Industrially produced high-purity recombinant human albumin as well as preparation method and application thereof

Abstract

The invention provides industrially produced high-purity recombinant human albumin as well as a preparation method and application thereof. Only three-step chromatography is adopted, and high-yield and high-purity recombinant human albumin in gene editing pig plasma can be prepared. Chromatographic packing suitable for protein purification is screened through different purification small-scale experiments, and meanwhile, three types of chromatographic packing are finally confirmed by considering that the requirement for later-stage industrial amplification is met. By regulating and controlling conditions such as sample loading amount, sample loading concentration, pH of samples in different chromatography steps, conductivity value and the like, the recovery rate of albumin is reserved to the greatest extent, the purity of albumin is guaranteed by using three-time chromatography, and the method has the characteristics of high recovery rate, high purity and simplicity in operation, and can be suitable for large-scale preparation of high-purity plasma albumin.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical separation, and specifically relates to industrially produced high-purity recombinant human albumin, a preparation method and application thereof. Background Art

[0002] Human serum albumin (HSA) is a single-chain non-glycosylated soluble polypeptide composed of 585 amino acids, with a molecular weight of 66.6 kDa and an isoelectric point between 4.7 and 4.9. The entire single-chain protein molecule has 17 pairs of disulfide bonds, 1 cysteine ​​site and 1 tryptophan site. As the most abundant protein in plasma, HSA plays an important role in maintaining plasma blood pressure and anticoagulation. It can protect and stabilize immunoglobulins in the blood, has important medical value, and is in great demand clinically.

[0003] At present, HSA used in clinical practice is mainly extracted and separated from human blood. However, this method has the disadvantage of insufficient supply of raw materials, and the blood itself may carry the risk of hepatitis virus or HIV virus, which makes people have great concerns about the use of HSA extracted from plasma. Therefore, the production of human serum albumin using DNA recombinant technology has been developed and utilized.

[0004] Chuangguan researchers used CRISPR / Cas9 technology to edit the genome of pig fertilized eggs and inserted the human serum albumin gene into the pig albumin gene region, so that only recombinant human serum albumin (rHSA) was produced in pig blood. A large amount of high-purity rHSA was obtained by purifying pig plasma (Reference: Li L, Meng H, Zou Q, Zhang J, Cai L, Yang B, Weng J, Lai L, Yang H, Gao Y. Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use. J Gastroenterol Hepatol. 2019 Oct; 34(10): 1851-1859. doi: 10.1111 / jgh.14666. Epub 2019 Apr 10. PMID: 30884543.). Since the rHSA separated and purified from transgenic pig blood must meet the requirements of clinical medication and biochemical research, separation and purification of rHSA is a key step in preparing rHSA from transgenic pig blood.

[0005] Biochemical separation technology is also known as the downstream engineering of biotechnology. It is mainly divided into the following five types according to the different separation mechanisms: (1) precipitation separation (2) chromatography separation (3) electrophoresis separation (4) centrifugation separation (5) membrane separation technology. Among them, precipitation and chromatography technology are the two main separation technologies.

[0006] The patent application with publication number CN110317260A discloses a purification process for preparing porcine albumin, but this process has strict temperature requirements and is complicated. The patent application with publication number CN102190722A discloses a method for purifying recombinant human albumin, including one-step chromatography of the fermentation broth with an expanded bed anion column to achieve three chromatographic purification effects of solid-liquid separation, concentration and preliminary purification. The sample is then subjected to heating, hydrophobic chromatography, cation exchange, ultrafiltration, chelation, precipitation and other steps to obtain ultra-high purity recombinant human albumin. This method has many steps, involving operations such as heating and precipitation, large sample loss, and complicated operation. The patent application with publication number CN112210002A discloses a method for purifying recombinant human serum albumin, which comprises the following steps: removing impurities from the fermentation broth containing recombinant human serum albumin, first concentration and liquid exchange, first cation exchange chromatography, hydrophobic chromatography, second concentration and liquid exchange, anion exchange chromatography, affinity chromatography, third concentration and liquid exchange, and second cation exchange chromatography. After five steps of chromatography and purification, the purity of recombinant human serum albumin can reach 99.97%. However, the purification method used in this study is relatively complicated and has many steps. The patent application with publication number CN102532254A discloses a method for separating and purifying recombinant human serum albumin from transgenic rice, but the production of rice in this method is affected by seasonality, and a complex process is required from obtaining rice seeds to processing and loading samples, with a long production cycle and many purification steps. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing recombinant human albumin from transgenic pig blood. This patent only uses three steps of chromatography to prepare high-yield and high-purity recombinant human albumin in gene-edited pig plasma. After different purification pilot experiments, the chromatography fillers suitable for the purification of the protein were screened. At the same time, the need to meet the later industrial amplification was considered, and the three chromatography fillers in the application were finally confirmed. By adjusting the conditions such as the sample amount, sample concentration, pH and conductivity of samples in different chromatography steps, the recovery rate of albumin is retained to the greatest extent. The purity of albumin is guaranteed by the three-step chromatography method. It has the characteristics of high recovery rate, high purity and simple operation, and can be used for large-scale preparation of high-purity plasma albumin.

