CN115286716B - Method for large-scale purification of anti-PD-1 antibody - Google Patents

Abstract

The invention relates to a method for purifying an anti-PD-1 antibody on a large scale, which specifically comprises the following steps: (1) Taking cell supernatant containing anti-PD-1 antibody, clarifying and filtering; (2) affinity chromatography; (3) low pH virus incubation; (4) middle deep filtration; (5) cation chromatography; (6) anion chromatography; and (7) removing viruses and filtering. By optimizing the parameters of affinity chromatography, cation chromatography and anion chromatography, the purification scale of the anti-PD-1 antibody is successfully enlarged to 200L, the total purification yield of three batches is similar, the process quality parameters are stable, the purity of an antibody product is high, the method is suitable for commercial large-scale production of the anti-PD-1 antibody, and the production cost is obviously reduced.

Description

Method for large-scale purification of anti-PD-1 antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for purifying an anti-PD-1 antibody on a large scale.

Background

Antibody drugs are biological drugs that have been rapidly developed in recent years, and as the market share of new drug varieties has increased, they play an increasingly important role in the treatment of diseases such as cancer and autoimmune diseases. Therapeutic antibody drugs, due to their extremely high purity requirements, ensure the safety of their injections to the patient's body. For the whole biological medicine industry, the biological antibody medicine obtained by industrial-grade purification has important economic benefits. The anti-PD-1 antibody is a kind of antibody which attracts attention, and at present, a plurality of overseas and overseas companies are involved in development, and the antibody has good safety, so the reported clinical research results show that the antibody has a remarkable inhibitory effect on certain tumors.

In the prior art, a purification process for an antibody generally involves multiple steps, common steps include affinity chromatography, low-pH virus incubation, intermediate depth filtration, cation chromatography, anion chromatography, virus removal filtration and the like, and each step has a specific purpose and function, so that the high-purity antibody is finally obtained. Because the physicochemical properties of antibody drug molecules are similar, each company uses a plurality of purification steps in series according to self conditions to establish a self antibody purification platform so as to ensure the stability of the platform process and the high efficiency of the purification yield. However, as the large scale-up is the bottleneck of most enterprises, the production scale is generally maintained at 50-100L, and the greater economic benefit cannot be realized.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for purifying a PD-1 antibody on a large scale aiming at the defects of the prior art.

In order to solve the technical problem, the invention discloses a method for purifying an anti-PD-1 antibody on a large scale, which specifically comprises the following steps:

(1) Taking cell supernatant containing anti-PD-1 antibody, clarifying and filtering;

(2) Subjecting the clarified filtered collection liquid obtained in the step (1) to affinity chromatography, and collecting eluent;

(3) Incubating the eluate obtained in step (2) with a low pH virus;

(4) Filtering the system treated in the step (3) through intermediate deep layer filtration to obtain a filtered solution;

(5) Performing cation chromatography on the filtered solution obtained in the step (4), and collecting eluent;

(6) Carrying out anion chromatography on the eluent obtained in the step (5), and collecting a sample flow-through liquid;

(7) And (4) performing virus removal and filtration on the sample flow-through liquid obtained in the step (6) to obtain the purified anti-PD-1 antibody.

Wherein, in the step (2), the affinity chromatography comprises the operations of balancing, loading, first leaching, second leaching, third leaching and eluting; wherein the formulation of the second leaching buffer solution is 60-80 mM NaAc-HAc, 0.9-1.1M NaCl, and the pH value is 5.3-5.8; the formulation of the elution buffer solution is 60-80 mM NaAc-HAc, and the pH value is 3.5-3.8; the loading capacity is 18-38 g/L;

preferably, the second elution buffer formulation is 70mM NaAc-HAc, 1M NaCl, pH 5.6; the formulation of the elution buffer solution is 70mM NaAc-HAc, and the pH value is 3.7; the loading was 28 g/L.

Wherein, in the step (5), the cation chromatography comprises the operations of balancing, loading, leaching and eluting; wherein the formula of the equilibrium buffer solution and the elution buffer solution is 60-80 mM NaAc-HAc, and the pH value is 4.9-5.2; the formulation of the elution buffer solution is 60-80 mM NaAc-HAc, and the pH value is 6.4-6.7; the loading capacity is 28-52 g/L;

preferably, the equilibration buffer and the elution buffer are formulated at 70mM NaAc-HAc, pH 5.0; the formulation of the elution buffer solution is 70mM NaAc-HAc, and the pH value is 6.6; the loading was 40 g/L.

Wherein, in the step (6), the anion chromatography comprises the operations of balancing, loading and rinsing; wherein the formula of the equilibrium buffer solution and the elution buffer solution is 60-80 mM NaAc-HAc, and the pH value is 5.5-6.1; the loading capacity is 80-135 g/L;

preferably, the equilibration buffer and the elution buffer are formulated at 70mM NaAc-HAc, pH 5.8; the loading was 110 g/L.

Wherein the large scale is 200L or more, and the preferred large scale is 200L.

In the step (1), the anti-PD-1 antibody is a novel anti-PD-1 antibody which can block the binding of a PD-1 ligand and a programmed death molecule 1 (PD-1), so that the inhibition effect of the PD-1 ligand on a T cell expressing PD-1 is blocked (the detailed construction process of the anti-PD-1 antibody is disclosed in a patent CN 106432494B).

Wherein in the step (1), the clarification filtration specifically comprises the following steps: the clear harvest obtained after the production culture reached harvest standard (at 14 days of culture or at cell densities below 60%) was filtered through depth filters and bag filters and stored in corresponding containers.

Specifically, the clarification filtration is to remove CHO cells and cell debris during cell culture, and is favorable for the following purification operation.

