WO2020147298A1 - Cell strain constructing method for preparing fully human anti-egfr monoclonal antibody - Google Patents

Abstract

Provided is a cell strain constructing method for preparing fully human anti-EGFR monoclonal antibody, wherein, in a POOL screening step after cells of dihydrofolate reductase negative Chinese hamster ovary cell lines (CHO/DHFR-) complete a transfection step, in the first stage, the cell lines integrated with the heavy chain gene are screened using a SFM4CHO culture medium without HT, and the cell lines integrated with the light chain gene are screened simultaneously using a Blasticidin in the culture medium; and in the second stage, MTX is added to increase copy numbers of the light and heavy chain genes integrated into the cell line genomes to improve the expression level. A stable cell line with an expression level of 2.0g/L-3.0g/L, which is suitable for a large trial production process, is cloned and screened out with improved product SEC, pI-cIEF, de-sugaring molecular weight and antibody activity based on the cells.

Description

A cell line construction method for preparing anti-EGFR fully humanized monoclonal antibody Technical field

The present invention relates to the field of biomedicine technology, in particular to a method for constructing a cell line for preparing an anti-EGFR fully humanized monoclonal antibody.

Background technique

Monoclonal antibody (mAb) is a highly uniform antibody produced by a single B-cell clone and only directed against a specific epitope. It has specific targeting. The indications are mainly for tumors, autoimmune diseases, Infectious diseases and other aspects. Since the world's first therapeutic monoclonal antibody Rituxin was approved by the FDA in 1997, monoclonal antibodies have rapidly developed in the global biomedicine field in just 20 years and have become one of the research hotspots in recent years.

In recent years, a number of companies and research institutions researching therapeutic monoclonal antibody drugs have also emerged in China. Among them, therapeutic antibodies targeting epidermal growth factor receptor (EGFR) are particularly concerned. EGFR is a transmembrane glycoprotein, which is highly expressed in solid tumors. Tumors with positive EGFR expression have the characteristics of high malignancy and strong invasiveness, and the level of EGFR expression is related to the prognosis, thus becoming an important target of current tumor molecular targeted therapy. Monoclonal antibody drugs targeting EGFR can compete with endogenous ligands to bind to EGFR, and produce anti-tumor effects by inhibiting the activation of tyrosine kinases and promoting EGFR internalization.

At present, three anti-EGFR monoclonal antibodies have been marketed at home and abroad. Compared with other chemotherapeutics, they have the characteristics of clear target, significant curative effect, and low side effects, and they have achieved good clinical effects. The first anti-EGFR monoclonal antibody marketed was cetuximab, which was approved by the US FDA (the Food and Drug Administration) in 2004. It is a recombinant anti-EGFR human-mouse chimeric monoclonal antibody for the treatment of metastatic colon. Cancer, this product has been approved to enter the domestic market in 2009, Chinese trade name: Erbitux; Panitumumab is the first recombinant anti-EGFR fully humanized monoclonal antibody for the treatment of advanced colorectal cancer Its immunogenicity is lower than that of chimeric monoclonal antibodies such as cetuximab, and its potential clinical application side effects are lower. At present, panitumumab has been approved for marketing in more than 40 countries such as Europe, America and Japan, but domestic needs still rely on imports. Imported original research drugs are expensive, which limits the chances of curing most colorectal cancer patients.

At present, domestic research institutions and pharmaceutical companies have jointly developed panitumumab biosimilar drugs. However, the research and development of biosimilar drugs is different from the research and development of chemical generic drugs. In comparison, the research and development of biosimilar drugs has a considerable technical threshold. Small changes in each production step will affect the expression or activity of the final product; high-expressing cell lines and cell culture process development, protein purification technology and process control, antibody drug quality standards, etc., all restrict biological analogs R&D and industrialization.

Summary of the invention

In order to solve the limitations and defects of the prior art, the present invention uses the original drug panitumumab as a reference control, constructs a high-expressing cell line, and develops a pilot scale culture and purification process to obtain recombinant antibodies with similar properties and functions. EGFR humanized monoclonal antibody. The present invention screens out stable cell strains with high expression of Vectibix through POOL construction, batch feeding experiment screening and plating, clone picking, HTRF, clone amplification, clone batch feeding experiment. Through process optimization, the expression level reached 2.05g/L, and the product SEC, pI-cIEF, the molecular weight of desaccharides, and cell-based antibody activity were significantly improved.

A cell line construction method for preparing a fully humanized monoclonal antibody against EGFR, which is characterized in that the cells of the Chinese hamster ovary cell dihydrofolate reductase deficient strain CHO/DHFR- complete the POOL screening after the transfection step In the first stage, SFM4CHO medium without HT is used to screen cell lines that integrate heavy chain genes, and blasticidin in the medium is used to screen out cell lines that integrate light chain genes; in the second stage, MTX is added to increase The copy number of light and heavy chain genes integrated into the genome of the cell line to increase expression.

Preferably, in the POOL screening step, in the first stage, the concentration of GlutaMAX-I in the culture medium is 4mM, and the concentration of blasticidin is 10mg/l. Passage twice a week until the cell viability is restored to more than 85%; in the second stage, culture The concentration of GlutaMAX-I in the base is 4mM, the concentration of blasticidin is 10mg/l, and the concentration of MTX is 500nM, and the cell viability is restored to more than 90% after passage twice a week.

Preferably, in the transfection step, after adjusting the cell density of the Chinese hamster ovary cell dihydrofolate reductase deficient strain CHO/DHFR- to a cell density of 10E5 cells/ml through complete medium, the transfection volume is 10ml, and then linear The plasmid pCHO1.0 invitrogen 20ug, FreeStyle ^TM MAX Reagent 16.7ul and OptiPro ^TM SFM 0.67ml were cultured, and 10ml CD DG44 complete medium was added after 6 hours. In the CD DG44 complete medium, the concentration of GlutaMAX-I was 4mM, PLURONIC F68 (PF68) The concentration is 18ml/L.

Preferably, clone amplification is performed after the POOL screening step is completed, and 2X103 feed culture is used. The concentration of FM012 in the 2X103 feed is 50 g/l, and sugar is supplemented to a final concentration of 10-12 g/l each time.

Preferably, the plated culture is carried out after the feed culture, the cells are calculated at a density of 300cells/ml, and the corresponding cell volume is added to a centrifuge tube containing a semi-solid medium, and then plated. The volume of each well is 2.5ml, and the temperature is 36.5℃. , 6% CO ₂ constant temperature incubator for static culture.

Preferably, the preparation method of semi-solid medium used for plating culture is as follows: take 90ml CloneMedium CHO DHFR from -40°C 24 hours before plating and put it in a 2-8°C refrigerator, and put 2ml GlutaMAX-I, 1ml Clone Detect antihuman FITC, Add 2ml of Clone XL Reagent to the prepared Clone Medium CHO DHFR, then add MTX and blasticidin to make the final volume of 100ml at 500nM and 10mg/l, and use the host cell supernatant to adjust the volume to 100ml.

Preferably, the cell culture conditions during the construction process: temperature 36.5°C, humidity 75%-85%, carbon dioxide concentration 6%, rotation radius 2.5cm, rotation speed 110-225rpm, volume 20-30ml.

The present invention also relates to a vectibix antibody purification method, which is characterized in that the vectibix antibody cell culture solution is subjected to the following treatments: two-stage deep filtration clarification, affinity chromatography, low pH incubation, virus inactivation, anion exchange chromatography, and cation exchange Chromatography, concentration and dialysis.