[0008] The method for preparing recombinant human albumin from transgenic pig blood provided by the present invention comprises the following steps:

[0009] (1) purifying the plasma supernatant through an anion chromatography filler to obtain a first chromatography product and performing ultrafiltration on the first chromatography product; the purification through anion chromatography filler sequentially includes CIP treatment of the chromatography column before loading, balancing, loading, rebalancing, eluting and collecting the target product, and column regeneration;

[0010] (2) purifying the first chromatography product after ultrafiltration and liquid exchange through anionic hydrophobic composite chromatography filler to obtain a second chromatography product and ultrafiltration and liquid exchange of the second chromatography product; the anionic hydrophobic composite chromatography filler purification sequentially includes CIP treatment of the chromatography column before sample loading, equilibration, sample loading, elution and collection of target substances, and column regeneration;

[0011] (3) Purifying the second chromatography product after ultrafiltration and liquid exchange through a cationic hydrophobic composite chromatography filler to obtain a third chromatography product, and ultrafiltration and liquid exchange, concentration, freeze-drying and packaging of the third chromatography product to obtain transgenic pig blood recombinant human albumin; the purification through cationic hydrophobic composite chromatography filler includes, in sequence, disinfecting the chromatography column before loading, CIP treatment, balancing, loading, eluting and collecting the target product.

[0012] In step (1) of the above method, the plasma supernatant is prepared by the following method: naturally thawing the transgenic humanized albumin plasma, centrifuging the thawed plasma and obtaining the supernatant;

[0013] Among them, the transgenic edited humanized albumin plasma is prepared by the method described in the reference (Li L, Meng H, Zou Q, Zhang J, Cai L, Yang B, Weng J, Lai L, Yang H, Gao Y. Establishment of gene-edited pigsexpressing human blood-coagulation factor VII and albumin for bioartificial liver use. J Gastroenterol Hepatol. 2019 Oct; 34(10): 1851-1859. doi: 10.1111 / jgh.14666. Epub 2019 Apr 10. PMID: 30884543.).

[0014] The natural thawing is to take the transgenic edited humanized albumin plasma out of the -80°C refrigerator and place it in a 4°C refrigerator overnight for natural thawing;

[0015] The centrifugal conditions are: 8000g±500g, 40min±5min, 4°C;

[0016] The supernatant is first diluted, pH adjusted, and membrane filtered or clarified before the anion chromatography filler purification operation in step (1).

[0017] Wherein, the dilution is diluted with buffer A to a conductivity of 5.0±0.2 mS / cm, and the buffer A can be specifically 50 mM Tris-HCl, pH 8.0±0.1;

[0018] The pH adjustment is to adjust the pH of the supernatant after dilution to 8.0±0.1;

[0019] The membrane filtration is carried out by using a 0.45 μm filter membrane, and the clarification is carried out by using a hollow fiber / capsule filter.

[0020] In step (1) of the above method, the anion chromatography filler may be an anion chromatography filler Maxtar Q;

[0021] Before loading, the column was rinsed with NaOH solution, then pre-balanced with buffer B, and then balanced with buffer A.

[0022] Specifically, firstly wash with NaOH solution for 2-3 column volumes (specifically 3 column volumes), then use buffer B for pre-equilibration for 1-2 column volumes (specifically 1 column volume), and then use buffer A for equilibration for 5-6 column volumes (specifically 5 column volumes);

[0023] The concentration of the NaOH solution may be 0.8-1.2 M, specifically 1 M;

[0024] The buffer B may specifically be 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0±0.1;

[0025] The loading speed may be 28-32 mL / min, specifically 30 mL / min;

[0026] The loading volume can be 7-7.5 mg / mL filler (the volume is calculated based on the albumin content in the plasma, that is, the mass of albumin contained in the diluted plasma sample added in step (1) is 7-7.5 mg / mL filler);

[0027] Collect flow-through samples during loading;

[0028] After the loading is completed, the buffer A is used for re-equilibrating. After the UV280 baseline is balanced, the gradient elution is started, and five sections of washed impurity samples are collected respectively. When the collection of the fifth impurity peak is completed, the yellow on the chromatographic column gathers downward, and the UV280 increases, the target peak is collected;

[0029] Wherein, the buffer A is used to rebalance for 1-2 column volumes (specifically 1 column volume);

[0030] The elution gradient of the gradient elution is: 0-60% buffer B, 6-8 column volumes (specifically 7 column volumes);

[0031] After the target is collected, it is regenerated using the buffer B, and after the regeneration is completed, it is rinsed with a NaOH solution;

[0032] The concentration of the NaOH solution may be 0.8-1.2 M, specifically 1 M, and the flow rate of the NaOH solution is 20 mL / min;

[0033] The column was washed with NaOH solution for 2-3 column volumes (specifically 3 column volumes).

[0034] The above method step (1) further comprises ultrafiltration of the obtained first chromatography product;

[0035] Specifically, at least 5 equal volumes of the second step chromatography buffer C were added for ultrafiltration, and the pH of the sample obtained after ultrafiltration was 6.0±0.1, Cond 18.5±0.5mS / cm, and the concentration of the diluted sample was 5.0±0.5mg / mL;

[0036] The second step chromatography buffer C may specifically be 50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0±0.1.

[0037] In step (2) of the above method, the anionic hydrophobic composite chromatography filler can specifically be a composite chromatography filler MaXtar MMA;

[0038] Before loading, the column was rinsed with NaOH solution, then pre-balanced with buffer D, and then balanced with buffer C.