Specifically, the depth filter is a Millipore D0HC depth filter, a Corblist PurcisAF filter L20, and the capsule filter is a Millipore 30-inch 0.5/0.2 μm capsule filter, preferably a Millipore D0HC depth filter (cat # MD0 HC) and a Millipore 30-inch 0.5/0.2 μm capsule filter (cat # KHGEA).

Wherein, in the step (2), the affinity chromatography is protein A affinity chromatography, and the chromatographic column packing adopts MabSelect SuRe (product number: 17-5438) of Cytiva as an affinity medium.

Specifically, the media consists of a rigid, high flow rate agarose backbone and an alkali-resistant protein a ligand. The protein A ligand after engineering modification has better stability than the traditional protein A ligand under the alkaline condition, and the economical efficiency and the product quality of the process are improved. In affinity chromatography the process solution is applied to the column at room temperature and the protein binds to the packing while most process related impurities, such as host protein (HCP), host DNA and other clear harvest components flow through.

Wherein, in the step (2), the affinity chromatography is the first step in the production and purification process. The column was first rinsed with first rinse buffer 70mM Tris-HAc,120 mM NaCl, pH 7.0 to remove column storage buffer, sterilized with 0.1M NaOH prior to use, and then equilibrated with equilibration buffer 70mM Tris-HAc,120 mM NaCl, pH 7.0 to prepare for loading. Performing affinity sample loading on the clarified harvest liquid; the first elution is carried out by using 70mM Tris-HAc and 120 mM NaCl as a first elution buffer solution at the pH of 7.0, then the second elution buffer solution is used for 70mM NaAc-HAc and 0.9-1.1M NaCl as a second elution buffer solution at the pH of 5.3-5.8, and finally the third elution buffer solution is used for 70mM NaAc-HAc and is used for elution at the pH of 5.5. Elution was performed with elution buffer 60-80 mM NaAc-HAc, pH 3.5-3.8. After the elution was completed, the affinity column was regenerated with 1M HAc and rinsed with 70mM Tris-HAc,120 mM NaCl, pH 7.0, second rinsing buffer; then the whole harvest is purified by disinfection and equilibration before use in the next cycle. Until the end of the last cycle, the column was sterilized after use with 0.1M NaOH solution, rinsed with 70mM Tris-HAc,120 mM NaCl, pH 7.0, third rinsing buffer and finally stored in 20% ethanol. AKTA was sterilized with 1M NaOH and then stored in 0.1M NaOH solution.

Preferably, the equilibration buffer is 70mM Tris-HAc,120 mM NaCl, pH 7.0, the first elution buffer is 70mM Tris-HAc,120 mM NaCl, pH 7.0, the second elution buffer is 70mM NaAc-HAc, 1M NaCl, pH 5.6, the third elution buffer is 70mM NaAc-HAc, pH 5.5, and the elution buffer is 70mM NaAc-HAc, pH 3.7.

Wherein, in the step (2), the affinity chromatography comprises a column packing method of an affinity chromatography column as follows: the affinity filler was first replaced three times with water for injection to remove the original preservation buffer of the filler, and then the filler concentration in the homogenate was measured. And taking out the column head, emptying bubbles on a screen at the bottom of the chromatographic column, uniformly mixing the filler in the container, and transferring the required filler suspension into the chromatographic column. After the filler is settled to the upper layer of clear solution, putting the column head into the column and starting to pack the column. Setting the initial linear flow rate to be less than or equal to 60 cm/h, reducing the column head after the column bed is stabilized, and increasing the flow rate until the target height of the column bed is reached.

Wherein, in the step (3), the incubation of the low-pH virus is specifically as follows: after completing the affinity chromatography, the eluent of each cycle of affinity chromatography is separately collected in a disposable liquid mixing bag, 1M HAc is added to adjust the pH value according to the requirement, and the mixture is incubated for 60-240 min at the pH value of 3.6-3.8 at the temperature of 15-25 ℃ for virus inactivation. After the inactivation is finished, the virus inactivation intermediate product is adjusted to pH 4.9-5.1 by using 1M Tris-HCl and pH 9.0.

Wherein, in the step (4), the main function of the intermediate depth filtration is to remove insoluble particles and impurities, such as host proteins (HCP), aggregates, residual Protein a and DNA.

Specifically, the affinity collection solution is subjected to virus inactivation at low pH, and then the pH is adjusted to 4.9-5.1, so that precipitation can occur. Filtering with a deep filter to remove precipitate, and filtering the middle deep filtration collected liquid with a bag filter.

Specifically, the depth filter is A1HC depth filter of Millipore, a PurciseSAF filter L10 of cobott, and the capsule filter is a 20 inch 0.5/0.2 μm capsule filter of Millipore, preferably A1HC depth filter of Millipore (cat # MA1 HC) and a 20 inch 0.5/0.2 μm capsule filter of Millipore (cat # KHGEA).

In the step (5), the cation chromatography adopts a chromatographic column of POROS XS (product number 1-2559) of Life Tech as a chromatographic packing.

Specifically, the filler consists of rigidly crosslinked polystyrene divinylbenzene and sulfopropyl ligand with negative charge. The cation exchange chromatography process is a purification step for further purifying protein after middle deep filtration, and removing process and product related impurities and pollutants.

Wherein, in the step (5), the cation Chromatography (CEX) is the second chromatography step in the production process. Cationic chromatography the intermediate depth-filtered solution was subjected to cationic protein purification at room temperature in a bind/elute manner. The cation column was first sterilized with 1M NaOH before use, and then equilibrated with an equilibration buffer 60-80 mM NaAc-HAc, pH 4.9-5.2, to prepare for loading. After the sample loading is finished, the chromatographic column is firstly leached by using 60-80 mM NaAc-HAc of leaching buffer solution and pH 4.9-5.2, and then is eluted by using 60-80 mM NaAc-HAc of eluting buffer solution and pH 6.4-6.7. After the product elution is completed, the chromatographic column is regenerated, sterilized and balanced before use, and then enters the next cycle until the purification of the whole sample loading is completed. Until the end of the last cycle of elution, the column was regenerated with 70mM NaAc-HAc, 1M NaCl, pH 5.5 and sterilized with post-use 1M NaOH, and finally stored in 0.1M NaOH buffer. AKTA Process was sterilized with 1M NaOH and then stored with 0.1M NaOH. The cation harvest was filtered using a Millipore 20 inch 0.5/0.2 μm capsule filter (cat. KHGEA).