Preferably, in the two-stage deep filtration and clarification step, the filter device is first rinsed with 100L/m2, 600LMH, and the filter device is connected in parallel with two Millipore Pod D0HC, 2×540cm2 and then connected in series with Millipore Pod A1HC, 540cm2; Equilibrate the filter device with 2 times the dead volume of the D0HC system with 600LMH until the pH value of the A1HC effluent is consistent with the balance buffer, and then start to filter the vectibix antibody cell culture solution. Discard the A1HC effluent end 0.5-0.7 times the entire system dead After the volume of the filtrate, connect the outflow end to the sterile filter in series, start collecting the filtrate, keep the feed rate at ≤200LMH, and maintain the pressure of each filter at ≤15psi; after the end of the sample filtration, use at least 2 times the dead volume of the entire system The buffer replaces the feed solution remaining in the depth filter membrane, and all the filtrates are combined. The balance buffer has a Tris-HAc concentration of 50 mM, a NaCl concentration of 150 mM, and a pH of 7.4.

Preferably, in the affinity chromatography step, the filler used is MabSelect, the column is 5.0×25cm, and the CV is 491mL. After washing, disinfecting and balancing the chromatography system, the combined filtrate obtained from the two-stage deep filtration and clarification step is loaded. , Flow rate 250cm/h, DBC≤35mg/mL packing, retention time≥5min, balance solution used for elution: 50mM Na-Acetate, pH 6.0, elution after elution, elution balance: 50mM Na-Acetate, pH Value 3.5, collection UV280: 150-150mAU/mm.

Preferably, in the low pH incubation step of virus inactivation, the affinity chromatography elution peak is adjusted to pH 3.5-3.8 and incubated for 2-4 hours at room temperature for virus inactivation, and then neutralized to a pH of 5.5-5.8. The combined product is filtered through sterilization.

Preferably, in the anion exchange chromatography step, the filler used is Super Q 650M (TOSOH), the chromatography column is 2.6×22.8 cm, CV 121 mL, and the product obtained in the low pH incubation step of virus inactivation is adjusted to pH 6.9. After the chromatography system is sterilized and equilibrated, load the sample, flow rate 250cm/h, DBC≤189mg/mL filler, retention time≥5min, start collecting UV280: 50mAU/mm, balance solution used for elution: 100mM Tris-HAc, pH 6.9 , End collection UV280: 50mAU/mm.

Preferably, in the cation exchange chromatography step, the filler used is Poros XS, the chromatography column is 2.6×25.4cm, CV 135mL, the chromatography system is sterilized and equilibrated, and the sample is loaded, the flow rate is 250cm/h, DBC≤65mg/mL Filler, retention time ≥5min, balance solution used for elution: 50mM Na-Acetate, pH 5.0, eluted after leaching, elution balance solution: 50mM Na-Acetate, 140mM NaCl, pH 5.0, collection UV280: 150- 150mAU/mm.

Preferably, during the concentration dialysis step, product concentration and liquid exchange are performed, the osmotic pressure is 276 mOsm/kg, the feed flux is not more than 360LMH, and the feed end pressure is not more than 20psi and the transmembrane pressure is not more than 15psi. Adjust the concentration of the buffer solution to 19-21mg/mL, and then filter it with a sterile filter.

The beneficial contributions of the present invention are:

1. For the first time cloned and screened a high-efficiency and stable cell line with an expression level of 2.0g/L-3.0g/L suitable for large-scale production processes. The product SEC, pI-cIEF, deglycosidated molecular weight, and cell-based antibody activity are all significantly improved. Glycosylation has also been significantly improved after process optimization, which provides the possibility for the industrialization of recombinant anti-EGFR humanized monoclonal antibodies in China.

2. Independently research and develop a four-step purification process, innovatively integrate two-stage deep filtration clarification, affinity chromatography, low pH incubation virus inactivation, anion exchange chromatography, cation exchange chromatography, concentration dialysis and other processes. The purification process is various The reaction conditions are mild, easy to industrialize, and the purification yield is ≥70%, and the purity of the original solution is ≥95%, which greatly reduces the industrialization cost. The innovative low pH incubation method can not only inactivate pathogens without introducing exogenous residues, but also separate NGF subunits in an acidic environment, which has the advantage of "one step, two effects".

BRIEF DESCRIPTION

Figure 1 is a logical diagram of the overall concept of the present invention.

Figure 2 is a schematic diagram of the cell line construction in Example 1.

Fig. 3 is a graph of cell viability in the POOL batch feeding experiment in Example 1.

Figure 4 is a graph of cell density in the POOL batch feeding experiment in Example 1.

Fig. 5 is a graph showing the viability curve of the batch feeding experiment of the candidate cell lines in Example 1.

Fig. 6 is a graph of the density curve of the batch feeding experiment of the candidate cell lines in Example 1.

Fig. 7 is a histogram of the expression level of a batch feeding experiment of candidate cell lines in Example 1.

Fig. 8 is a graph of cell density in a batch feeding experiment of selected cell lines in Example 1.

Fig. 9 is a graph of cell viability in a batch feeding experiment of selected cell lines in Example 1.

FIG. 10 shows the use of PCB, MCB, WCB, and EOPC cell cDNA as templates to amplify HC (M: Marker III; lane1: cDNA) in Example 2, and the cDNA obtained by reverse transcription of the extracted total RNA as the template for PCR amplification; lane2: RNA, using unreverse transcribed original RNA as a negative control for amplification; lane3: H ₂ O, using H ₂ O as a negative control for PCR amplification).

Figure 11 is the use of PCB, MCB, WCB, EOPC cell cDNA as a template to amplify LC (M: Marker III; lane1: cDNA, and cDNA obtained by reverse transcription of extracted total RNA as a template for PCR amplification in Example 2; lane2: RNA, using unreverse transcribed original RNA as a negative control for amplification; lane3: H ₂ O, using H ₂ O as a negative control for PCR amplification).

Fig. 12 is a graph showing the change of living cell density with time in Example 3.

Fig. 13 is a graph showing changes in cell viability with time in Example 3.

Fig. 14 is a graph showing the change of glucose concentration with time in Example 3.

Figure 15 is a graph showing the variation of lactate concentration with time in Example 3.

Figure 16 is a graph showing the change in protein yield over time.

Figure 17 is the affinity chromatogram in Example 5.

Figure 18 is an anion exchange chromatogram in Example 5.

Figure 19 is a cation exchange chromatogram in Example 5.

detailed description

In order to enable those skilled in the art to better understand the technical solutions of the present invention, a cell line construction method for preparing anti-EGFR fully humanized monoclonal antibodies provided by the present invention will be described in detail below in conjunction with examples.

As shown in Figures 1 and 2, the present invention uses the original product Vectibix (panitumumab) panitumumab as a reference product to develop a biosimilar drug with the same amino acid sequence as Vectibix. On the basis of the preliminary small-scale test process, explore and optimize the 200L-scale pilot-scale process methods and conditions, determine the key process parameters, and form a complete quality control system and standard, which is a further development of the recombinant anti-EGFR fully humanized monoclonal antibody. Lay the foundation for large-scale industrialization. The present invention adopts independent innovation technology to carry out the development and research of the pilot production process of recombinant anti-EGFR humanized monoclonal antibody, and intends to break through the three key technologies of efficient and stable cell line construction, cell culture technology, and purification technology; it intends to pass technological transformation and process innovation, While the product quality meets or exceeds the quality of the original foreign drug, its preparation cost is greatly reduced, and more colorectal cancer patients can be cured.

In the following examples of the present invention, the standard products used for the control are all commercially available Vectibix 400mg/20ml (panitumumab intravenous infusion solution, Amgen).