[0039] Specifically, firstly wash with NaOH solution for 2-3 column volumes (specifically 3 column volumes), then use buffer D for pre-equilibration for 1-2 column volumes (specifically 1 column volume), and then use the second step chromatography buffer C for equilibration for 4-5 column volumes (specifically 5 column volumes);

[0040] The concentration of the NaOH solution may be 0.8-1.2 M, specifically 1 M;

[0041] The buffer D may specifically be 50 mM NaAC-HAC, 1 M NaCl, pH 6.0±0.1;

[0042] The loading speed may be 9-11 mL / min, specifically 10.6 mL / min;

[0043] The loading amount can be 7-8.5 mg / mL filler (specifically 8-8.5 mg / mL filler) (volume calculated based on albumin content) of the first chromatography product after ultrafiltration and liquid exchange;

[0044] Collect flow-through samples during loading;

[0045] After loading, re-equilibrate using the second step chromatography buffer C;

[0046] Specifically, the second step chromatography buffer C is used to equilibrate for 1-2 column volumes (specifically 1 column volume);

[0047] The elution is performed by isocratic elution using 50% of the buffer D, and the first impurity peak and the second target peak are collected;

[0048] Elution for 5-6 column volumes (specifically 5 column volumes);

[0049] After the collection is completed, 100% of the buffer D is used to elute and collect the impurity peak, and CIP cleaning and flushing are performed to remove impurities on the chromatography column to achieve column regeneration;

[0050] Wherein, 100% of the buffer D is used for elution for 2-3 column volumes (specifically 3 column volumes),

[0051] CIP cleaning was performed using 1 M HAC at a flow rate of 10.6 mL / min for 2 column volumes.

[0052] The above method step (2) further comprises ultrafiltration of the obtained second chromatography product;

[0053] Specifically, an equal volume of buffer E was added for ultrafiltration five times to change the solution. The pH of the sample obtained after ultrafiltration was 5.5±0.1, and the cond was 15±0.5 mS / cm. The sample was diluted to a concentration of 5.0±0.5 mg / mL;

[0054] The buffer E may specifically be 50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5±0.1.

[0055] In step (3) of the above method, the cationic hydrophobic composite chromatography filler can specifically be a composite chromatography filler MaXtar MMC;

[0056] Before loading the sample, the chromatography column was first rinsed with a NaOH solution, then rinsed with a HAC solution to lower the pH value of the system, and then balanced with the buffer E.

[0057] Specifically, first use NaOH solution to wash 1-2 column volumes (specifically 2 column volumes), then use HAC solution to wash 0.5-1 column volume (specifically 1 column volume), and then use the buffer E to balance 5-6 column volumes (specifically 5 column volumes);

[0058] The concentration of the NaOH solution may be 0.8-1.2 M, specifically 1 M;

[0059] The concentration of the HAC solution may be 0.8-1.2M, specifically 1M;

[0060] The loading speed may be 9-11 mL / min, specifically 10.6 mL / min;

[0061] The loading amount can be 7-8.5 mg / mL filler (volume calculated based on albumin content) of the second chromatography product after ultrafiltration and fluid dilution;

[0062] Collect flow-through samples during loading;

[0063] After the sample loading is completed, the buffer E is used for re-equilibration;

[0064] Use the buffer E to re-equilibrate for 1-2 column volumes (specifically 1 column volume);

[0065] The elution is performed by isocratic elution using 100% buffer F, and the first impurity peak and the second target peak are collected;

[0066] The buffer F is 50 mM NaAC-HAC, 1 M NaCl, pH 5.5 ± 0.1;

[0067] Elution: 6-8 column volumes (specifically 7 column volumes);

[0068] After the collection, 1 M NaOH was used to perform CIP cleaning for 2 column volumes to achieve column recovery.

[0069] The above method further comprises the operation of ultrafiltration and liquid replacement of the third chromatography product obtained in step (3): adding equal volume of ultrapure water for liquid replacement 5 times until the conductivity detection is less than 20 μs / cm.

[0070] The application of the above-mentioned method for preparing recombinant human albumin from transgenic pig blood in the preparation of recombinant human albumin from transgenic pig blood and the recombinant human albumin from transgenic pig blood prepared by the above-mentioned method also fall within the protection scope of the present invention.

[0071] Compared with the prior art, the present invention has the following advantages:

[0072] 1) The present invention does not require traditional ethanol sample pretreatment, and only uses chromatography separation, which has a simple process, high separation efficiency, and good purification effect, and can be facilitated for factory-scale preparation;

[0073] 2) The present invention uses the latest Maxtar series of fillers from Bailinke. The improved MaXtar base frame has stronger rigidity, so it can achieve higher process flow rate under lower back pressure, improve process efficiency and facilitate scale-up;

[0074] 3) The process of the present invention is stable, has low requirements on temperature and pH value, and is highly controllable;

[0075] 4) During the purification process of the process of the present invention, albumin is always in solution, which can reduce the step of adding dissolving solution and avoid loss;

[0076] 5) The raw materials of the present invention are derived from gene-edited humanized albumin pigs. After purification, the protein does not contain animal-derived immune proteins, thereby avoiding immune reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the chromatogram of the purification process of the anion chromatography filler Maxtar Q in Example 1 of the present invention.

[0078] Figure 2 This is the electrophoresis diagram of the purification of the anion chromatography filler Maxtar Q in Example 1 of the present invention, wherein 1: sample loading; 2: flow-through; 3: wash 1; 4: wash 2; 5: wash 3; 6: wash 4; 7: wash 5; 8: elution.

