protein and HCP have similar properties, a part of HCP protein remains in the process of capturing the target protein, and most of the impure protein and most of the HCP flow through and are relatively disordered from the peak shape, which indicates that a part of impurities can be captured at the stage of capturing the target protein, and can be removed by the subsequent chromatography step.
(2) Carrying out hydrophobic chromatography on the eluent obtained in the step (1):
the instrument comprises the following steps: AKTA pure 150 chromatography system;
a chromatographic column: BXP16/30;
filling: phenyl bestarose FF (HS);
and (3) an equilibrium buffer: 21mM NaAc-HAc,1M (NH) 4 ) 2 SO 4 pH5.5；
Elution buffer: 21mM NaAc-HAc, pH5.5;
the chromatographic column is firstly balanced by the balance buffer solution, in the loading stage, the balance buffer solution is used for continuously washing 2 column volumes,
linear elution: 0-100% elution buffer;
flow rate: 2.0mL/min;
and (3) detection: a wavelength of 280nm;
and collecting an elution peak, and determining the protein yield.
The sampled protein concentration was (1.4 mg/mL), the protein yield was 87.5%, and the residual amount of HCP was 800 (ng/mg).
The method mainly comprises the steps of loading high salt, changing exposed groups of protein by high salt, exposing hydrophobic groups of protein, so that the hydrophobic groups can be combined on hydrophobic filler, eluting by reducing the salt concentration in the later period, exposing hydrophilic groups of the protein after the salt concentration is reduced, hiding the hydrophobic groups into the protein, and enabling the protein to flow along with the polarity change of solution according to the polarity compatibility principle, so that HCP can be further removed.
Referring to FIG. 2, a small portion of the impure protein flows through the loading stage (the first small drum in the figure), while most of the target protein and a small portion of the HCP are bound to the hydrophobic packing, and a small portion of the HCP remains on the packing during the elution process and cannot be eluted, while most of the target protein remains on the packing of the chromatographic column, thereby completing the separation of the HCP.
(3) Performing CHT chromatography on the eluent obtained in the step (2), firstly, balancing a chromatographic column by using an equilibrium buffer solution, wherein the chromatographic conditions are as follows:
the instrument comprises: AKTA pure 150 chromatography system;
filling: CHT;
a chromatographic column: BXP16/30;
and (3) an equilibrium buffer: 20mM PB buffer, pH7.2;
eluting the equilibrium solution: 400mM PB buffer, 1.0M sodium chloride, pH7.2;
the parameters and requirements of the loading stage are as follows:
flow rate: 2.0mL/min;
and (3) detection: a wavelength of 280nm;
collecting: flow through the peak;
the step adopts the principle of combining metal chelation and cation exchange for separation, mainly plays a role of metal chelation, can separate proteins with similar properties, certain proteins cannot be carried on the filler when being loaded, but target proteins flow through in the flow-through stage, HCP is hung on the filler, and therefore HCP can be removed.
The collected protein concentration was (0.9 mg/mL), the protein yield was 64.3%, and the residual amount of HCP was 231 (ng/mg).
Referring to FIG. 3, the flow of the sample through a portion of HCP reduces the residual HCP content, and the linear elution phase is divided into two parts, the first part is the major portion of the target protein and a small amount of HCP protein, while the second step shows more peaks of HCP protein, which further reduces the HCP content based on the previous step.
(4) Carrying out cation exchange chromatography on the eluent obtained in the step (3), firstly balancing a chromatographic column by using a balance buffer solution,
the instrument comprises the following steps: AKTA pure 150 chromatography system;
the chromatographic column is BXP16/30;
filling: SP Bestrose HP;
and (3) an equilibrium buffer: 50mM sodium acetate-acetic acid, pH4.2;
eluting the equilibrium solution: 50mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
in the sample loading stage, continuously washing 2 column volumes with an equilibrium buffer solution, eluting with 3.2%,7.5%,11.2%,24.5% and 100% of elution buffer solutions at the flow rate of 2.0mL/min, collecting elution peaks, and detecting the wavelength of 280nm;
this step is similar to step (1), again removing HCP;
the sampled protein concentration was 0.8 (mg/mL), the protein yield was 88.9%, and the residual amount of HCP was 89ppm (ng/mg).
Referring to fig. 4, the step is performed according to the chromatography principle of CHT, i.e. chelating metal calcium ions, and can adsorb a large amount of HCP protein and a small amount of target protein, while most of the target protein cannot adsorb the filler, so that the filler flows down, and the target protein with low HCP content can be obtained by collecting.