Cell line construction

The cell line construction includes Examples 1 and 2. The construction of a stable and efficient cell line is the key to the domestic industrialization of recombinant anti-EGFR fully humanized monoclonal antibodies. This example intends to prepare 50 primary cell banks (PCB, Primary Cell Bank) and 100 primary cell banks by constructing a CHO/DHFR-engineered cell line produced by recombinant anti-EGFR humanized monoclonal antibody (vectibix) antibody drugs. MCB, Master Cell Bank) and 200 working cell banks (WCB, Work Cell Bank) to screen high-expressing Vectibix cell lines suitable for large-scale production processes. Through process optimization, the expression level can reach more than 2.0g/L, and the expression level of the oldest cells is not less than 70%, which is easy to scale up and production; product SEC, pI-cIEF, desaccharide molecular weight, cell-based antibody activity and glycosylation are all Reach the level of the original research drug.

Example one

Transfection experiment:

Experimental Materials:

Host cell: DG44 (Chinese hamster ovary cell dihydrofolate reductase deficient strain CHO/DHFR-, ATCC)

Transfection reagent: Freestyle Max Reagent (Invitrogen 16447-100)

OPTI PRO SFM (GIBCO 12309-019)

Linearized plasmid: pCHO1.0 invitrogen

DG44 complete medium: CD DG44 (Invitrogen 12610-010) contains

4mM GlutaMAX-I (Invitrogen 35050-061)

18ml/L PLURONIC F68 (PF68) (Invitrogen 24040-032)

Consumables and related instruments

CORNING 125ml Erlenmeyer Flask, item number: 431143;

Shaker INFORS HT Model: Multitron Ⅱ

Parameter setting: 110rpm, 36.5℃, 6% CO ₂ , humidity 75%;

Cell counter BECKMAN COULTER model: VI-CELL XR

Biological safety cabinet Shanghai Shangjing Model: BSC-Ⅱ A2

Transfection experiment steps:

1. 24 hours before transfection, the spare transfected DG44 cells were passaged at a density of 8E5/ml, and the medium was CD DG44 complete medium.

2. On the day of transfection, count and test the viability of spare transfected cells.

3. Use CD DG44 complete medium to adjust the cell density to 10E5cells/ml, the transfection volume is 10ml, and use a 125ml shake flask.

4. Add 20ug of plasmid (pCHO1.0 invitrogen) and 16.7ul of FreeStyle ^TM MAX Reagent respectively to OptiPro ^TM SFM containing 0.67ml and mix.

5. After incubating the mixture of plasmid and transfection reagent at room temperature for 10 minutes, add it to the previously prepared transfected cells and place it in a shaker for culture.

6. Add 10ml DG44 complete medium after 6h to continue culturing.

POOL's screening experiment:

Consumables and related instruments

CORNING 125ml Erlenmeyer Flask, Item No.: 431143

Shaker INFORS HT Model: Multitron Ⅱ

Parameter setting: 110rpm, 36.5°C, 6% CO2, humidity 75%.

Cell counter BECKMAN COULTER model: VI-CELL XR

Biological safety cabinet Shanghai Shangjing Model: BSC-Ⅱ A2

Centrifuge EPPENDORF model: Centrifuge 5810R

The batch feed experiment medium information is as follows:

Table 1

Medium name	Brand	Item No.
SFM4CHO	Hyclone	SH30518.01
Glutamine	Sigma	G8540
MTX	Sigma	A6770-1
Blasticidin	Invitrogen	A11139-03

POOL's screening experiment steps

In the first step after transfection, SFM4CHO medium without HT is used to screen cell lines that integrate heavy chain genes. At the same time, the blasticidin contained in the medium can screen cell lines that integrate light chain genes. The second step of adding MTX is to increase the copy number of light and heavy chain genes integrated into the cell line genome and increase the expression level. Two-step pressurization can be screened to obtain stable cell lines with high expression that simultaneously integrate light and heavy chain sequences.

step one:

Basic medium: SFM4CHO

And contains: 4mM GlutaMAX-I, 10mg/l Blasicidin

Passaging: Cells are passaged twice a week in 125ml shake flasks until the cell viability recovers to over 85%. Freeze the cells twice, 2 each time.

Step two:

Basic medium: SFM4CHO

And contains: 4mM GlutaMAX-I, 10mg/l Blasicidin, 500nM MTX

Passaging: Cells are passaged twice a week in 125ml shake flasks until the cell viability recovers to more than 90%. Freeze the cells twice, 2 each time.

POOL batch feeding

Consumables and related instruments

CORNING 125ml Erlenmeyer Flask, Item No.: 431143

Shaker INFORS HT Model: Multitron Ⅱ

Parameter setting: 110rpm, 36.5°C, 6% CO2, humidity 75%.

Cell counter BECKMAN COULTER model: VI-CELL XR

Biological safety cabinet Shanghai Shangjing Model: BSC-Ⅱ A2

See Table 2 below for the information of batch feeding experiment medium:

Table 2

Medium name	Brand	Item No.
SFM4 CHO	Hyclone	SH30518.01
Cell Boost 5 (CB5)	Hyclone	SH30865.03
FM012	Sheffield	5X00483
2X103	Donin	F103-201
GlutaMAX-I	GIBCO	35050-061

Glucose

Sigma

49139

The experimental plan is as follows:

When the cell viability recovered to more than 90%, the batch feeding experiment was carried out in a 125ml shake flask.

POOL name: JN-XBK(10mg/l Blasticidin+500nM MTX)

The cells were inoculated and cultured at 5E5cells/ml for 4 days in SFM4CHO medium. After 4 days of growth, it was diluted 1:5 into Fed-batch basic medium 90% SFM4 CHO+10% CB5.

Batch feeding experiment plan:

Replenishment: 2X103; 50g/l FM012 in 2X103;

Each feeding will supplement the sugar to a final concentration of 10-12g/l.

The experimental conditions are shown in Table 3:

table 3

The results of the batch feeding experiment are shown in Figures 2 and 3. After the supernatant was harvested in the Pool batch feeding experiment, Titer was detected by HPLC-Protein A. The result: the Titer on the 13th day of JN-XBK was 1.67 g/L.

Semi-solid medium plating:

Equipment and consumables:

6-well plate Greiner Bio-One Cell Star Item No: 657185

Cell counter BECKMAN COULTER model: VI-CELL XR

Carbon dioxide static incubator THERMO SCIENTIFIC Model: HERA cell240i

Constant temperature water bath Thermo, Microprocessor controlled 280, 475524

Biological safety cabinet Shanghai Shangjing Model: BSC-Ⅱ A2

The medium and reagents are shown in Table 4:

Table 4

Formula components	supplier	Item No.
CloneMedium CHO DHFR	Genetix	K8712
Clone XL Reagent	Genetix	K8521
GlutaMAX-I	Gibco	35050-061
MTX	Sigma	A6770-1
Blasticidin	Invitrogen	A11139-03
CloneDetect anti-human FITC	Genetix	K8200

Preparation steps of semi-solid medium:

Take a 90ml bottle of CloneMedium CHO DHFR from -40°C 24 hours before plating and place it in a refrigerator at 2-8°C for later use.

Add 2ml GlutaMAX-I, 1ml Clone Detect antihuman FITC, 2ml Clone XL Reagent to the prepared CloneMedium CHO DHFR.

MTX and blasticidin were added to the prepared CloneMedium CHO DHFR to make the concentration of 500nM and 10mg/l in the final volume of 100ml, respectively.

Adjust the volume to 100 ml with the host cell supernatant.

Laying steps:

1. Cell count, take the cultured Pool JN-XBK and count it with Vicell-XR for use.

2. Take out two prepared and mixed semi-solid medium into a sterile 50ml centrifuge tube, each 10ml.

3. Add the counted cells to the centrifuge tube of step 2 according to the density of 300 cells/ml and mix.

4. Use a pipette to transfer the prepared cells to be plated into a 6-well plate with a volume of 2.5ml per well.

5. After the plating is finished, move the plate to a temperature of 36.5°C and a 6% CO ₂ constant temperature incubator for static culture.