Preferably, the equilibration buffer and the elution buffer used in the cation chromatography are 70mM NaAc-HAc, pH 5.0, and the elution buffer is 70mM NaAc-HAc, pH 6.6.

Wherein, in the step (5), the cation chromatography comprises a cation chromatography column packing method: the chromatographic packing was first replaced three times with water for injection to remove the original preservation buffer of the packing and then the concentration of the packing homogenate was determined. And (4) taking out the column head, emptying bubbles of a screen at the bottom of the chromatographic column, uniformly mixing the filler in the container, and transferring the required filler suspension into the chromatographic column. After the filler is settled to the upper layer of clear solution, putting the column head into the column and starting to pack the column. High flow rate until the target bed height is reached.

In the step (6), in the anion chromatography, the chromatographic column adopts POROS 50HQ (product number 4404334) of Life Tech as chromatographic packing.

Specifically, the filler consists of a rigid cross-linked polystyrene divinylbenzene pedestal and quaternized polyethylene imine ligand. The anion exchange chromatography process is mainly characterized in that after the cation exchange chromatography process, protein is further purified, and related impurities and pollutants of the process and the product are removed.

Wherein, in the step (6), the anion chromatography (AEX) is the third chromatography step in the production process. Anion chromatography the cation chromatography eluate was subjected to protein purification at room temperature in flow-through mode. The column was first sterilized with 1M NaOH prior to use, then pre-equilibrated with 700 mM NaAc-HAc pre-equilibration buffer, pH 5.5, and then equilibrated with 60-80 mM NaAc-HAc equilibration buffer, pH 5.5-6.1, in preparation for loading. During loading, the protein flows through the column, while negatively charged impurities bind to the column. After the sample loading is finished, the chromatographic column is rinsed by 60-80 mM NaAc-HAc of rinsing buffer solution and pH 5.5-6.1, and unbound products in the chromatographic column are recovered. After the product collection is completed, the AEX column enters the next cycle through regeneration, rinsing, disinfection before use, pre-balancing and balancing until the purification of the whole AEX sample loading is completed. After the end of the production run, the column was sterilized after use with 1M NaOH and finally stored in 10 mM NaOH. AKTA Process was sterilized with 1M NaOH and stored in 0.1M NaOH solution.

Preferably, the pre-equilibration buffer used for anion chromatography is 700 mM NaAc-HAc, pH 5.5, and both the equilibration buffer and the elution buffer are 70mM NaAc-HAc, pH 5.8.

Wherein, in the step (6), the anion chromatography comprises a column packing method of an anion chromatography column as follows: the chromatographic packing was first replaced three times with water for injection to remove the original preservation buffer of the packing and then the concentration of the packing homogenate was determined. And (4) taking out the column head, emptying bubbles of a screen at the bottom of the chromatographic column, uniformly mixing the filler in the container, and transferring the required filler suspension into the chromatographic column. After the filler is settled to the upper layer of clear solution, putting the column head into the column and starting to pack the column. High flow rate until the target bed height is reached.

In the step (7), the virus removal filtering is a step of removing viruses based on the principle of size interception.

Specifically, the anion-collected solution was subjected to virus filtration using a Modus 1.3 virus removal prefilter (cat # VPPS) and a Modus 1.3 virus removal filter (cat # VPMD). The virus filtration prefilter was first rinsed with water for injection, then the prefilter and the virus filter were rinsed with water for injection, and finally the virus prefilter and the virus filter were equilibrated with 60-80 mM NaAc-HAc, pH 5.5-6.1 buffer. During the product filtration process, the virus filter is passed under a certain pressure difference (Δ P) while the potential virus is retained. The product after virus filtration was collected in a collection bag, and the system was rinsed with 60-80 mM NaAc-HAc, pH 5.5-6.1, and the product remaining in the system was collected. Viral filtration harvest was filtered using a Millipore 20 inch 0.5/0.2 μm capsule filter (cat. KHGEA).

Preferably, 70mM NaAc-HAc, pH5.8, is used for both the equilibration buffer and the post-viral filtration rinse system.

Has the advantages that:

(1) The method for purifying the anti-PD-1 antibody provided by the invention has the advantages of simple process, good stability and high purity of the produced antibody product.

(2) The present invention successfully scales up the purification of anti-PD-1 antibodies to 200L by optimizing the parameters of affinity chromatography, cation chromatography and anion chromatography, relative to a production scale of 50-100L.

(3) Through the determination of a series of experimental results, the quality parameters of the purified product under the process flow are stable, the method can be applied to commercial large-scale production, and the company benefits can be further improved while the production cost is remarkably reduced.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a graph showing the trend of SEC-HPLC monomers (%) in each process step among different batches.

FIG. 2 is a diagram showing the trend of the CEX-HPLC main peak (%) in each process among different batches.

FIG. 3 is a graph showing the tendency of the reducing CE-SDS purity (%) in each step among different batches.

FIG. 4 is a graph showing the tendency of purity (%) of non-reducing CE-SDS in each step among different batches.

FIG. 5 is a summary of the affinity chromatography DoE study.

FIG. 6 is a summary of the cation chromatography DoE study.

FIG. 7 is a summary of the anion chromatography DoE study.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The first rinsing and rinsing 1, the second rinsing and rinsing 2, the third rinsing and rinsing 3, the second rinsing and rinsing 2 and the third rinsing and rinsing 3 have the same meaning.

