Method for removing impurities in sample containing tri-specific antibodies
By optimizing conditions through steps such as filtration, affinity chromatography, low pH incubation, and ion exchange chromatography, impurities in trispecific antibody samples were removed, solving the problem of insufficient purity, achieving the production of high-purity antibodies, and reducing the risk of immunogenicity.
Patent Information
- Application Number
- CN202411126560.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-16
- Publication Date
- 2026-03-03
AI Technical Summary
Existing technologies are insufficient to effectively remove aggregates and fragments from trispecific antibody samples, leading to increased immunogenicity risk and insufficient purity.
Impurities are removed by employing steps such as filtration, affinity chromatography, low pH incubation, deep filtration, anion exchange chromatography, and cation exchange chromatography, combined with specific packing materials and buffer conditions, to optimize sample loading and elution conditions.
The purity of the trispecific antibody was significantly improved, and the risk of immunogenicity was reduced. The purity increased from about 74% to over 98%, and the residual HCP decreased from 1312.0 ppm to 22.6 ppm. The process is robust and easy to scale up.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibody purification technology, specifically relating to a method for removing impurities from a sample containing trispecific antibodies and a method for producing trispecific antibodies. Background Technology
[0002] Antibody-based targeted cancer therapy has become one of the main treatment methods for cancer. Among them, trispecific antibody (referred to as "tri-antibody") targeted cancer therapy is an important conceptual advancement. It adds a specific antigen-binding site to the bispecific antibody, resulting in three specific antigen-binding sites. These sites can simultaneously interact with target cells and functional cells (generally T cells or NK cells), thereby enhancing the killing effect on target cells. Compared to monoclonal antibodies, tri-antibodies add two specific antigen-binding sites, which is beneficial for redirecting drugs to the tumor site, increasing binding specificity, improving the accuracy of drug targeting tumor cells, and reducing off-target toxicity. Tri-antibodies can specifically deliver combinations of immune regulatory signals (such as co-stimulatory signals and checkpoint blockers) in the tumor microenvironment, potentially making them safer and more effective than the combination of multiple specific immunomodulatory monoclonal antibodies.
[0003] Currently, trispecific antibodies generate a large number of aggregates and fragments during the expression process. In order to reduce the risk of immunogenicity in patients and improve product purity, it is necessary to select appropriate purification methods and conditions to achieve the effect of removing a large number of aggregates and fragments. Summary of the Invention
[0004] The purpose of this invention is to provide a method for removing impurities from a sample containing trispecific antibodies, aiming to remove impurities such as aggregates and fragments to obtain high-purity trispecific antibodies and reduce the risk of trispecific antibodies producing immunogenicity in the human body.
[0005] In one aspect, the present invention provides a method for removing impurities from a sample containing trispecific antibodies, comprising:
[0006] The sample containing the trispecific antibody was filtered, and the filtrate was subjected to affinity chromatography to obtain the affinity chromatography eluent.
[0007] The affinity chromatography eluent was incubated at low pH, and the pH was adjusted back after incubation to obtain the intermediate product.
[0008] The intermediate product was subjected to deep filtration, and the resulting filtrate was subjected to anion exchange chromatography and cation exchange chromatography in sequence to obtain a cation exchange chromatography eluent.
[0009] The cation exchange chromatography eluent was then filtered to remove viruses.
[0010] In some embodiments, the step of filtering the sample containing trispecific antibodies is a cell harvest fluid sample; and the filtering process is clarification filtration.
[0011] In some embodiments, the affinity chromatography packing material is MabSelect SuRe LX, and the sample loading is preferably no more than 40 mg / mL.
[0012] In some embodiments, the affinity chromatography includes the steps of equilibration with a equilibration buffer, washing with a first eluent, a second eluent, and a third eluent in sequence, and elution with an elution buffer.
[0013] In some preferred embodiments, the equilibration buffer is a mixed solution of 20 mM to 25 mM phosphate and 100 mM to 150 mM sodium chloride, with a pH of 6.8 to 7.6, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or any value within the range thereof.
[0014] In some preferred embodiments, the first rinsing solution is a mixed solution of 20 mM to 25 mM phosphate and 100 mM to 150 mM sodium chloride, with a pH of 6.8 to 7.6, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or any value within the range thereof.