Example 2

Equipment and consumables

Constant temperature water bath Julabo Model: TW20

Biological safety cabinet Shanghai Shangjing Model: BSC-Ⅱ A2

Carbon dioxide static incubator THERMO SCIENTIFIC Model: HERA cell240i

Cell counter BECKMAN COULTER model: VI-CELL XR

Centrifuge EPPENDORF model: Centrifuge 5810R

ClonePix GENETIX model: Clonepix FL

96-well plate (greiner bio-one CELLSTAR, catalog number: 655185)

24-well plate (greiner bio-one CELLSTAR, item number: 662102)

6-well plate (greiner bio-one CELLSTAR, item number: 657185)

Shaker KUHNER Model: Climo-Shaker ISF1-XC

Parameter setting: 225rpm, 36.5°C, 6% CO2, humidity 85%.

Shaking tube TPP TUBESPIN BIOREACTOR 50, item number: 87050

The culture medium and reagents are as follows in Table 5:

table 5

Medium name	Brand	Item No.
SFM4 ADCF	Hyclone	SH3A2156.01
Glutamine	Sigma	G8540
MTX	Sigma	A6770-1
Blasticidin	Invitrogen	A11139-03
Cell Boost5(CB5)	Hyclone	SH30865.03
FM012	Sheffield	5X00483
2X103A	Donin	F103-201.A
Glucose	Sigma	49139
BR7	Donin	F008-007

Clone picking

Observe the growth status of the clones, and select the clones after they form a uniform spherical shape.

1. Take a 96-well plate and add 100ul of fresh medium SFM4ADCF to each well (containing 4mM Glutamine, 1g/L PF68, 10mg/l blasticidin and 500nM MTX).

2. Use ClonePix to pick Pool JN-XBK clones and transfer them to a 96-well plate (SFM4 ADCF+10mg/l Blasicidin+500nM MTX).

3. Move the 96-well plate to a temperature of 36.5°C and 6% CO _{2 in a} constant temperature incubator for static culture.

HTRF clone screening and amplification

Medium: SFM4ADCF (contains 4mM Glutamine, 1g/L PF68, 10mg/l blasticidin and 500nM MTX)

1. On the 7th day after clone selection, add 100ul of fresh medium to the 96-well plate.

2. Take the supernatant for HTRF detection, select the clone with higher expression and transfer it to a 24-well plate, and add 400ul of fresh medium to each well.

3. Cultivate for 3-4 days, transfer the clones in the 24-well plate to the 6-well plate, and add 2ml of fresh medium to each well.

4. On the 3-4 days after transfer to the 6-well plate, transfer the clones in the 6-well plate to a shaker tube, and add 8ml of fresh medium to each clone.

5. Take two higher HTRF clones according to the above operation to plate and pick subclones, and screen for amplification.

Batch feeding clone screening

After the cells entered the shake tube and passaged twice, they were inoculated and cultured at 3E5cells/ml for 4 days with the medium SFM4ADCF. After 4 days of growth, it was diluted 1:5 into Fed-batch basal medium 90% SFM4ADCF+10% CB5, and the batch feeding experiment was carried out in a 50ml shake tube. The subclones of Pool JN-XBK are named JK-I, JK-II, JK-III. The Top3 clones selected were JK-I, JK-II, and JK-III.

The batch feeding experiment protocol is shown in Tables 6 and 7.

Supplement: 30g/l FM012 in 2X103A; BR7.

Each feeding will supplement the sugar to a final concentration of 10-12g/l.

Table 6

Table 7

The results of the batch-feeding clone screening experiment are shown in Figures 4 to 5. The results of the clone screening batch feeding experiment using Protein A-HPLC to detect Titer are shown in Figure 6. The output of JK-I is the highest at 1.82g/l. It is selected for further shake flask optimization and quality analysis, and process development for upstream.

Selected clone shake flask optimization experiment and quality analysis

Selected clone shake flask optimization experiment

Clone name: JK-I

The cells were inoculated and cultured at 3E5cells/ml for 4 days with SFM4ADCF medium. After 4 days of growth, it was diluted 1:5 into self-prepared Fed-batch basal medium. The batch feeding experiment was performed in a 50ml shake tube.

The batch feeding experiment scheme is shown in Tables 8 and 9:

Medium: SFM4 ADCF+supplements

Supplement: 30g/L FM012 in 2X103A; BR7.

Each feeding will supplement the sugar to a final concentration of 10-12g/l.

Table 8

Table 9

The experimental results are shown in Figures 7 and 8. The protein A-HPLC Titer test results after the optimization of the selected clone batch feeding experiment: the yield on the 14th day is 2.05g/l.

Selected clone quality analysis results and optimization

The supernatant of fed-batch cells is purified by Protein A in one step and then sent for quality analysis.

Table 10 Purity SEC-HPLC

Through Purity SEC-HPLC analysis and testing, the test results are close to the original drug (Vectibix), and the indicators meet the requirements.

Table 11 pI-cIEF

Through the detection of pI-cIEF, the isoelectric point of the antibody is consistent with the original drug and meets the requirements.

Glycan

Before optimization: Table 12

After optimization: Table 13

The glycosylation test report shows that G0F, G1F, and G2F are basically the same as the original drug and meet the requirements.

Table 14 Cell Based Assay

Sample ID	Test Result
JK-I	88%

Through cell-based antibody activity detection, the antibody activity is close to the original drug and meets the requirements.

Table 15 Deglycosylated mass

Through Deglycosylated mass analysis, the molecular weight of antibody deglycosylated is consistent with the theoretical value.

The present invention screens out the stable cell line JK-I with high expression of Vectibix through POOL construction, batch feeding experiment screening and plating, clone picking, HTRF, clone amplification, clone batch feeding experiment. Through process optimization, the expression level reached 2.05g/L, and the product SEC, pI-cIEF, the molecular weight of desaccharides, and cell-based antibody activity were all significantly improved.

Example 3 Cell line stability test

The genetic stability of cell lines is the basis for subsequent stable production processes and product quality. Use molecular biology methods to study the genetic stability of the cells in the PCB (Primary Cell Bank), MCB (Master Cell Bank), and WCB (Work Cell Bank) cell banks, including the heavy chain (HC) and Light chain (LC) DNA sequence stability and copy number stability. The project intends to conduct cell line stability studies for no less than 12 weeks, including cell growth stability, product expression stability and molecular level cell line stability evaluation.

By molecular biology methods, the PCB (Primary Cell Bank), MCB (Master Cell Bank), WCB (Work Cell Bank) cells in the three cell banks and the pilot-scale production of the final cell EOPC (End of Production Cell) conducts research on genetic stability, including heavy chain (HC) and light chain (LC) DNA sequence stability and copy number stability. The stability of the copy number is the extraction of genomic DNA from PCB, MCB, WCB cell banks and pilot production end-of-production cells (EOPC) (the number of cells is about 1x107 each), and the heavy chain or light chain is compared to the internal reference gene B2M ( β2-microglobulin) compares the different amplification results in qPCR to determine the changes in the copy number of heavy and light chains in the three cell banks of PCB, MCB, and WCB and in the final cells of the pilot production. The stability of the heavy and light chain DNA sequence is investigated by obtaining PCB, MCB, WCB and EOPC cells (the total number of cells is about 1x107 each). After extracting the total RNA, the total RNA is used as a template to reverse transcription to obtain the cDNA, which contains heavy Chain and light chain cDNA. Then use the cDNA as a template to amplify HC and LC using synthetic primers, and finally sequence the PCR products to detect whether the cell bank cells and terminal cells have mutations at the gene level, so as to evaluate the stability of the cell bank Sex.