[0079] Figure 3 This is the chromatogram of the purification process of the composite chromatography filler MaXtar MMA in Example 2 of the present invention.

[0080] Figure 4 This is the electrophoresis diagram of the composite chromatography filler MaXtar MMA purification in Example 2 of the present invention, wherein 1: sample loading; 2: flow-through; 3: wash 1; 4: target; 5: wash 2.

[0081] Figure 5 This is the chromatogram of the purification process of the composite chromatography filler MaXtar MMC in Example 3 of the present invention.

[0082] Figure 6 This is the electrophoresis diagram of the purification of the composite chromatography filler MaXtar MMC in Example 3 of the present invention, wherein 1: loading; 2: flow-through; 3: target; 4: regeneration; 5: flow-through; 6: washing; 7: target; 8: regeneration; 9: target with half loading.

[0083] Figure 7This is a comparison chart of the electrophoresis test of the purified albumin product prepared by the method of the present invention and the purified albumin products from other sources. Among them, 1: the third chromatography product of the present invention; 2: Sigma albumin standard; 3: Jetbelin (imported) commercially available HSA; 4: North China Pharmaceutical (yeast) rHSA; 5: Wuhan Heyuan (rice) rHSA; 6: the freeze-dried product of the present invention; 7: pig IgG.

[0084] Figure 8 The present invention is a flow chart for preparing recombinant human albumin from transgenic pig blood.

[0085] Fig. 9 This is the HPLC test result of the freeze-dried sample prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0086] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0087] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0088] In the following examples, the anion chromatography filler Maxtar Q was purchased from Bio-Pharmaceuticals (Shanghai) Co., Ltd. under the trade name Q ion exchange filler, the column used is Chrom-LinX TM 50 / 1000, column volume 450 mL;

[0089] The composite chromatography filler MaXtar MMA was purchased from Bio-Pharmaceuticals (Shanghai) Co., Ltd. under the trade name MMA multi-mode packing, the column used is Chrom-LinX TM 26 Column volume 110 mL;

[0090] The composite chromatography filler MaXtar MMC was purchased from Bio-Pharmaceuticals (Shanghai) Co., Ltd. under the trade name MMC multi-mode packing, the column used is Chrom-LinX TM 26 column volume 110 mL.

[0091] The plasma samples in the following examples were prepared by the method described in the literature (Li L, Meng H, Zou Q, Zhang J, Cai L, Yang B, Weng J, Lai L, Yang H, Gao Y. Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use. J Gastroenterol Hepatol. 2019 Oct; 34(10): 1851-1859. doi: 10.1111 / jgh.14666. Epub 2019 Apr 10. PMID: 30884543.).

[0092] Example 1: Exploration of process conditions for anion exchange chromatography medium (Maxtra Q)

[0093] 1.1 Study on plasma sample processing methods

[0094] Method 1: The plasma sample was taken out of the -80℃ refrigerator and thawed naturally in a 4℃ refrigerator overnight, and the supernatant was centrifuged and diluted with buffer A (50mM Tris-HCl, pH 8.0±0.1) to a conductivity of 10±0.2mS / cm, pH 8.2±0.1, and filtered with a 0.45μm filter membrane before direct loading;

[0095] Method 2: The plasma samples were taken out of the -80℃ refrigerator and naturally thawed at 4℃ overnight, and then centrifuged to obtain the supernatant, diluted with buffer A (50mM Tris-HCl, pH 8.0±0.1) to a conductivity of 5.0±0.2mS / cm, pH 8.0±0.1, and filtered with a 0.45μm filter membrane before direct loading;

[0096] The sample loading capacity was 10 mg / mL filler and the gradient elution was 0-100% for 10 cv. The yield results are shown in Table 1.

[0097] Table 1

[0098] Parallel 1 (yield %) Parallel 2 (yield %) Parallel 3 (yield %) Average yield% Method 1 64.13％ 59.82％ 70.32％ 64.75％ Method 2 71.4％ 73.5％ 72.21％ 72.37％

[0099] Conclusion: Among the two sample processing methods, method 2 has the most stable sample yield.

[0100] 1.2 Study on the purification loading capacity of anion exchange chromatography medium (Maxtra Q)

[0101] Method 1: Sample loading 10-10.5 mg / mL filler

[0102] Method 2: Sample loading 7-7.5 mg / mL filler

[0103] Method 3: Sample loading 5-5.5 mg / mL filler

[0104] Confirm the sample processing method and use gradient elution 0-100% elution yield results, as shown in Table 2.

[0105] Table 2

[0106]

[0107] According to the above results, the chromatography yield is best when the sample load is 7-7.5 mg / mL;

[0108] 1.3 Study on purification and elution methods of anion exchange chromatography medium (Maxtra Q)

[0109] Method 1: Gradient elution, 0-100% buffer B (50 mM Tris-HCl, 0.5 M NaCl, pH 8.0 ± 0.1) 10CV gradient elution, humanized albumin at 50% buffer B concentration, the target peak;

[0110] Method 2: Referring to the results of method 1, use 40% buffer B to isocratically remove impurities, and then use 50% buffer B to isocratically elute the target;

[0111] Mode 3: Gradient elution, 0-60% buffer B for 7 column volumes (3150 mL), collecting impurity peaks 1, 2, 3, 4, 5 respectively, and the last peak is the target peak.