Different from the prior art, the method can remove HCPs (with similar properties to target proteins) remained in extracellular secretory proteins in yeast, and the removal can reach relevant standards through detection, thereby being beneficial to development of relevant biological processes and progress of the whole biological industry.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (1)
1. A method for removing HCP residues from yeast cells, comprising:
(1) Carrying out cation exchange chromatography on the yeast cell sap, eluting, and collecting an elution peak to obtain an elution sample solution;
(2) Carrying out hydrophobic chromatography on the elution sample liquid obtained in the step (1), eluting, and collecting an elution peak to obtain an elution sample liquid;
(3) Performing CHT chromatography on the elution sample liquid obtained in the step (2), eluting, and collecting flow-through peaks to obtain an elution sample liquid;
(4) Performing cation chromatography on the elution sample liquid obtained in the step (3), eluting, and collecting an elution peak;
the method comprises the following steps:
(1) Carrying out cation chromatography on yeast cell sap, firstly balancing a chromatographic column by using a balance buffer solution, continuously washing 2 column volumes by using the balance buffer solution in a sample loading stage, eluting at the flow rate of 2.0mL/min by using 2.8-3.6%, 8.2-11.8%, 23.8-27.3%, 47.9-51.5% and 100% gradient elution buffer solution respectively, collecting an elution peak, and detecting at the wavelength of 280nm to obtain an elution sample solution;
the balance buffer solution: 52mM sodium acetate-acetic acid, pH4.2;
the elution buffer: 52mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
(2) Performing hydrophobic chromatography on the elution sample liquid obtained in the step (1), firstly balancing a chromatographic column by using an equilibrium buffer solution, in a sample loading stage, continuously washing 2 column volumes by using the equilibrium buffer solution, performing linear elution by using 0-100% of elution buffer solution, eluting at the flow rate of 2.0mL/min, collecting an elution peak, and detecting at the wavelength of 280nm to obtain an elution sample liquid;
and (3) an equilibrium buffer: 21mM NaAc-HAc,1M (NH) 4 ) 2 SO 4 pH5.5；
Elution buffer: 21mM NaAc-HAc, pH5.5;
(3) Performing CHT chromatography on the elution sample liquid obtained in the step (2), firstly balancing a chromatographic column by using an equilibrium buffer solution, in a sample loading stage, continuously washing 2 column volumes by using the equilibrium buffer solution, eluting by using the elution equilibrium solution at the flow rate of 2.0mL/min, collecting flow-through peaks, and detecting at the wavelength of 280nm to obtain an elution sample liquid;
the balance buffer solution: 20mM PB buffer, pH7.0;
the elution equilibrium solution: 400mM PB buffer, 1.0M sodium chloride, pH7.0;
(4) Carrying out cation exchange chromatography on the elution sample liquid obtained in the step (3), firstly balancing a chromatographic column by using a balance buffer solution, continuously washing 2 column volumes by using the balance buffer solution in a sample loading stage, eluting at the flow rate of 2.0mL/min, respectively at the flow rate of 2.1-4.8%, at the flow rate of 5.9-8.6%, at the flow rate of 9.3-12.7%, at the flow rate of 23.5-27.6% and at the flow rate of 100% of the elution buffer solution, collecting elution peaks, and detecting the wavelength of 280nm;
the balance buffer solution: 50mM sodium acetate-acetic acid, pH4.2;
the elution equilibrium solution: 50mM sodium acetate-acetic acid, 1.5M sodium chloride, pH4.2;
the steps (1) to (4) all adopt an AKTApure 150 chromatography system, and all the used chromatography columns are BXP16/30;
the chromatographic column packing in the step (1): any one of NanoSP 30L, nuvia and HR-S;
the chromatographic column packing in the step (2): phenyl bestarose FF (HS);
the chromatography column packing in the step (3): CHT;
the chromatographic column packing in the step (4): SPBestrose HP.
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| CN107793469A (en) * | 2017-10-16 | 2018-03-13 | 上海药明生物技术有限公司 | A kind of affinity purification technique for removing host cell proteins content |
Non-Patent Citations (3)
| Title |
|---|
| pH-based cation exchange chromatography in the capture and elution of monoclonal antibodies;Paul K.Ng等;《J.Sep.Sci》;20121231;第35卷;第29-35页 * |
| 双柱连续流层析亲和分离抗体的过程设计与应用;高宗晔等;《高校化学工程学报》;20181218;第117-127页 * |
| 抗体药物分离纯化中的层析技术及进展;卢慧丽等;《化工学报》;20181231;第69卷(第1期);第341-351页 * |
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