[0015] In some preferred embodiments, the second rinsing solution is a mixture of 20 mM to 30 mM citrate-sodium citrate buffer solution and 0.5 M to 1 M arginine hydrochloride, with a pH of 5.0 to 6.0, such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or any value within the range thereof.
[0016] In some preferred embodiments, the third rinsing solution is a 20mM to 30mM citrate-sodium citrate buffer solution with a pH of 5.0 to 6.0, such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or any value within the range thereof.
[0017] In some preferred embodiments, the elution buffer is a 20mM to 30mM citrate-sodium citrate buffer solution with a pH of 3.0 to 3.4, such as 3.0, 3.1, 3.2, 3.3, 3.4 or any value within the range thereof.
[0018] In some preferred embodiments, the elution pH is 3.1 to 3.3, for example 3.1, 3.2, 3.3 or any value within the range thereof.
[0019] In some embodiments, the pH of the low pH incubation is 3.5 to 3.7, for example, 3.5, 3.6, 3.7 or any value within the range thereof.
[0020] In some embodiments, the incubation time for the low pH incubation is 45 min to 90 min, for example, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min or any value within the range thereof.
[0021] In some implementations, the pH callback step is performed to a pH of 5.0 to 6.0, for example, to 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or any value within the range thereof.
[0022] In some preferred embodiments, 1M Tris is used for pH correction.
[0023] In some embodiments, the depth filtration is performed using a depth filtration membrane, preferably Millistak A1HC, with a filtration capacity of no more than 1606.1 g protein / m³. 2 .
[0024] In some embodiments, the packing material for the anion exchange chromatography is Capto Q. Preferably, the sample loading is no more than 100 g / L, and the pH of the loaded sample is adjusted to 8.0–8.6, for example, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or any value within the range thereof.
[0025] In some embodiments, the packing material for the cation exchange chromatography is Eshmuno CPX. Preferably, the sample loading capacity is 20–40 mg / mL, for example, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or any value within the range thereof. More preferably, the sample loading capacity is 25–35 mg / mL. In some embodiments, the pH of the loaded sample is adjusted to 6.0 ± 0.2.
[0026] In some implementations, the virus removal filtration is performed using a virus removal filtration membrane pack. Preferably, pre-filtration is performed using Pod Viresolve Prefilter, followed by nanofiltration using Viresolve Pro Modus.
[0027] In some embodiments, after the anion exchange chromatography and before the cation exchange chromatography, a step of adjusting the pH and conductivity of the anion exchange chromatography flow-through solution is included. Preferably, the pH of the anion exchange chromatography flow-through solution is adjusted to 6.0 ± 0.2, and the conductivity is adjusted to ≤ 5 mS / cm.
[0028] In some preferred embodiments, the trispecific antibody is a trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF. More preferably, the trispecific antibody is the trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF disclosed in PCT / CN2023 / 121074, which is incorporated herein by reference in its entirety.
[0029] In some implementations, the impurities include at least one of aggregates, cell debris, host cell proteins, and DNA.
[0030] On the other hand, the present invention also provides a method for producing trispecific antibodies, which includes a purification step, wherein the purification step uses the method provided by the present invention for removing impurities from a sample containing trispecific antibodies.
[0031] The method for removing impurities from samples containing trispecific antibodies provided by this invention optimizes affinity chromatography, low-pH virus inactivation, intermediate deep filtration, and ion exchange chromatography. Through optimization of chromatographic packing materials, sample loading, elution, and washing conditions, it effectively removes impurities (especially aggregates and cell debris) from samples containing trispecific antibodies, controlling the host cell protein content at a low level. This method also offers the advantage of process robustness, significantly improving the quality of antibody products and facilitating large-scale production. Samples treated using this method show improved purity (from approximately 74% to over 98% as detected by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC)) and purity (from approximately 76% to over 93% as detected by non-reducing sodium dodecyl sulfate capillary electrophoresis (NR-CE-SDS). Attached Figure Description
[0032] Figure 1 Affinity chromatography spectrum of MabSelect SuRe LX;
[0033] Figure 2 Capto Q anion chromatography pattern;
[0034] Figure 3 ESHMUNO CPX cation chromatography cycle 1 spectrum;
[0035] Figure 4ESHMUNO CPX cation chromatography cycle 2 spectrum;
[0036] Figure 5 The results show that the purity of the sample obtained by the method of the present invention, as detected by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), is increased from about 74% to over 98%.