Example 1 Process for purifying anti-PD-1 antibody

(1) Cell supernatants containing anti-PD-1 antibodies obtained after production culture reached harvest criteria (harvest criteria: 14 days of culture or at cell densities below 60%) and were clarified, filtered through Millipore's D0HC depth filter (cat # MD0 HC) and Millipore's 30 inch 0.5/0.2 μm capsule filter (cat # KHGEA) and stored.

(2) Affinity chromatography: the clarified filtered pool (concentration 9.0-10.0 g/L) was applied to a MabSelect Sure column. The column was first rinsed with first rinse buffer 70mM Tris-HAc,120 mM NaCl, pH 7.0 to remove column storage buffer, sterilized with 0.1M NaOH prior to use, and then equilibrated with equilibration buffer 70mM Tris-HAc,120 mM NaCl, pH 7.0 to prepare for loading. Performing affinity sample loading on the clarified harvest liquid; the first elution is carried out with 70mM Tris-HAc,120 mM NaCl, pH 7.0, the second elution is carried out with 70mM NaAc-HAc, 1M NaCl, pH 5.6, and the third elution is carried out with 70mM NaAc-HAc, pH 5.5. Elution was performed with elution buffer 70mM NaAc-HAc, pH 3.7. After the elution was completed, the affinity column was regenerated with 1M HAc and rinsed with 70mM Tris-HAc,120 mM NaCl, pH 7.0, second rinsing buffer; then the whole harvest is purified by disinfection and equilibration before use in the next cycle. Until the end of the last cycle, the column was sterilized after use with 0.1M NaOH solution, rinsed with 70mM Tris-HAc,120 mM NaCl, pH 7.0, third rinsing buffer and finally stored in 20% ethanol. AKTA was sterilized with 1M NaOH and then stored in 0.1M NaOH solution. The specific process parameters are detailed in table 1.

TABLE 1 Process parameters for affinity chromatography

The experimental results are shown in table 2 below, and it can be seen that the pH and the conductance of the three experimental samples are stable, the microbial load is less than 1 CFU/10mL, the endotoxin is less than 0.25 EU/mL, and the host protein residue of batch 1 is higher than that of the other two experiments. Since affinity chromatography is a crude purification step, host protein residues will be effectively removed in a subsequent fine purification step. The monomer proportion of three experimental SEC-HPLC is more than 95%, the proportion of CEX-HPLC main peak is more than 50%, the proportion of reducing capillary gel electrophoresis CE-SDS is more than 98%, and the proportion of non-original capillary gel electrophoresis CE-SDS is more than 98%, showing that the operation of the affinity chromatography process is stable.

TABLE 2 results of affinity chromatography sample detection

(3) Low pH virus incubation: after completing the affinity chromatography, the eluent of each cycle of affinity chromatography is separately collected in a disposable liquid mixing bag, 1M HAc is added to adjust the pH value according to the requirement, the pH value of the sample solution is adjusted to 3.6-3.8 under the condition of 15-25 ℃, and the incubation is carried out for 60-240 min for virus inactivation. After inactivation, the virus-inactivated intermediate product was adjusted to pH4.9 with 1M Tris-HCl, pH 9.0. The experimental results are shown in table 3, and it can be seen that the pH of the three experimental samples is 4.9, and the conductance and protein concentration are stable.

TABLE 3 incubation test results for low pH virus

(4) Middle deep filtration: after the affinity collection liquid is subjected to low-pH virus inactivation, the pH is adjusted to 4.9-5.1, and precipitation can occur. After filtration at room temperature using Millipore's A1HC depth filter (cat # MA1 HC), millipore's 20 inch 0.5/0.2 μm capsule filter (cat # KHGEA) was used. Specific intermediate depth filtration process parameters are shown in table 4.

TABLE 4 intermediate depth filtration Process parameters

The results are shown in table 5, and it can be seen that the pH and the conductance of the three experimental samples are relatively stable, and the host protein residue of batch 1 is effectively removed in the operation process. The residual quantity of DNA of three batches of experiments is also obviously reduced compared with the residual quantity after affinity chromatography, the monomer proportion of SEC-HPLC is more than 95%, the main peak proportion of CEX-HPLC is more than 50%, the reducing capillary gel electrophoresis CE-SDS is more than 98%, the non-original capillary gel electrophoresis CE-SDS is more than 98%, and the operation of the intermediate deep layer filtration process is stable.

TABLE 5 detection results of middle depth filtration experiment

(5) Cation chromatography: cationic chromatography the intermediate depth-filtered solution was subjected to cationic protein purification at room temperature in a bind/elute manner. The cation column was first sterilized with 1M NaOH prior to use and then equilibrated with 70mM equilibration buffer NaAc-HAc, pH 5.0, to prepare for loading. After the loading was completed, the column was first eluted with 70mM NaAc-HAc, pH 5.0, followed by 70mM NaAc-HAc, pH 6.6. After the product elution is completed, the chromatographic column is regenerated, sterilized and balanced before use, and then enters the next cycle until the purification of the whole sample loading is completed. Until the end of the last cycle of elution, the column was regenerated with 70mM NaAc-HAc, 1M NaCl, pH 5.5 and sterilized with 1M NaOH and finally stored in 0.1M NaOH buffer. AKTA Process was sterilized with 1M NaOH and then stored with 0.1M NaOH. The cation pool was filtered using a 0.5/0.2 μm pore size type filter. Other cation process parameters are shown in table 6.