[0112] Confirm the loading method, the loading concentration is 7-7.5 mg / mL filler, and the yield results are shown in Table 3.

[0113] Table 3

[0114] Parallel 1 (yield %) Parallel 2 (yield %) Parallel 3 (yield %) Average yield% Method 1 80.25％ 79.34％ 80.12％ 79.90％ Method 2 65.32％ 64.33％ 65.19％ 64.94％ Method 3 83.50％ 83.17％ 86.29％ 84.32％

[0115] There are three elution methods, and method 3 is the best. In this plasma sample, there is a foreign protein close to the target peak. If isocratic elution is used, the separation effect cannot be achieved. 0-60% 7 column volumes can reduce the overall experimental time.

[0116] Based on the above three experimental conditions, the first step of anion exchange chromatography medium (Maxtra Q) purification process was confirmed as follows:

[0117] ① Buffer preparation: Buffer A: 50 mM Tris-HCl, pH 8.0 ± 0.1; Buffer B: 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0 ± 0.1;

[0118] ② Sample preparation: Plasma was taken out of the -80℃ refrigerator and placed in a 4℃ refrigerator overnight for natural thawing; after thawing, the plasma was centrifuged (8000g±500g), 40±5min, 4℃) to obtain the supernatant; the supernatant was diluted with buffer A (50mM Tris-HCl, pH 8.0±0.1) to a conductivity of 5.0±0.2mS / cm, the pH was adjusted to 8.0±0.1, and filtered through a 0.45μm filter membrane (or clarified with a hollow fiber / capsule filter), and a large number of bubbles were avoided during filtration.

[0119] ③ Experimental process, as shown in Table 4.

[0120] Table 4

[0121]

[0122]

[0123] ④Chromatographic spectrum of purification process Figure 1 shown.

[0124] ⑤Sample collection: After Conc 50%, the sample can be collected automatically by the sample collector (the color of the chromatography column can be observed and the collection tube can be manually switched when Slope_UV1 changes from decreasing to increasing), and all the eluates of the last large peak are combined. The collection is stopped when Slope_UV1 is greater than -20.

[0125] ⑥Chromatographic electrophoresis diagram Figure 2 As shown, sequence number 8-eluted as the target peak (the first chromatography product) with a yield of 84.21%.

[0126] Example 2: Exploration of purification process conditions for composite chromatography medium (MaXtar MMA)

[0127] 2.1 Study on ultrafiltration method of the first chromatography product sample

[0128] Method 1: The first step chromatography product is ultrafiltered using a tangential flow membrane package, and ultrapure water is added three times with equal volumes to remove Tris salt and NaCl in the first step eluent;

[0129] Method 2: The first step chromatography product was ultrafiltered using a tangential flow membrane package, and two equal volumes of ultrapure water were added for ultrafiltration to remove the Tris salt and NaCl in the first step eluate, and then two equal volumes of buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) were added;

[0130] Method 3: The first step chromatography product was ultrafiltered using a tangential flow membrane bag, and an equal volume of buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) was added five times;

[0131] The ultrafiltration results are shown in Table 5.

[0132] Table 5

[0133]

[0134] Method 3: Ultrafiltration is the best method.

[0135] 2.2 Study on pH and different NaCl concentrations (cond values) of the buffer system (referring to the buffer C used for ultrafiltration in 2.1)

[0136] Method 1: The buffer system pH is 5.5, and no NaCl is added to Buffer C;

[0137] Method 2: The pH value of the buffer system is 6.0, and no NaCl is added to Buffer C;

[0138] Method 3: Buffer system pH 5.5, Buffer C added with 0.15M NaCl;

[0139] Method 4: Buffer system A pH 6.0, Buffer C added with 0.15M NaCl;

[0140] The sample concentration was 5 mg / mL, the sample loading was 5 mg / mL filler, and the experimental phenomena and results are shown in Table 6.

[0141] Table 6

[0142]

[0143] According to the determination and selection of the above four methods, Buffer C does not add NaCl, ultrafiltration will produce precipitation, filtration clarification, and the eluent will still have precipitation after sample elution. The yield of method three is low. Method four, buffer system pH 6.0, Buffer C 0.15M NaCl concentration, not only will no precipitation be produced during ultrafiltration, but also the purification yield and purification effect are the best.

[0144] 2.3 Study on the sample loading capacity of the second-step composite chromatography medium MaXtar MMA

[0145] Method 1: Refer to the first step of loading, the loading capacity is 7.0-7.5 mg / mL filler

[0146] Method 2: Increase the sample loading to 8-8.5 mg / ml filler

[0147] Method 3: Sample loading 9-9.5 mg / mL filler

[0148] The ultrafiltration method was determined as Buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) and the loading concentration was 5 mg / mL.

[0149] The experimental results are shown in Table 7.

[0150] Table 7

[0151]

[0152] According to the above results, the sample loading capacity can be 7-8.5 mg / mL of filler. Considering the purification efficiency, 8-8.5 mg / mL is the best.

[0153] 2.4 Study on the loading concentration of the second step composite chromatography medium MaXtar MMA:

[0154] Method 1: After the first chromatography product is ultrafiltrated, the albumin sample is diluted to a concentration of 10-10.5 mg / mL;

[0155] Method 2: After the first chromatography product is ultrafiltered, the albumin sample is diluted to a concentration of 7-7.5 mg / mL;

[0156] Method 3: After the first chromatography product is ultrafiltered, the albumin sample is diluted to a concentration of 5-5.5 mg / mL;

[0157] The sample loading capacity was determined to be 8-8.5 mg / ml filler. The experimental results at different sample loading concentrations are shown in Table 8.