[0037] Figure 6 The results show that the purity of the sample obtained by the method of the present invention, as detected by non-reduction sodium dodecyl sulfate capillary electrophoresis (NR-CE-SDS), was increased from about 76% to over 93%. Detailed Implementation
[0038] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experiments were conducted in accordance with the description herein or with conventional methods.
[0039] To better understand this invention, some terms are first defined. Other definitions are listed throughout the Detailed Description section. The term "PB" refers to phosphate; the term "CV" refers to column volume; and the term "Na-Citrate" refers to citrate-sodium citrate buffer solution.
[0040] The following abbreviations are used in this article:
[0041] pI: Isoelectric point;
[0042] SEC-HPLC: Size Exclusion Chromatography-High Performance Liquid Chromatography;
[0043] HCP: Host cell protein;
[0044] NR-CE-SDS: Non-reducing sodium dodecyl sulfate capillary electrophoresis;
[0045] HMW: High molecular weight component;
[0046] MP: Main peak;
[0047] LMW: Low molecular weight component.
[0048] Unless otherwise specified, all reagents and products used in the examples are commercially available, and all methods and conditions used in the examples are conventional methods and conditions in the art.
[0049] Example 1: Affinity chromatography to capture trispecific antibodies from the filtrate
[0050] Cell harvesting fluid containing trispecific antibodies (obtained using methods described in, for example, PCT / CN2023 / 121074) was clarified and filtered, and the clarified filtrate was collected. The clarified filtrate was loaded onto a MabSelect SuRe LX chromatography column to capture the trispecific antibodies. The MabSelect SuRe LX column was packed with a diameter of 5.0 cm, a bed height of 20.8 cm, and a column volume (CV) of approximately 408 ml. The system flow rate was set to 81.6 ml / min (retention time: 5 min). The column was first sterilized by washing with 0.1 M NaOH for 3 CV, and then equilibrated with equilibration buffer (20 mM PB + 150 mM NaCl, pH 7.0) for 5 CV. The filtrate, after two stages of deep filtration, was then loaded onto the column. The loading concentration was 40 mg of protein per ml of resin. The column was then eluted 3 CV each with equilibration buffer and the following eluents: a first eluent (a mixture of 20 mM phosphate and 150 mM sodium chloride, pH 6.8–7.6), a second eluent (a mixture of 20 mM citrate-sodium citrate buffer and 0.5 M arginine hydrochloride, pH 5.0–6.0), and a third eluent (20 mM citrate-sodium citrate buffer, pH 5.0–6.0). Eluting was then performed with 20 mM citrate-sodium citrate buffer at pH 3.1. The chromatogram is shown below. Figure 1 Collect the elution peaks and measure the host cell protein content, purity, and concentration of each collected elution sample.
[0051] Affinity chromatography is effective in removing process-related impurities (such as HCP and DNA), but its ability to remove product-related impurities (such as cell debris and aggregates) is limited. Relevant quality data for affinity-eluted samples and the recovery rates of affinity chromatography steps are shown in Table 1.
[0052] Table 1. Sample quality data and yield of affinity chromatography elution step.
[0053]
[0054] Example 2: Low pH incubation for virus inactivation and deep filtration of intermediate products
[0055] The collected affinity elution samples were pH-adjusted and incubated to inactivate potential viruses. The pH of the affinity elution samples was first adjusted to 3.6 with 1M Tris, and then incubated at this pH for 60 min. After incubation, the pH was adjusted to 5.5 with 1M Tris.
[0056] Connect the Millistak A1HC depth filtration membrane pack, vent the air, rinse the pack with purified water, and then equilibrate it with 20 mM Na-Citrate, pH 5.5 solution. Connect the sterile filter, and then filter the virus-inactivated sample after low-pH incubation. The filtration capacity of the Millistak A1HC membrane pack should not exceed 1606.1 g protein / m³. 2 After filtration, the membrane was washed with 20 mM Na-Citrate, pH 5.5 solution and collected.
[0057] The low-pH incubation virus inactivation step effectively inactivates enveloped viruses, ensuring product safety. Deep filtration of intermediate products effectively removes process-related impurities, such as HCP. The purity and HCP-related results of the collected filtrate are shown in Table 2.