Tests showed that in the PCB, MCB, WCB, and pilot-produced EOPC of Example 1, there was no significant change in the copy number of the heavy chain and light chain; the target product was amplified at the cDNA level, and the PCR product was sent to the sequencing company for sequencing. Detect whether the cell bank cells and terminal cells have mutations at the genetic level.

laboratory apparatus

7500 Fast Real-Time PCR System (Applied Biosystems, Model#7500)

BioSafety Cabinet (AIRTECH, Model#BSC-1300II A2)

Experimental supplies

Dneasy Blood&Tissue Kit(250) (QIAGEN, Cat.#69506)

TaqMan Universal Master Mix II with (AB, Cat.#4426710)

UNG

RNeasy mini kit (QIAGEN, Cat.#74104)

Superscript III First-Strand Synthesis (Invitrogen, Cat.#No18080-051)

System for RT-PCR kit

PrimeSTAR DNA polymerase (TaKaRa, Cat.#DR044A)

Experimental sample

Example 1 JK-I-PCB, Example 1 JK-I-MCB and Example 1 JK-I-WCB were cultured after thawing and resuscitation, and the 1E7 cell pellet was centrifuged for use. The names, batch numbers and sources of tertiary cell banks and EOPC are shown in Table 16.

Table 16 Name, batch number and source of experimental samples

The stability test of copy number of heavy and light chain in PCB, MCB, WCB and EOPC cells in Example 1

1. Genomic DNA extraction

Genomic DNA was extracted from the PCB, MCB, and WCB cells of the three cell banks of JK-I in Example 1 and the pilot production terminal cells. For specific steps, refer to the Dneasy Blood&Tissue Kit user manual.

2. Design of primers and probes

Design primers and probe combinations that specifically bind to heavy chain, light chain and internal reference genes, see Table 17.

Table 17

3. qPCR reaction setup

Choose 50ng/5μl, 10ng/5μl and 5ng/5μl of three concentrations of PCB, MCB, WCB and EOPC gDNA as the substrate, and configure a 25μl reaction system according to the instructions of TaqMan Universal Master Mix II with UNG. ABI 7500 Fast Real Time System selects the Comparative-Ct (ΔΔCt) method to set up qPCR and run the experiment.

Example 1: Detection of the stability of heavy and light chain gene sequences in PCB, MCB, WCB and EOPC cells

1. Extraction of total RNA from cell bank cells

The total RNA of each cell was extracted from the PCB, MCB, and WCB cell bank cells of JK-I in Example 1 and the pilot production terminal cells. For specific steps, refer to RNeasy minikit.

2. Synthesis of first strand cDNA of antibody gene

Use OligodT to amplify cDNA fragments by reverse transcription, and the experimental steps refer to the first Script III First-Strand Synthesis System for RT-PCR kit operation flow.

3. Amplification of HC and LC sequences

The cDNA obtained by reverse transcription was used as a template to amplify the HC and LC fragments of the antibody respectively. The primers used are shown in Table 18. The amplified PCR products were delivered to the sequencing company for sequencing.

Table 18 Amplification primers and sequences of HC and LC fragments in cDNA

Different amplification reactions of heavy and light chains in PCB, MCB, WCB and EOPC

Example 1 The calculation of the difference between the Ct value of the heavy chain HC and light chain LC in each cell bank of JK-I and EOPC relative to the internal reference gene B2M is summarized in the following table. The average value and standard deviation of ΔCt calculated under different concentration samples are also listed in Table 19 below.

Table 19 Calculation of ΔCt (HC-Ref) and ΔCt (LC-Ref)

Changes in gene copy number of heavy chain and light chain in PCB, MCB, WCB and EOPC

Standardize the relative copy number of heavy chain HC and light chain LC in PCB relative to the internal reference gene B2M to 1, in Example 1 JK-I, the change in the gene copy number of heavy chain HC and light chain LC relative to PCB in MCB, WCB and EOPC It is summarized in Table 20.

Table 20 Example 1 JK-I Changes in the copy number of heavy chain and light chain genes in PCB, MCB, WCB and EOPC

HC	PCB	MCB	WCB	EOPC
-ΔΔCt	0.00	-0.23	-0.29	0.02
2 -ΔΔCt	1.00	0.85	0.82	1.01
LC	PCB	MCB	WCB	EOPC
-ΔΔCt	0.00	-0.09	-0.09	0.18
2 -ΔΔCt	1.00	0.94	0.94	1.14

Analyzed by the Comparative-Ct (ΔΔCt) method, the copy number of the heavy chain HC gene in MCB is reduced by 15% compared to PCB, the copy number of heavy chain HC gene in WCB is reduced by 18% compared to PCB, and the copy number of HC in EOPC Compared with PCB, the gene copy number of light chain LC in MCB is reduced by 6% compared to PCB, and the copy number of heavy chain LC gene in WCB is reduced by 6% compared to PCB. The copy number of LC in EOPC The number has increased by 14% compared to PCB. Example 1 The copy number changes of the heavy chain and light chain in MCB, WCB and EOPC were all within the allowable error range of the experiment.

Example 1: Results of stability test of heavy and light chain gene sequences in PCB, MCB, WCB and EOPC cells

Using the cDNA obtained by reverse transcription as a template, the HC (about 1395 bp) and LC fragments (711 bp) of the antibody were respectively amplified, and the amplified PCR products were subjected to agarose gel electrophoresis. The electrophoresis results are shown in Figure 10 and Figure 11.

Using Vector NTI software, the obtained sequencing results of HC and LC in PCB, MCB, WCB and EOPC cells were compared with the theoretical sequence. The comparison of all sequencing results with the theoretical sequence was 100% consistent.

The comparative-Ct (ΔΔCt) method in quantitative PCR analysis showed that the copy numbers of HC and LC in PCB, MCB, WCB and EOPC in Example 1 were relatively stable. Sequencing analysis showed that the HC and LC cDNAs in PCB, MCB, WCB and EOPC in Example 1 remained relatively stable at the gene level, and there was no gene mutation, base deletion or increase in the HC and LC sequences. . In summary, the genetic stability of PCB, MCB, WCB and EOPC in Example 1 is good.

Example 4 Optimization of cell culture pilot test process

After obtaining a monoclonal cell line, the culture conditions are optimized to achieve higher expression levels and meet the specified product quality. The depletion of nutrients and the accumulation of a large number of metabolic by-products in the cell culture process are often the main factors that limit the cell growth density and affect the culture process. Especially in the batch culture process, the continuous consumption of nutrients often becomes an influence on cell density and cell density. The limiting factors of viability and cell expression product yield.

In this embodiment, on the basis of the small-scale cultivation process, the scale is expanded, and different parameter control ranges are optimized to design product quality. Study the kinetics of cell growth and monoclonal antibody production, set physical parameters (including temperature, gas flow, and stirring speed), chemical parameters (including dissolved oxygen and carbon dioxide, pH, osmotic pressure, and redox potential), and metabolite levels ( Including the influence of the basic material of the culture medium), amino acids and waste (by-products), as well as the feeding time and foam control on cell growth and protein production, determine the best environmental conditions for pilot cell culture.