[0158] Table 8

[0159]

[0160] Based on the experimental results of the above three loading concentrations, the loading concentration of 5-5.5 mg / mL is the best.

[0161] Based on the above four experimental conditions, the second step of the composite chromatography medium MaXtar MMA purification process was confirmed as follows:

[0162] ① Buffer preparation: Buffer C: 50mM NaAC-HAC, 0.15M NaCl, pH 6.0±0.1 (cond about 18mS / cm) Buffer D: 50mM NaAC-HAC, 1M NaCl, pH 6.0.

[0163] ②Sample preparation: The MaXtar Q eluate (the first chromatography product) was fully ultrafiltered and replaced with buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) was added at least 5 times with equal volume (Note: Failure to fully ultrafilter and replace the solution will affect the purity and recovery rate of the second step chromatography product). After ultrafiltration, the pH was 6.0 ± 0.1, the cond was 18.5 ± 0.5 mS / cm (buffer or water can be added for fine adjustment), and the concentration was 5.0 ± 0.5 mg / mL.

[0164] ③The experimental process is shown in Table 9.

[0165] Table 9

[0166]

[0167] ④Chromatographic spectrum of purification process Figure 3 shown.

[0168] ⑤Sample collection: Start collecting when Slope_UV1 is greater than 20 after elution begins. Switch the collection tube when Slope_UV1 changes from rising to falling and then rising again, and merge the elution peak (elute until Slope_UV1 is greater than -20).

[0169] ⑥Chromatographic electrophoresis diagram Figure 4 As shown, sequence number 4-eluted as the target peak (second chromatography product) with a yield of 95.63%.

[0170] Example 3: Exploration of purification process conditions of composite chromatography filler MaXtar MMC

[0171] 3.1 The filler used in the third step is similar to that in the second step, which is the composite chromatography medium MaXtar MMC. The ultrafiltration method, sample loading volume, and sample loading concentration are the same as those in the second step chromatography method. The buffer system refers to the MaXtar MMC manual. The results of the three chromatography experiments are shown in Table 10.

[0172] Table 10

[0173]

[0174] The process of the third step composite chromatography medium MaXtar MMC purification was confirmed as follows:

[0175] ① Buffer preparation: Buffer E: 50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5 ± 0.1 (cond15.0 ± 0.5 mS / cm) Buffer F: 50 mM NaAC-HAC, 1 M NaCl, pH 5.5 ± 0.1;

[0176] ②Sample preparation: The MaXtar MMA eluate (second chromatography product) was fully ultrafiltrated and replaced with buffer E (50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5 ± 0.1) with an equal volume in 5 times. After ultrafiltration, the pH was 5.5 ± 0.1, the cond was 15.0 ± 0.5 mS / cm (buffer or pure water can be added for adjustment), and the concentration was 5.0 ± 0.5 mg / mL.

[0177] ③The experimental process is shown in Table 11.

[0178] Table 11

[0179]

[0180] ④Chromatographic spectrum of purification process Figure 5 shown.

[0181] ⑤Sample collection: Start collecting after Slope_UV1 is greater than 20 after elution begins, and collect until the peak is the highest and Slope_UV1 is greater than -15. The elution peak is Figure 5 The two peaks shown are connected, and the other elution peaks are not combined.

[0182] ⑥Chromatographic electrophoresis diagram Figure 6 As shown, sequence number 9 is the target peak (the third chromatography product) with a yield of 93.04%.

[0183] 3.2 Sample processing of the third chromatography product: The third step of chromatography collects samples and uses ultrapure water for ultrafiltration. The first purpose is to replace the buffer in the chromatography process; the second purpose is to concentrate the sample. The sample concentration can reach 50-60 mg / mL through ultrafiltration. After the ultrafiltration is completed, the sample is filtered through a 0.22μm sterile filter head for storage, or freeze-dried into freeze-dried powder for storage.

[0184] 3.3 Freeze-dry the sample and calculate the final recovery rate. Total recovery rate: 74%.

[0185] Obtaining high-purity recombinant human serum albumin is the key to meeting clinical drug use. The plasma in this protocol is derived from gene-edited pig plasma, which is consistent with human plasma species and belongs to mammals. The purified sample does not contain pig IgG. This protocol does not require a crude purification step. The plasma is directly extracted by chromatography after centrifugation, with fewer steps, high yield, and easy operation. Figure 7 The electrophoresis test of the patented purified albumin product was compared with that of purified albumin products from other sources. Figure 7 It can be seen that the sample purified by this patent has better purity.

[0186] Example 4: Purification of recombinant human serum albumin

[0187] according to Figure 8The process flow chart shown is for purifying and extracting recombinant human serum albumin from humanized frozen plasma of gene-edited pigs.

[0188] (1) Plasma was taken out of a -80°C freezer and placed in a 4°C refrigerator overnight for natural thawing. After thawing, the plasma was centrifuged (8000 g, 40 min, 4°C) to obtain the supernatant, which was diluted to a conductivity of 5.0 mS / cm by adding buffer A (50 mM Tris-HCl, pH 8.0±0.1) and adjusted to pH 8.0±0.1. The supernatant was filtered through a 0.45 μm filter membrane to avoid generating a large number of bubbles during filtration.