[0058] Table 2. Sample quality data collected by deep filtration of intermediate products after low pH incubation.
[0059]
[0060] Example 3: Anion exchange chromatography further removes impurities.
[0061] The pH of the intermediate sample after deep filtration was adjusted to 8.0–8.6, followed by anion exchange chromatography. A 2.6 cm diameter Capto Q packing column was used, with a bed height of 20.5 cm and a column volume (CV) of approximately 108.84 mL. The system flow rate was set to 21.77 mL / min (retention time: 5 min). The column was first sterilized with 0.5 M NaOH for at least 3 column volumes, then pre-equilibrated with a pre-equilibration solution for at least 3 column volumes, and then equilibrated with an equilibration solution for at least 5 column volumes. The pH at the column outlet was checked and found to be within the pH range of the equilibration solution. The pH-adjusted intermediate sample was then deep filtered and collected, and loaded onto the column. The loading rate was 100 mg protein per mL of resin, followed by head washing with an equilibration solution for at least 3 column volumes. The flow-through peak was monitored using UV light during loading and head washing, with a collection range of 100 mAu / 2 mm – Max – 100 mAu / 2 mm. The chromatogram is shown below. Figure 2 .
[0062] Anion exchange chromatography effectively removes process-related impurities such as HCP and DNA. Table 3 shows the results regarding the purity, HCP, and recovery rates of the collected anion exchange chromatography samples.
[0063] Table 3. Sample loading and collection data and yield of anion exchange chromatography steps.
[0064]
[0065] Example 4: Cation exchange chromatography further improves product quality.
[0066] The pH and conductivity of the samples collected by anion exchange chromatography were adjusted, with pH adjusted to 6.0 ± 0.2 and conductivity adjusted to ≤ 5 mS / cm. After adjustment, Eshmuno CPX cation exchange chromatography was performed. Eshmuno CPX packing material was used to pack a 2.6 cm diameter column with a bed height of 17.5 cm and a column volume (CV) of approximately 92.91 mL. The system flow rate was set to 18.58 mL / min (retention time: 5 min). The column was first sterilized with 0.5 M NaOH for at least 3 column volumes, then equilibrated with equilibration solution for at least 5 column volumes. The pH of the column outlet was checked and found to be within the pH range of the equilibration solution. The adjusted anion exchange chromatography samples were then loaded onto the column at a rate of 35 mg protein per mL of resin. The column was then eluted with equilibration solution for at least 3 column volumes, followed by elution with 20 mM citrate-sodium citrate buffer + 150 mM NaCl at pH 6.0. Elution peak collection range: 700 mAu / 2 mm - Max - 700 mAu / 2 mm. See the chromatography chromatogram. Figure 3 and Figure 4 .
[0067] Cation exchange chromatography effectively removes product-related impurities such as debris and aggregates. The purity, HCP, and recovery rate of the collected cation exchange chromatography samples were measured; the results are shown in Table 4.
[0068] Table 4. Sample loading and collection data and recovery rates for cation exchange chromatography steps.
[0070]
[0071] Example 5: Virus Removal Filtration Further Removes Viruses
[0072] According to the "General Principles for Technical Review of Viral Safety Evaluation of Biological Tissue Extracts and Eukaryotic Cell Expression Products" and the requirements of ICH Q5, the production process must include effective process steps for virus removal / inactivation. Therefore, the antibodies expressed in CHO cell culture are filtered to remove viruses.
[0073] Except for virus pre-filters, Merck's Viresolve is used. Connect the PodViresolve Prefilter to the virus removal filter, then rinse with purified water. Connect the Viresolve Pro Modus filter, and rinse again with purified water. Equilibrate the membrane with 20mM citrate-sodium citrate buffer at pH 6.0 until the pH and conductivity of the effluent section match the equilibration buffer. Add the sample collected by cation exchange chromatography and filter, ensuring the filter loading does not exceed 1094.2 g / m³. 2 Then wash the membrane pack with equilibration solution and collect it.
[0074] Virus removal filtration can significantly reduce the titer of the model virus without affecting product quality. The purity and HCP of the collected virus removal filtered samples were tested, and the relevant results are shown in Table 5.