TABLE 6 cation chromatography Process parameters

The results are shown in table 7, and it can be seen that the pH and conductance of the three experimental samples are stable, and the host protein residue is less than 10 ng/mg. The residual quantity of DNA of three batches of experiments is further reduced compared with that of DNA after deep filtration, the monomer proportion of SEC-HPLC is more than 97%, the main peak proportion of CEX-HPLC is more than 50%, the reducing capillary gel electrophoresis CE-SDS is more than 98%, and the non-original capillary gel electrophoresis CE-SDS is more than 98%, thus showing that the cation chromatography process is stable in operation.

TABLE 7 detection results of cation chromatography experiments

(6) Anion chromatography: anion chromatography the cation chromatography eluate was subjected to protein purification at room temperature in flow-through mode. The column was first sterilized with 1M NaOH prior to use, then pre-equilibrated with pre-equilibration buffer 700 mM NaAc-HAc, pH 5.5, and then equilibrated with equilibration buffer 70mM NaAc-HAc, pH5.8, to prepare for loading. During loading, the protein flows through the column, while negatively charged impurities bind to the column. After the loading is finished, the chromatographic column is rinsed by 70mM NaAc-HAc of rinsing buffer solution and pH5.8, and the unbound products in the chromatographic column are recovered. After the product is collected, the AEX column enters the next cycle through regeneration, rinsing, disinfection, pre-balancing and balancing before use until the purification of the whole AEX sample is completed. After the end of the production run, the column was sterilized after use with 1M NaOH and finally stored in 10 mM NaOH. AKTA Process was sterilized with 1M NaOH and stored in 0.1M NaOH solution. Other anion process parameters are shown in table 8.

TABLE 8 anion chromatography Process parameters

The results are shown in table 9, and it can be seen that the pH and conductance of the three experimental samples are relatively stable, the residual amounts of host proteins and DNA in the three experimental samples have reached detectable lower limits, the monomer proportion of SEC-HPLC is >97%, the main peak proportion of CEX-HPLC is >50%, the reducing capillary gel electrophoresis CE-SDS is >98%, and the non-native capillary gel electrophoresis CE-SDS is >98%, indicating that the anion chromatography process is stable in operation.

TABLE 9 anion chromatography test results

(7) Virus removal and filtration: the anion pools were virus filtered using a Modus 1.3 virus removal prefilter (cat # VPPS) and a Modus 1.3 virus removal filter (cat # VPMD). The virus filtration prefilter was first rinsed with water for injection, then the prefilter and the virus filter were rinsed with water for injection, and finally the prefilter and the virus filter were equilibrated with 70mM NaAc-HAc, pH5.8 buffer. During the product filtration process, the virus passes through the virus filtration membrane at a certain pressure difference (Δ P) while the potential virus is retained. The product after virus filtration was collected in a collection bag, the system was rinsed with 70mM NaAc-HAc, pH5.8, and the product remaining in the system was collected. Viral filtration harvest was filtered using a Millipore 20 inch 0.5/0.2 μm capsule filter (cat. KHGEA). The specific virus-removal filtration process parameters are shown in table 10.

TABLE 10 Virus removal filtration Process parameters

As shown in Table 11, the results show that the pH and the conductance of the three experimental samples are stable, the monomer ratio of SEC-HPLC of the three experiments is more than 97%, the main peak ratio of CEX-HPLC is more than 50%, the reducing capillary gel electrophoresis CE-SDS is more than 98%, and the non-native capillary gel electrophoresis CE-SDS is more than 98%, which shows that the virus-removing filtration process is stable in operation.

TABLE 11 detection results of the virus removal filtration experiment

From fig. 1 to fig. 4, the trend of the intermediate quality data of each process among different batches can be seen, and the detection results of three batches of experiments can show that the trends of the SEC-HPLC monomer (%), the CEX-HPLC main peak (%), the reducing CE-SDS purity (%) and the non-reducing CE-SDS purity (%) of the three batches of intermediates are more concentrated, wherein the SEC-HPLC monomer is more than 97%, the CEX-HPLC main peak is more than 50%, the reducing CE-SDS purity (%) >98%, and the non-reducing CE-SDS purity (%) >98%, and no large deviation phenomenon occurs, so that the product quality attribute under the 200L scale production process is stable, the scale amplification is successful, and the product can be used for commercial stable production.

Example 2 affinity chromatography Process parameter optimization

The specific process parameters of the affinity chromatography study are shown in Table 12.

TABLE 12 Process parameters for affinity chromatography

1.1 Experimental design (DoE)

The 5 factors to be studied in the DoE experiment were (1) the pH of the buffer of elution 2, (2) the sodium chloride concentration of the buffer of elution 2, (3) the pH of the elution buffer, (4) the concentration of the elution buffer, and (5) the loading capacity, respectively.

The DoE experiment will be performed by a 5-factor, 3-level, 4-resolution fractional factorial experimental design, with 4 additional centroids (median), and a number of experiments of 20 (16 experiments +4 centroids). Specific experimental protocols are shown in tables 13 and 14.

TABLE 13 DoE factors and levels thereof

TABLE 14 DoE study protocol-partial factorization

1.2 Results and discussion

1.2.1 Results of DoE experiments

The analytical results of the DoE study are summarized in table 15.

TABLE 15 analysis results of the affinity DoE study

1.3 DoE summary

Through JMP software, a statistical model of the response value of each factor of the affinity chromatography is established, and the summary of the DoE result is shown in FIG. 5. All factors that statistically had a significant effect on response values are indicated by the star boxes.

The pH of the elution buffer has a significant effect on the purity of the product SEC, the volume of the eluent and the recovery. Higher elution pH can result in higher SEC purity, but also increases elution volume and decreases recovery.

The loading has a significant effect on the product SEC purity, relative content of the CEX-HPLC main peak, volume of the eluate. Lower loadings help to increase SEC purity and CEX-HPLC main peak relative content and lower elution volumes.