[0189] (2) Before loading the sample, the MaXtar Q-450 mL column was flushed with 1 M NaOH for three column volumes (1350 mL), equilibrated with buffer B (50 mM Tris-HCl, 0.5 M NaCl, pH 8.0 ± 0.1) for one column volume (450 mL), and then equilibrated with buffer A (50 mM Tris-HCl, pH 8.0 ± 0.1) for five column volumes (2250 mL).

[0190] (3) After equilibration, load 3150 mg (volume calculated based on albumin content) of the diluted plasma sample in step (1) above at a rate of 30 mL / min, and collect the flow-through sample during the process; after loading, use buffer A 50 mM Tris-HCl, pH 8.0±0.1 to equilibrate another column volume (450 mL), and after UV280 baseline equilibrium, start gradient elution, the elution gradient is 0-60% buffer B, 3150 mL, collect five sections of eluted samples respectively, when the collection of the fifth impurity peak is completed, the yellow color on the chromatographic column gathers downward, and the UV280 increases, start collecting the target peak.

[0191] (4) After the target is collected, use buffer B 50mM Tris-HCl, 0.5M NaCl, pH 8.0±0.1, regenerate for 3 column volumes (1350mL). After regeneration, use 1M NaOH at 20mL / min to flush for three column volumes (1350mL).

[0192] (5) The target collected in step (3) is the first chromatography product. The sample is loaded, flow-through, washed, and the target sample is subjected to concentration and electrophoresis detection, and the yield is calculated, which is 84.29%.

[0193] (6) The first chromatography product was ultrafiltered, and the second step chromatography buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) was added five times with equal volume to perform ultrafiltration and change the buffer. After ultrafiltration, the pH was 6.0 ± 0.1, Cond 18.5 mS / cm, and the sample concentration was diluted to 5.0 mg / mL by adding buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1).

[0194] (7) Before loading the sample, the MaXtar MMA-110 mL column was rinsed with 1 M NaOH for 2 column volumes (220 mL), equilibrated with buffer D (50 mM NaAC-HAC, 1 M NaCl, pH 6.0 ± 0.1) for 1 column volume (110 mL), and then equilibrated with buffer C (50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1) for 5 column volumes (550 mL).

[0195] (8) After equilibration, 880 mg (volume calculated based on albumin content) of the first chromatography product after ultrafiltration in step (6) above was loaded at a rate of 10.6 mL / min, and the load flow-through was collected. After loading, buffer C 50 mM NaAC-HAC, 0.15 M NaCl, pH 6.0 ± 0.1 was used to equilibrate for another 1 column volume (110 mL).

[0196] (9) Target elution: use 50% buffer D 50mM NaAC-HAC, 1M NaCl, pH 6.0±0.1 for isocratic elution over 5 column volumes (550 mL). Collect the first impurity peak and the second target peak.

[0197] (10) After the collection, 100% buffer D 50mM NaAC-HAC, 1M NaCl, pH 6.0±0.1 was used to elute for 3 column volumes (330 mL) to collect the impurity peak. 1M HAC, 10.6mL / min, was used for CIP cleaning and flushing for 2 column volumes (220 mL) to remove impurities on the chromatography column.

[0198] (11) The target collected in step (9) is the second chromatography product. The sample is loaded, flow-through, washed, and the target sample is subjected to concentration and electrophoresis detection, and the yield is calculated, which is 94.88%.

[0199] (12) The second chromatography product was ultrafiltered and an equal volume of buffer E (50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5 ± 0.1) was added five times. After ultrafiltration, the pH was 5.5 ± 0.1 and the cond was 15 mS / cm. The sample concentration was diluted to 5.0 mg / mL by adding buffer E (50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5 ± 0.1).

[0200] (13) Before loading the sample on the MaXtar MMC-110 mL column, flush it with 1 M NaOH for 2 column volumes (220 mL). After that, flush it with 1 M HAC for 1 column volume (110 mL) to lower the pH value of the system. After the pH value drops, use buffer E 50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5 ± 0.1 to equilibrate it for 5 column volumes (5*110 mL).

[0201] (14) After equilibration, 880 mg (albumin) of the second chromatography product after ultrafiltration and liquid exchange in step (12) above was loaded at a rate of 10.6 mL / min, and the load flow-through was collected. After loading, 1 column volume (110 mL) was equilibrated with buffer E 50 mM NaAC-HAC, 0.12 M NaCl, pH 5.5±0.1.

[0202] (15) Target elution: use 100% buffer F, 50 mM NaAC-HAC, 1 M NaCl, pH 5.5±0.1 for isocratic elution over 7 column volumes (770 mL). Collect the first impurity peak and the second target peak.

[0203] (16) After collection, CIP cleaning was performed using 1 M NaOH for 2 column volumes (220 mL).

[0204] (17) The target collected in step (15) is called the third chromatography product. The sample is loaded, flow-through, washed, and the target sample is subjected to concentration and electrophoresis detection. The yield is calculated and the yield is 93.04%.

[0205] (18) The third chromatography product was ultrafiltered and replaced with an equal volume of ultrapure water for 5 times. After the end, the conductivity test was <20 μs / cm, and the product was concentrated, freeze-dried and packaged. The HPLC test results of the freeze-dried sample were as follows: Fig. 9 As shown, the purity is 99.23%.