[0075] Table 5. Virus filtration, sample loading, and sample collection quality data.
[0076]
[0077]
[0078] Combining Table 1, Table 5, and Figure 5 and Figure 6 As can be seen, at the time of antibody cell harvesting, the SEC-HPLC purity is approximately 74%, the NR-CE-SDS purity is approximately 76%, and the residual HCP is approximately 1312.0 ppm. After downstream purification processes, up to the virus removal filtration step, the SEC-HPLC purity can be increased to approximately 98%, the NR-CE-SDS purity to approximately 93%, and the residual HCP to approximately 22.6 ppm. Therefore, the antibody purification process of this invention is very robust and can significantly improve product quality. Furthermore, the antibody purification process of this invention is easy to scale up and suitable for the production of clinical samples.
Claims
1. A method for removing impurities from a sample containing trispecific antibodies, comprising: The sample containing the trispecific antibody was filtered, and the filtrate was subjected to affinity chromatography to obtain the affinity chromatography eluent. The affinity chromatography eluent was incubated at low pH, and the pH was adjusted back after incubation to obtain the intermediate product. The intermediate product was subjected to deep filtration, and the resulting filtrate was subjected to anion exchange chromatography and cation exchange chromatography in sequence to obtain a cation exchange chromatography eluent. The cation exchange chromatography eluent was then filtered to remove viruses.
2. The method according to claim 1, wherein, The affinity chromatography packing material is MabSelect SuRe LX, preferably with a loading capacity of no more than 40 mg / mL; and / or The affinity chromatography includes the steps of equilibration with equilibration buffer, washing with first eluent, second eluent and third eluent in sequence, and elution with elution buffer. Preferably, the equilibration buffer is a mixed solution of 20mM-25mM phosphate and 100mM-150mM sodium chloride, with a pH of 6.8-7.6; The first rinsing solution is a mixed solution of 20 mM to 25 mM phosphate and 100 mM to 150 mM sodium chloride, with a pH of 6.8 to 7.6; The second rinsing solution is a mixed solution of 20mM-30mM citrate-sodium citrate buffer solution and 0.5M-1M arginine hydrochloride, with a pH of 5.0-6.0; The third rinsing solution is a 20mM to 30mM citrate-sodium citrate buffer solution with a pH of 5.0 to 6.0; The elution buffer is a 20mM–30mM citrate-sodium citrate buffer solution with a pH of 3.0–3.4; and / or The elution pH is 3.1 to 3.
3.
3. The method according to claim 1 or 2, wherein, The pH of the low pH incubation is 3.5–3.7; The incubation time for the low pH incubation is 45 min to 90 min; and / or In the pH callback step, the pH is adjusted back to 5.0-6.0; preferably, 1M Tris is used for pH callback.
4. The method according to any one of claims 1 to 3, wherein, The deep filtration is performed using a deep filtration membrane, preferably Millistak A1HC, with a filtration capacity of no more than 1606.1 g protein / m³. 2 ; The packing material for the anion exchange chromatography is Capto Q; preferably, the sample loading is no more than 100 g / L, and the pH of the loaded sample is adjusted to 8.0–8.6; and / or The packing material for the cation exchange chromatography is Eshmuno CPX; preferably, the sample loading is 20-40 mg / mL, more preferably 25-35 mg / mL; the pH of the sample loaded is adjusted to 6.0±0.
2.
5. The method according to any one of claims 1 to 4, wherein, The virus removal filtration is performed using a virus removal filtration membrane package, preferably using Pod Viresolve Prefilter for pre-filtration, followed by nanofiltration using Viresolve Pro Modus.
6. The method according to any one of claims 1 to 5, wherein, The process includes adjusting the pH and conductivity of the anion exchange chromatography flow-through solution after the anion exchange chromatography and before the cation exchange chromatography.
7. The method according to any one of claims 1 to 6, wherein, The trispecific antibody is a trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF.
8. The method according to any one of claims 1 to 7, wherein, The impurities include at least one of aggregates, cell debris, host cell proteins, and DNA.
9. Use of the method according to any one of claims 1 to 8 in the production of trispecific antibodies.
10. A method for producing a trispecific antibody, comprising a purification step, wherein the purification step uses the method according to any one of claims 1 to 8.