Elution 2 buffer pH and sodium chloride concentration had a significant effect on the removal of residual DNA and there was an interaction. The best removal of residual DNA was achieved at a combination of high sodium chloride concentration (1100 mM) and low pH level (pH 5.3). Furthermore, sodium chloride concentration is a more important factor for residual DNA removal than pH, at low sodium chloride concentration levels (900 mM), pH levels can have a significant effect on residual DNA removal, while at high sodium chloride concentration levels, different levels of pH have little effect on residual DNA removal.

As for the SEC purity, the quality standard of the final product is more than or equal to 95 percent, while in the affinity chromatography research, the SEC purity of the elution sample is 96.5 to 97.7 percent, and in consideration of the subsequent process, the anion chromatography also has the capability of improving the SEC purity, so in the affinity chromatography research, the influence of each parameter on the SEC purity in the research range is acceptable.

For HCPs, none of the parameters set had a significant effect on HCPs over the range studied.

For the relative content of the main peak of CEX-HPLC, the data of the relative content of the main peak of CEX-HPLC are not greatly different (51.2% -48.6%) within the research range of each factor, and the standard of the detection item is not less than 40% considering the final product, so that the variation of the relative content of the main peak of CEX-HPLC is acceptable in the research of affinity chromatography, and the relative content of the main peak of CEX-HPLC is low risk to the final product within the research range of each parameter.

The worst case for residual DNA occurs at low sodium chloride concentration levels (900 mM) and high pH (5.9) in elution 2 buffer. The model was highly fitted (adjustment R2= 0.94) and the worst case DNA residual was predicted by the model to be 92.50pg/mg. Whereas in the worst case of possible running values (sodium chloride concentration level 900mM, pH 5.8), the predicted DNA residue is 85.15pg/mg, pH5.3-5.8 being considered to be an acceptable pH range for the elution 2 buffer.

For recovery, recovery rates greater than 90% were acceptable in the affinity DoE study, within the set parameters.

In summary, suggested ranges for the affinity chromatography parameters are shown in Table 16.

Table 16 suggested affinity chromatography parameters and ranges thereof

Example 3 cation chromatography Process parameter optimization

The specific process parameters of the cation chromatography process study are shown in Table 17.

TABLE 17 cationic chromatography Process parameters

2.1 design of experiment (DoE)

According to the pre-experimental results, the 6 factors to be studied in the DoE experiment were (1) the equilibration buffer pH, (2) the equilibration buffer concentration, (3) the loading sample pH, (4) the elution buffer pH, (5) the elution buffer concentration, (6) the loading load, respectively.

The DoE experiment will be performed by a partial factorial design of experiments with 6 factors, 3 levels, resolution 4, for a total number of experiments of 20 (16 +4 center points). Specific experimental protocols are shown in tables 18 and 19.

TABLE 18 DoE factors and levels thereof

TABLE 19 DoE study protocol-partial factorization

2.2 results and discussion

2.2.1 Results of DoE experiments

The analytical results of the DoE study are summarized in table 20.

TABLE 20 analytical results of the cation chromatography DoE study

2.3 DoE summary

Statistical models of the response values of the various factors of cation chromatography were established by JMP software, and a summary of the DoE results is shown in fig. 6. All factors that statistically had a significant effect on response values are indicated by the star boxes.

The concentration of the elution buffer has a significant influence on the SEC-HPLC purity of the product, the relative purity of the main peak of the CEX-HPLC, the elution volume and the recovery rate. Lower elution buffer concentrations can give higher SEC and CEX-HPLC purity, but also increase elution volume and decrease recovery.

The pH of the elution buffer has a significant influence on the SEC-HPLC purity of the product, the relative purity of the CEX-HPLC main peak and the recovery rate. Lower elution buffer pH gives higher SEC and CEX-HPLC purity, but also results in lower recovery.

The pH value of the equilibration buffer, the concentration of the equilibration buffer and the pH value of a sample to be loaded have obvious influence on the purity and the recovery rate of the SEC-HPLC product. Higher pH of equilibration buffer and higher concentration of equilibration buffer, lower pH of the sample load, can result in higher SEC purity and recovery.

For relative content of the CEX-HPLC main peak, the influence of the change of the research parameter on the result of the relative content of the CEX-HPLC main peak is not large. The minimum value in DOE experimental study is 53.3%, the standard of the test item can be achieved to be not less than 40%, so that in cation chromatography study, the change of the relative content of the CEX-HPLC main peak of the product in the study range of each parameter is acceptable and low risk.

For elution volume, elution buffer concentration and loading capacity had a significant effect on elution volume, with no interaction between factors. The worst case occurred at a combination of high loading (52 g/L) and low elution buffer concentration (30mM, cond, 2.3 mS/cm), with a maximum elution volume of 5.4CV achieved in the DoE experiment. Since the elution volume does not have an effect on the product quality of the product itself, it is believed that each parameter is of low risk to the process performance within the investigated range.

For the recovery rate, the pH value of the equilibrium buffer solution, the concentration of the elution buffer solution, the concentration of the equilibrium buffer solution, the pH value of the elution buffer solution and the pH value of the sample loading have obvious influence on the recovery rate. Wherein there is an interaction between the equilibration buffer pH and the equilibration buffer concentration. At low equilibration buffer pH levels (pH 4.8), the level of equilibration buffer concentration may have a significant effect on recovery, while at high equilibration buffer pH levels, different levels of equilibration buffer concentration have little effect on recovery. The worst case recovery (72%) occurs under a combination of low equilibration buffer pH (pH 4.8), low elution buffer concentration (50mm, cond 2.3 ms/cm), low equilibration buffer concentration (50mm, cond 1.4 ms/cm), low elution buffer pH (pH 6.4), and high loading sample pH (pH 5.2). The fit of the model was acceptable (adjustment R2= 0.78), and the recovery for each parameter combination could be predicted by the model. When the pH value of the equilibrium buffer solution is adjusted to be pH4.9, the concentration low level of the elution buffer solution is adjusted to be 60mM, the concentration low level of the equilibrium buffer solution is adjusted to be 60mM, and the rest parameters reach the worst condition in the DoE design range, the model predicts that the worst recovery rate is 80%. See table 21. In the downstream process, the five factors have low probability of reaching the worst condition at the same time, so that the recovery rate of the eluted product within the range of the adjusted factors is acceptable after the risk factors are comprehensively considered.