In the early stage, 2 rounds of reactor experiment and 3 rounds of shake flask experiment were used to determine the medium, and it was verified that 35℃ is the best culture temperature in the whole process; the best feed is 2×104 (including 50g/L FM012), and feed time It is the 4th, 7th, 9th, and 11th days; at the same time, through the FG01 feeding experiment, it is determined that 0.28%, 0.4%, 0.4%, and 0.4% are fed on the 4th, 7th, 9th and 11th days respectively. Finally, the cell culture pilot process was determined. The scale-up experiment of this embodiment intends to adopt the medium flow-add culture method to carry out the culture in a 200L reactor scale. Observe cell viability, cell metabolism, protein production, etc. to optimize the pilot process, investigate the culture volume and inoculation density, reactor parameter control, culture conditions, and feed strategy (day 3, day 6, day 9 and day 11) The effects of feed concentration and ratio on cell growth and protein yield were tested and compared with the original research products to determine the key process parameters and the pilot reactor process.

The cell line used in the cell culture technology research in this report is Example 1 JK-I, which is the cell line determined by the cell line development group through the clone screening experiment, hereinafter referred to as "JK-I clone".

The following mainly introduces three batches of 15L reactor experiments to verify the optimized process of the small test. The experimental data showed that the cell growth and metabolic parameters in the three batches were basically parallel. The protein yields were 2.35g/L, 2.39g/L, and 2.45g/L at harvest; the acid peaks in the CEX test results were all lower than 13.3%, and the glycoform In the distribution result, G0F is 47-48%. 3 batches of 15L experiment repeat results are better, thus verifying the stability of the small-scale test to determine the process, according to this process for 3 batches of 200L production.

Table 21 Explanation of professional terms

Table 22 Equipment

Table 23 Consumables

Table 24 Medium composition

Medium	Brand	Item No.
2x104	Duoning Biotechnology	F104-001
CB5	Hyclone	SH30865.03
SFM4 ADCF	Hyclone	SH3A2156.01
FM012	Sheffield	5X00483
L-Glutamine	Sigma	G8540
Sodium bicarbonate	Sigma	S5761
Sodium carbonate	Sigma	S7795
glucose	Sigma	G7021
PF68	Spectrum	P1169
MTX	Sigma	M9929
BS(Blasticidin S HCl)	Gibco	A11139-03

The general parameter settings of the shaker involved in this experiment are shown in the following table. If it is not a general setting, see the description in the corresponding section for details.

Table 25 Parameter setting table of general shaker

Shaker parameters	Set up	unit
CO 2	6	%
temperature	36.5	°C
humidity	80	%
Shaking speed	125	RPM

The sampling plan used in the fed-batch cultivation stage is shown in the following table. If it is not a general sampling plan, see the description in the corresponding section for details.

Table 26 Sampling and retention sample plan

15L reactor process verification experiment

Preliminary experiment results

Through 2 rounds of reactor experiment and 3 rounds of shake flask experiment, it was verified that 35℃ is the best culture temperature in the whole process; the best feed is 2×104 (including 50g/L FM012), and the feed time is 4th, 7th and 9th , 11 days; at the same time, through the FG01 feeding experiment, it is determined that 0.28%, 0.4%, 0.4%, 0.4% will be fed on the 4th, 7th, 9th and 11th days respectively. Finally determine the cell culture process. In this round of experiments, three batches of process stability were verified on a 15L reactor scale.

Seed cultivation information

Cell source

Table 27 Clone information table

Passage and expansion information:

Table 28 Seed passage and amplification medium information table

Table 29 N-2, N-1 generation seed amplification information table

Shaker parameter setting is shown in Table 25 General Shaker Parameter Setting Table.

Sampling plan

N-2 generation shake flasks are sampled every 3-4 days to detect viable cell density, cell viability and cell diameter. N-1 generation reactors are used to detect viable cell density, cell viability, cell diameter and biochemical parameters (pH, pO ₂ , PCO ₂ , Gln, Glu, Gluc, Lac, NH4 ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Osmo).

Fed-batch culture

Culture volume and seeding density

Table 30 Basic information table of flow plus training

Culture process settings

Table 31 Reactor parameter control in the fed culture stage

Feed flow adding table

Table 32 Feed flow addition table during the cultivation phase

Sugar supplement strategy

Check the glucose content every day. When it is lower than 3g/L, add 400g/L glucose mother liquor to 3-5g/L.

Harvest standard

Harvested at 15th day or when the cell viability rate is lower than 50%.

The sampling and retention sample plan is shown in Table 26, the general sampling and retention sample plan.

Experimental results

Cell growth

From the viable cell density data, the trends of the three batches of 15L over time are basically the same, and the highest viable cell density was obtained on the 11th day, respectively 27.06×10 ⁶ cells/ml, 29.47×10 ⁶ cells/ml, 28.02× 10 ⁶ cells/ml, the density of the 3 batches of viable cells was basically the same at harvest on the 15th day, about 17×10 ⁶ cells/ml. The density curve of viable cells is shown in Figure 12.

From the cell viability data, the trend of cell viability in the three batches of experiments was basically the same, and the cell viability at harvest was 66.8%, 59.8%, and 63.8%, respectively. The cell viability curve is shown in Figure 13.

Cell metabolism

From the data of glucose concentration, the change trend of 3 batches of 15L is basically the same. When the glucose content is lower than 3.0g/L after the 11th day, the final glucose concentration after supplementation is between 3.0-5.0g/L. The glucose concentration curve is shown in Figure 14.

From the lactate concentration data, the change trend of the three batches of 15L was basically the same, and the three batches were 2.22g/L, 2.45g/L, and 2.12g/L at harvest. The lactate concentration curve is shown in Figure 15.

Protein yield results

Judging from the protein production data, the three batches of protein production are basically parallel, respectively 2.35g/L, 2.39g/L, and 2.45g/L. The protein yield curve is shown in Figure 16.

Protein quality results

The protein quality results are all quoted from the test data after the three-step purification of the downstream protein purification department. From the CEX test data, the CEX acid peak area of the three batches of 15L harvest on the 15th day was all lower than 13.3%. CEX test results are shown in Table 33.

Table 33 Protein CEX test results table

According to the sugar distribution data, the G0F of the three batches of 15L at the 15th day harvest was 47.4%, 48.2%, and 47.4%, respectively. The test results of glycoform distribution are shown in Table 34.

Table 34 Test results of protein glycoform distribution

The experimental data show that using the final culture process determined by the small test, the cell growth and metabolic parameters of each batch of 3 batches of 15L production are basically the same, and the final protein output range is 2.35-2.45g/L, and the CEX acid peak is lower than 13.3%, the G0F in the sugar type distribution result is basically 47-48%, which verifies the stability of the process. This process can carry out 200L production.

After 3 batches of 15L process experiments, the stability of the process was verified. The determined reactor process is as follows, and 3 batches of production will be carried out in a 200L reactor scale. The final reactor process is as follows:

Table 35 Reactor parameter setting table

Table 36 Reactor feed flow plus experiment scheme

Embodiment 5 Purification process

The temperature, pH, and organic solvents in the separation and purification steps will affect the activity of the target protein. The sequence of the steps and process control will affect the purity and activity of the target protein. Therefore, in the separation and purification process of biosimilar drugs, the separation and purification conditions used should be as reasonable and mild as possible, and the operation steps should be minimized. In addition, biological analogues are all derived from biologically active substances, so the potential hazard factors they carry, especially viral factors, must be strictly controlled.

The present invention explores the purification process through many experiments, and intends to adopt four steps for purification. The cell culture solution after the tank is clarified by two-stage deep filtration, sterilized and filtered, and then loaded onto the Protein A affinity chromatography column; the elution peak of affinity chromatography is incubated with low pH and the virus is inactivated, and after neutralization After the pH is adjusted, the sample is loaded on the anion exchange chromatography column, and the anion chromatography flow-through peak is collected; the anion chromatography flow-through peak is adjusted by pH and loaded on the cation exchange chromatography column, and the cation chromatography wash is collected Peak off, concentration and liquid exchange. The sample after the liquid change is subjected to final sterilization and filtration to obtain the product stock solution.