TABLE 21 acceptable recovery Rate Range Condition for each factor

For SEC-HPLC purity, elution buffer concentration, equilibration buffer pH, elution buffer pH, loading sample pH and equilibration buffer concentration all had significant effects on the elution product SEC-HPLC purity. There is an interaction between the elution buffer concentration and the equilibration buffer pH. At low elution buffer concentration levels, the level of equilibration buffer pH had no significant effect on SEC-HPLC purity, while at high elution buffer concentration levels, higher equilibration buffer pH gave higher SEC-HPLC purity. There is also an interaction between the pH of the equilibration buffer and the pH of the elution buffer, with different levels of elution buffer concentration having a greater effect on the SEC-HPLC purity at low pH levels of the elution buffer (pH 6.4), and with different levels of variation of elution buffer concentration having a lesser effect on the SEC-HPLC purity at high pH levels of the elution buffer. The SEC-HPLC worst case (95.7%) occurs under a combination of low equilibration buffer pH (pH 4.8), low equilibration buffer concentration (50 mM), high loading sample pH (pH 5.2), high elution buffer pH (pH 6.8) and high elution buffer concentration (90 mM). The fitting degree of the model is higher (adjusting R) ² = 0.99), the SEC purity at each parameter combination can be predicted by a model. When the lower limit of the pH of the equilibration buffer was adjusted to pH4.9, the concentration of the elution buffer was adjusted to 80mM at a high level, the pH of the elution buffer was adjusted to 6.7 at a high level, and the remaining parameters reached the worst conditions within the DoE design range, the model predicted the worst SEC-HPLC purity of 96.6%, see Table 22. In the downstream process, the five factors simultaneously reach the worst conditionThe probability of a condition is small, so that the SEC-HPLC purity results of the eluted product are acceptable over the adjusted range of factors, taking into account the risk factors.

TABLE 22 acceptable SEC-HPLC Condition of factors within the purity Range

In summary, the suggested ranges for the cation chromatography parameters are shown in Table 23.

Table 23 suggested cation chromatography parameters and ranges thereof

Example 4 anion chromatography Process parameter optimization

The specific process parameters of the anion chromatography process study are shown in Table 24.

TABLE 24 anion chromatography Process parameters

3.1 Experimental design (DoE)

The anion chromatography process is in a weak binding mode, and utilizes the charge property difference between antibody monomers and polymers to improve the monomer purity. The 5 factors studied in the DoE experiment were (1) the pH of the equilibration buffer, (2) the concentration of the equilibration buffer, (3) the pH of the sample, (4) the conductance of the sample, and (5) the load of the loaded sample, respectively. The DoE experiment was performed by a 5-factor, 3-level, 4-resolution fractional factorial experimental design, with 4 centroids (median) added, and a number of experiments of 20 (16 experiments +4 centroids). The specific experimental protocol is shown in tables 25 and 26.

TABLE 25 DoE factors and levels thereof

TABLE 26 DoE study protocol-partial factorization

3.2 Results and discussion

3.2.1 Results of DoE experiments

The results of the DoE study are summarized in table 27.

TABLE 27 detection results of the anion chromatography DoE study

3.3 DoE summary

Through JMP software, a statistical model of the response value of each factor of anion chromatography was established, and the summary of the DoE results is shown in FIG. 7. All factors that statistically had a significant effect on response values are indicated by the star boxes.

The equilibration buffer pH had a significant effect on product SEC purity, CEX-HPLC main peak relative content, and recovery. Higher equilibration buffer pH is beneficial for increasing SEC purity and relative content of the CEX-HPLC main peak, but at the same time results in a decrease in recovery.

The equilibration buffer concentration also had a significant effect on product SEC purity, CEX-HPLC main peak relative content, and recovery. As the equilibration buffer concentration increased, both SEC purity and the relative content of the CEX-HPLC main peak decreased, but resulted in an increase in recovery.

The sample pH only has an effect on the relative content of the main peak of CEX-HPLC. When the pH of the sample is increased, the relative content of the main peak of the CEX-HPLC is increased. However, the relative content of the CEX-HPLC main peak fluctuates between 54.1 and 57.7 percent, and considering the inherent system errors in the aspects of experiments and detection, the influence of the change of the research parameters on the relative content result of the CEX-HPLC main peak can be considered to be acceptable.

The sample conductivity value affects the SEC purity, the relative content of the CEX-HPLC main peak and the recovery rate of the product. As the conductivity of the sample increased, the SEC purity and relative content of the CEX-HPLC main peak decreased and the recovery increased, showing the same trend as the effect of equilibration buffer concentration on the product.

The loading capacity had a significant effect on HCP removal, CEX-HPLC main peak relative content and recovery. Under the condition of high load, the recovery rate is improved.

As for the SEC purity, the quality standard of the final product is more than or equal to 95%, in the anion chromatography process experiment, the SEC purity of the sample is 97.3-97.7%, and the SEC purity of the flow-through product is 97.8-99.3%. In general, the SEC purity was improved after anion chromatography process experiments relative to the sample as loaded. Considering that the main effect of the chromatography is to improve the SEC purity, according to a statistical model, the pH value of the equilibrium buffer solution and the conductivity of the sample loading have obvious influence on the SEC purity, the conductivity of the sample loading is adjusted to be less than or equal to 3mS/cm, the pH value of the equilibrium buffer solution is adjusted to be within the range of 5.7-6.1, and the SEC purity can be ensured to be more than or equal to 98.5%.