On the basis of the pilot process, 3 batches of 200L pilot scale-up were carried out, key process parameters were screened and optimized, the pilot-scale purification process was determined, and 3 batches of products were tested and compared with the original research products. It is expected that the average purification yield shall not be less than 65%, the purity of the original solution shall not be less than 95%, the protein A, DNA, HCP residues and other standards shall be met, and the activity shall be qualified.

Table 37 Abbreviation list

abbreviation	definition
ChP	Chinese Pharmacopoeia
USP	United States Pharmacopoeia
GE	General Electric Company
EQ	Equilibration
CV	Column Volume
WFI	Water for Injection
DBC	Dynamic Binding Capacity
UF/DF	Ultrafiltration/Diafiltration
NWP	Normalized Water Permeability
TMP	Transmembrane Pressure
LVIN	Low pH Virus Inactivation&Neutralization
SEC	Size Exclusion Chromatography
cIEF	Capillary Isoelectric Focusing
HMW	High Molecular Weight
LMW	Low Molecular Weight
HCP	Host Cell Protein
AEX	Anion Exchange Chromatography
CEX	Cation Exchange Chromatography
CE	Capillary Electrophoresis
LMH	Liters per Square Meter per Hour

The chemical reagents used in the downstream process are shown in Table 5. Unless otherwise specified, all reagents meet the Chinese Pharmacopoeia or the US Pharmacopoeia or equivalent levels.

Table 38: Reagents used in downstream processes

1. Clarification of cell fluid

The cell culture solution in the lower tank of Example 1 or 2 was clarified by a two-stage deep filtration method, and then subjected to sterilization filtration and then loaded onto the affinity chromatography column.

Materials and equipment:

Pressure sensor: PENDO TECH Pressure MAT

Peristaltic pump: LongerPump, BT300-2J

Sample: Three batches of 10L cell culture fluid

Equilibrium buffer: 50mM Tris-HAc, 150mM NaCl, pH 7.4

Depth filter: Millipore Pod D0HC, 2×540cm2 (first level); Millipore Pod A1HC, 540cm2 (second level)

Sterilization filter: Sartopore 2 300, Sartorius

The steps are as follows:

Wash the depth filter A1HC and D0HC with WFI at 100L/m2, 600LMH.

Connect the two DOHC in parallel and then in series with the A1HC, balance the depth filter with a balance buffer (at least 2 times the dead volume of the DOHC system), until the pH of the A1HC effluent is determined to be consistent with the balance buffer, 600LMH.

Start the filtration of the sample, the feed rate is gradually increased, and after 0.5-0.7 times the dead volume of the entire system at the A1HC outflow end is discarded, the outflow end is connected in series to the sterilization filter and the filtrate is collected.

Continue to filter and keep the feed rate at ≤200LMH, monitor and maintain the pressure of the depth filter at all levels at ≤15psi.

After the sample is filtered, the material liquid remaining in the depth filter membrane is replaced with a buffer solution of at least 2 times the dead volume of the entire system.

Drain the depth filter and combine all filtrates.

The protein A-HPLC method was used to determine the antibody content of the samples before and after the deep filtration, and the yield was calculated.

The results of three batches of 10L cell culture solution deep filtration clarification experiment are shown in Table 39:

Table 39

*The calculation of solid content is based on the percentage of solid content after two-stage centrifugation (1000g, 10min+8000g, 10min).

2. Affinity chromatography

After clarification, sterilization and filtration, the supernatant was purified by GE's Protein A affinity chromatography filler MabSelect, and the elution peaks were collected and sampled for detection.

Materials and equipment

Packing and chromatography column: MabSelect, 5.0×25cm, CV 491mL

equipment

avant, 569951, GE

pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

Sample: Three batches of clarified cell culture supernatant after sterilization and filtration

Buffer (see Table 40)

Methods and results

The affinity chromatography method is shown in Table 40 for details. The operation steps of this experiment are as follows:

Use 1M NaOH to

The system and pipeline are disinfected and depyrogenated (maintained for more than 30 minutes).

Rinse with WFI

The system and pipelines are neutral, and each buffer pipeline is filled with corresponding buffer.

Filter the sample and return to room temperature to prepare for loading.

Connect the column to the system and prepare to run the method.

Run the chromatographic method, collect the elution peaks, sample and send for inspection, use Nanodrop to determine the protein concentration and calculate the yield.

After the chromatography method runs, remove the chromatography column. Flush the system pipeline with water and store it in 20% ethanol.

Table 40: Affinity chromatography method

Table 41: Summary of affinity chromatography

3. Low pH incubation for virus inactivation and neutralization

Adjust the pH of the affinity chromatography elution peak to 3.5-3.8 and incubate for 2-4 hours at room temperature for virus inactivation, and then neutralize to pH 5.5-5.8, and then the neutralized product is sterilized and filtered.

Materials and equipment

equipment

pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

Sample: affinity chromatography elution collection peak

Titration solution: 1M Citric Acid (adjusting acid); 1M Tris Base (adjusting alkali)

Sterilization filter: Bottle Top Filter, Express Plus, 0.22 microns, Millipore

Methods and results

First take a sample of 3mL from the elution collection solution to determine the pH, and add 1M Citric Acid to adjust the pH to 3.5-3.8, and calculate the added percentage based on the amount of 1M Citric Acid added. According to this addition percentage, add 1M Citric Acid to the elution collection solution in proportion to adjust, and stir while adding. After mixing, take another 3 mL sample for pH measurement to confirm. Place the sample at room temperature and start timing. After 2-4h, add 1M Tris Base in proportion to the percentage of Tris added when the pH of the 3mL sample is adjusted to 5.5-5.8. After mixing, take another 3 mL sample for pH measurement to confirm. The neutralized sample is sterilized and filtered. Three batches of 10L low pH incubation for virus inactivation and neutralization are shown in Table 42.

Table 42: Summary of virus inactivation and neutralization in low pH incubation

Four, anion exchange chromatography

The low-pH virus inactivated and neutralized sample was adjusted to pH 6.9 and used as a sample for anion exchange chromatography. Collect anion chromatographic flow-through peaks and sample for detection.

Materials and equipment

Packing and chromatography column: Super Q 650M (TOSOH), 2.6×22.8cm, CV 121mL

equipment

avant, 569951, GE

pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

Sample: Adjust the pH of the sample after virus inactivation and neutralization to 6.9 before loading

Sterilization filter: Bottle Top Filter, Express Plus, 0.22 microns, Millipore

Buffer (see Table 43)

Methods and results

The anion exchange chromatography method is detailed in Table 43. The operation steps of this experiment are as follows: (Experiment results are shown in Table 44)

Use 1M NaOH to

The system and pipeline are disinfected and depyrogenated (maintained for more than 30 minutes).

Rinse with WFI

The system and pipelines are neutral, and each buffer pipeline is filled with corresponding buffer.

Filter the sample and return to room temperature to prepare for loading.

Connect the column to the system and prepare to run the method.

Run the chromatographic method, collect the protein peaks that flow through and sample for detection. Use Nanodrop to determine the protein concentration and calculate the yield.

After the chromatography method runs, remove the chromatography column. Flush the system pipeline with water and store it in 20% ethanol.

Table 43: Anion exchange chromatography method

Table 44: Summary of Anion Exchange Chromatography

5. Cation exchange chromatography

The anion chromatography flow-through collection peak was adjusted to pH 5.0 as the loading of cation exchange chromatography. Collect cation chromatography elution peak and sample for detection.