For HCPs, the parameters set for the rest, except loading, had no significant effect on HCPs over the range studied. Thus, in anion chromatography studies, where the parameters were run within the study range, the HCP content of the flow-through product was acceptable.

For the relative content of the main peak of CEX-HPLC, the data of the relative content of the main peak of CEX-HPLC are not greatly different (54.1% -57.7%) within the research range of each parameter, and the standard of the detection item is not less than 40% considering the final product, so that the variation of the relative content of the main peak of CEX-HPLC is acceptable in the research of anion chromatography, and the relative content of the main peak of CEX-HPLC is low risk for the final product within the research range of each parameter.

The worst case for the DoE experiment occurs for recovery under a combination of high equilibration buffer pH (pH 6.1), low equilibration buffer concentration (50 mM), low sample conductance (2 mS/cm) and low loading (65 g/L), which gives an actual recovery of 79.5% with low recovery. The model has high fitting degree (R2 =0.97, and R2=0.87 is adjusted), and the possible recovery rate under each condition can be predicted through the model. When each parameter range is in the worst condition, the worst recovery rate is predicted to be 78.8% through the model, and the model prediction is more accurate compared with the actual experimental data. Other parameters in the combination are set to be unchanged, and when the loading capacity is increased to 80g/L, the recovery rate predicted by the model can reach 80%. The recovery rate can be guaranteed to be above 80% considering that the probability of each parameter being in the worst condition at the same time is very small.

In summary, suggested ranges for the anion chromatography parameters are shown in Table 28.

Anion chromatography parameters and ranges thereof as suggested in Table 28

While the invention provides a novel concept and method for large scale purification of PD-1 antibodies, and a number of methods and approaches are contemplated for practicing the invention, the above description is merely a preferred embodiment of the invention, and it should be understood that modifications and adaptations can be made by one of ordinary skill in the art without departing from the principles of the invention and are intended to be considered within the scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims (1)

1. A method for large scale purification of an anti-PD-1 antibody, comprising the steps of:

(1) Taking cell supernatant containing anti-PD-1 antibody, clarifying and filtering;

(2) Subjecting the clarified filtered collection liquid obtained in the step (1) to affinity chromatography, and collecting eluent;

(3) Incubating the eluate obtained in the step (2) with low pH virus;

(4) Filtering the system treated in the step (3) through intermediate deep layer filtration to obtain a filtered solution;

(5) Performing cation chromatography on the filtered solution obtained in the step (4), and collecting eluent;

(6) Carrying out anion chromatography on the eluent obtained in the step (5), and collecting a sample flow-through liquid;

(7) Performing virus removal and filtration on the sample flow-through liquid obtained in the step (6) to obtain a purified anti-PD-1 antibody;

in the step (2), the affinity chromatography comprises the operations of balancing, loading, first leaching, second leaching, third leaching and eluting; wherein the balance buffer solution is 70mM Tris-HAc,120 mM NaCl, and pH 7.0; the first elution buffer is 70mM Tris-HAc,120 mM NaCl, and pH 7.0; the second leaching buffer solution is 70mM NaAc-HAc, 1M NaCl and has pH of 5.6; the third elution buffer is 70mM NaAc-HAc, and the pH value is 5.5; the elution buffer was 70mM NaAc-HAc, pH 3.7; the loading capacity is 18-37 g/L;

in the step (5), the cation chromatography comprises the operations of balancing, loading, leaching and eluting; wherein the balance buffer solution and the elution buffer solution are 70mM NaAc-HAc, and the pH value is 5.0; the elution buffer was 70mM NaAc-HAc, pH 6.6; the loading capacity is 30-50 g/L;

in the step (6), the anion chromatography comprises operations of pre-equilibrium, loading, leaching and elution; wherein the pre-equilibrium buffer solution is 700 mM NaAc-HAc, the pH value is 5.5, and the equilibrium buffer solution and the elution buffer solution are 70mM NaAc-HAc, the pH value is 5.8; the loading capacity is 80-120 g/L; the pH value of a sample is 5.7-6.1, and the conductivity value of the sample is 2-4mS/cm;

in step (1), the clarification filtration is carried out by a D0HC deep filter of Millipore and a 30-inch 0.5/0.2 μm capsule filter of Millipore; the concentration range of the anti-PD-1 antibody in the collected liquid obtained after clarification and filtration is 9.0-10.0 g/L;

in the step (2), the affinity chromatography is proteinA affinity chromatography, and a chromatographic column filler adopts MabSelect SuRe as an affinity medium;

in the step (3), the low-pH virus is incubated at pH 3.6-3.8 and 15-25 ℃ for 60-240 min, then virus inactivation is carried out, and the pH is adjusted to 4.9-5.1 after inactivation is finished;

in the step (4), the intermediate depth filtration is carried out by filtering at room temperature by using a A1HC depth filter of Millipore and then filtering by using a 20-inch 0.5/0.2 μm capsule filter of Millipore;

in the step (5), in the cation chromatography, a chromatographic column adopts POROS XS as a chromatographic filler, and a filtering solution obtained by middle deep filtration is subjected to cation chromatography at room temperature in a combination/elution mode;

in the step (6), in the anion chromatography, a chromatographic column adopts POROS 50HQ as a chromatographic filler, anion chromatography is carried out on the cation chromatography eluent at room temperature in a flow-through mode, and a product is recovered;

in the step (7), the virus removal filtration is carried out by using a Modus 1.3 virus removal prefilter and a Micro 1.3 virus removal filter to carry out virus filtration on the anion chromatography sample flow penetration solution; the balance buffer solution and the washing system after virus filtration are 60-80 mM NaAc-HAc, and the pH value is 5.5-6.1;

wherein the large scale is a 200L or more scale.

Images