Materials and equipment

Packing and chromatography column: Poros XS, 2.6×25.4cm, CV 135mL (the first batch of 10L); Poros XS, 5.0×21.8cm, CV 428mL (the third batch of 10L and the fourth batch of 10L)

equipment

avant, 569951, GE

pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

Sample: Adjust the pH of the flow-through peak of anion chromatography to 5.0 (when the third batch of 10L was loaded, the sample flowed through because the pH was not adjusted, and the remaining samples and the flow-through samples were adjusted to pH 5.0 again. Sample)

Sterilization filter: Bottle Top Filter, Express Plus, 0.22 microns, Millipore

Buffer (see Table 45)

8.2 Methods and results

The cation exchange chromatography method is detailed in Table 45. The operation steps of this experiment are as follows: (Experiment results are shown in Table 46)

Use 1M NaOH to

The system and pipeline are disinfected and depyrogenated (maintained for more than 30 minutes).

Rinse with WFI

The system and pipelines are neutral, and each buffer pipeline is filled with corresponding buffer.

Filter the sample and return to room temperature to prepare for loading.

Connect the column to the system and prepare to run the method.

Run the chromatographic method, sample the collected elution peaks, and use Nanodrop to determine the protein concentration and calculate the yield.

After the chromatography method runs, remove the chromatography column. Flush the system pipeline with water and store it in 20% ethanol.

Table 45: Cation exchange chromatography method

Table 46: Summary of Cation Exchange Chromatography

Table 47: Glycan-HPLC results and CEX-HPLC results of cation exchange chromatography products

*The sample is the filtrate after the cation elution peak is diluted and then subjected to nanofiltration.

Six, concentrated dialysis (UF/DF)

The elution peak of cation exchange chromatography was concentrated and buffer exchanged. After concentrating and changing the solution, adjust the protein concentration of the original solution to 19-21 mg/mL, and perform the final sterilization filtration.

Materials and equipment

equipment

Peristaltic pump: LongerPump, BT300-2J/YZ1515x, BT300-2J/YZ2515x

Pressure monitoring: Pendo TECH, Pressure MAT

pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

Sample: cation exchange chromatography elution collection peak

Buffer

UF/DF balance solution and dialysis buffer: 6.4g sodium acetate trihydrate/kg solution, 5.8g sodium chloride/kg solution, 0.182g glacial acetic acid/kg solution, pH 5.8, osmotic pressure 276mOsm/kg.

Membrane disinfectant: 1M NaOH

Membrane preservation solution: 0.1M NaOH

Sterilization filter: Bottle Top Filter, 0.22 micron, PES, Corning

Ultrafiltration membranes and fixtures: Pellicon XL Biomax 30, 50cm2, CAT No. PXB030A50, Lot No. C2MA64012-030 (the first batch of 10L concentration and liquid exchange); Pellicon 2mini 30k, 0.1m2, CAT No. P2B030A01, Lot No. C2PA14764-001, fixture Millipore ZJMP-14-001 (the third batch of 10L and the fourth batch of 10L concentration and liquid exchange)

Methods and results

See Table 48 for details of the concentration and exchange procedure. The UFDF operation steps of this experiment are as follows: (Experimental results are shown in Table 49 and Table 50)

Rinse the membrane with WFI (for new membranes more than 80L/m2, for used membranes more than 20L/m2).

Use 1M NaOH for disinfection before use (cycle 1h).

Rinse the membrane with WFI to neutrality and perform NWP measurement. Under the conditions of a feed flux of 360LMH, a feed end pressure of 1.0 bar, and a return end pressure of 0.5 bar, the water flow rate at the permeate end was measured to calculate the NWP.

Drain the water in the pipe and membrane to determine the dead volume of the system.

Equilibrate the membrane with an equilibration buffer of at least 2 times the dead volume of the system. Measure the pH and conductivity of the effluent at the return end and the permeate end, which need to be consistent with the equilibration buffer.

Perform sample concentration and liquid exchange. During this process, the feed flux is no more than 360LMH, and the pressure at the reflux end is adjusted to keep the feed end pressure no more than 20psi and transmembrane pressure no more than 15psi.

After the fluid is changed at least 6 times, appropriate over-concentration is necessary if necessary, then close the valve of the permeate port, and circulate at least 2 times the dead volume of the dialysis buffer for more than 5 minutes. The membrane is emptied and all products are recycled to the product storage tank.

Rinse the membrane with WFI.

Use 1M NaOH for post-use disinfection (cycle 1h).

Rinse the membrane with water, and save the membrane with 0.1M NaOH.

After the concentrated solution is changed, the product is adjusted to 19-21mg/mL with an appropriate amount of dialysis buffer, and then filtered with a sterile filter to calculate the UF/DF yield.

Table 48: UF/DF steps

Table 49: Stock solution test result 1 (UF/DF summary)

Table 50: Stock solution test results 2 (UF/DF summary)

It can be understood that the above embodiments are merely exemplary embodiments adopted for explaining the principle of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various variations and improvements can be made without departing from the spirit and essence of the present invention, and these variations and improvements are also regarded as the protection scope of the present invention.

Claims (6)

1. A cell line construction method for preparing a fully humanized monoclonal antibody against EGFR, which is characterized in that the cells of the Chinese hamster ovary cell dihydrofolate reductase deficient strain CHO/DHFR- complete the POOL screening after the transfection step In the first stage, SFM4CHO medium without HT is used to screen cell lines that integrate heavy chain genes, and blasticidin in the medium is used to screen out cell lines that integrate light chain genes; in the second stage, MTX is added to increase The copy number of light and heavy chain genes integrated into the genome of the cell line to increase expression.
2. The method for constructing a cell line for preparing an anti-EGFR fully humanized monoclonal antibody according to claim 1, wherein in the POOL screening step, in the first stage, the concentration of GlutaMAX-I in the culture medium is 4mM, and the concentration of Blasticidin In the second stage, the GlutaMAX-I concentration in the culture medium is 4mM, the blasticidin concentration is 10mg/l, and the MTX concentration is 500nM, twice a week until the cell viability is restored to more than 85%. After the second passage, the cell viability was restored to more than 90%.
3. The cell line construction method for preparing anti-EGFR fully humanized monoclonal antibodies according to claim 2, characterized in that, after the POOL screening step is completed, clonal expansion is performed, 2X103 feed culture is used, and 2X103 feed is FM012 The concentration is 50g/l, and the sugar is supplemented to a final concentration of 10-12g/l each time.
4. The method for constructing a cell line for preparing an anti-EGFR fully humanized monoclonal antibody according to claim 3, characterized in that the plated culture is carried out after the feed culture, and the cells are calculated at a density of 300 cells/ml. The amount is added to a centrifuge tube containing a semi-solid medium and mixed, and then plated, with a volume of 2.5 ml per well, and transferred to a temperature of 36.5°C, 6% CO _{2 in a} constant temperature incubator for static cultivation.
5. The cell line construction method for preparing anti-EGFR fully humanized monoclonal antibodies according to claim 4, wherein the preparation method of the semi-solid medium used for plating culture is: 90ml Clone Medium CHO DHFR before plating 24 Take it out from -40°C and put it in the refrigerator at 2-8°C in hours, add 2ml GlutaMAX-I, 1ml Clone Detect antihuman FITC, 2ml Clone XL Reagent to the prepared Clone Medium CHO DHFR, and then add MTX and Blasticidin to the final volume The concentration at 100ml was 500nM and 10mg/l, and the volume was adjusted to 100ml with the host cell supernatant.
6. The method for constructing a cell line for preparing an anti-EGFR fully humanized monoclonal antibody according to claim 5, wherein the cell culture conditions during the construction process: temperature 36.5°C, humidity 75% to 85%, carbon dioxide concentration 6%, rotation radius 2.5cm, rotation speed 110 to 225rpm, volume 20 to 30ml.

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