[0206] Examples 5 and 6 were carried out with reference to Example 4. The samples and chromatography conditions of Examples 5 and 6 were consistent with those of Example 4, and the data were statistics of the experimental results for each time.

[0207] Table 12

[0208]

[0209] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for preparing recombinant human albumin from transgenic pig blood, comprising the following steps: (1) purifying the plasma supernatant through an anion chromatography filler to obtain a first chromatography product and performing ultrafiltration on the first chromatography product; the purification through anion chromatography filler sequentially includes CIP treatment of the chromatography column before loading, balancing, loading, rebalancing, eluting and collecting the target product, and column regeneration; (2) purifying the first chromatography product after ultrafiltration and liquid exchange through anionic hydrophobic composite chromatography filler to obtain a second chromatography product and ultrafiltration and liquid exchange of the second chromatography product; the anionic hydrophobic composite chromatography filler purification sequentially includes CIP treatment of the chromatography column before sample loading, equilibration, sample loading, elution and collection of target substances, and column regeneration; (3) Purifying the second chromatography product after ultrafiltration and liquid exchange through a cationic hydrophobic composite chromatography filler to obtain a third chromatography product, and ultrafiltration and liquid exchange, concentration, freeze-drying and packaging of the third chromatography product to obtain transgenic pig blood recombinant human albumin; the purification through cationic hydrophobic composite chromatography filler includes, in sequence, disinfecting the chromatography column before loading, CIP treatment, balancing, loading, eluting and collecting the target product.

2. The method according to claim 1, characterized in that The plasma supernatant is prepared by the following method: naturally thawing the transgenic edited humanized albumin plasma, centrifuging the thawed plasma to obtain the supernatant; The supernatant is first diluted, pH adjusted, and membrane filtered or clarified before the anion chromatography filler purification operation in step (1). Wherein, the dilution is diluted with buffer A to a conductivity of 5.0±0.2 mS / cm, and the buffer A is 50 mM Tris-HCl, pH 8.0±0.1; The pH adjustment is to adjust the pH of the supernatant after dilution to 8.0±0.

1.

3. The method according to claim 1, characterized in that In step (1), the anion chromatography filler is anion chromatography filler Maxtar Q; In step (2), the anionic hydrophobic composite chromatography filler is a composite chromatography filler MaXtar MMA; In step (3), the cationic hydrophobic composite chromatography filler is a composite chromatography filler MaXtar MMC.

4. The method according to claim 1, characterized in that In step (1), before loading the sample, the chromatography column is first rinsed with a NaOH solution, then pre-balanced with buffer B, and then balanced with buffer A; The buffer B is 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0 ± 0.1; The loading speed is 28-32 mL / min; The sample loading amount is 7-7.5 mg / mL of filler; After the loading is completed, the buffer A is used for balancing first. After the UV280 baseline is balanced, the gradient elution is started, and five sections of eluted impurity samples are collected respectively. When the collection of the fifth impurity peak is completed, the yellow on the chromatography column gathers downward, and the UV280 increases, the target peak is collected; Equilibrate 1-2 column volumes with buffer A; The elution gradient of the gradient elution is: 0-60% buffer B, 6-8 column volumes; After the target peak is collected, the buffer B is used to regenerate the column.

5. The method according to claim 1, characterized in that Step (1) ultrafiltration of the obtained first chromatography product; Specifically, at least 5 equal volumes of the second step chromatography buffer C were added for ultrafiltration, the pH of the sample obtained after ultrafiltration was 6.0±0.1, Cond 18.5±0.5mS / cm, and the concentration of the diluted sample was 5.0±0.5mg / mL; Wherein, the second step chromatography buffer C is 50mM NaAC-HAC, 0.15M NaCl, pH 6.0±0.1; In step (2), the obtained second chromatography product is subjected to ultrafiltration and liquid exchange; Specifically, an equal volume of buffer E was added for ultrafiltration five times to change the solution. The pH of the sample obtained after ultrafiltration was 5.5±0.1, and the cond was 15±0.5 mS / cm. The sample was diluted to a concentration of 5.0±0.5 mg / mL; Wherein, the buffer E is 50mM NaAC-HAC, 0.12M NaCl, pH 5.5±0.1; The operation of ultrafiltration and liquid replacement of the third chromatography product obtained in step (3) is: adding equal volume of ultrapure water for liquid replacement 5 times until the conductivity detection is <20 μS / cm.

6. The method according to claim 1, characterized in that In step (2), before loading the sample, the chromatography column is first rinsed with NaOH solution, then pre-balanced with buffer D, and then balanced with the second step chromatography buffer C. The buffer D is 50 mM NaAC-HAC, 1 M NaCl, pH 6.0 ± 0.1; The loading speed is 9-11 mL / min; The sample loading amount is 7-8.5 mg / mL of filler; After loading, re-equilibrate using the second step chromatography buffer C; Use the second step chromatography buffer C to equilibrate for another 1-2 column volumes; The elution is performed by isocratic elution using 50% of the buffer D, and the first impurity peak and the second target peak are collected; Elution 5-6 column volumes; After the collection is completed, 100% of the buffer D is used to elute and collect the impurity peak.

7. The method according to claim 1, characterized in that In step (3), before loading the sample, the chromatography column is first rinsed with a NaOH solution, then rinsed with a HAC solution to lower the pH value of the system, and then balanced with the buffer E; The loading speed is 9-11 mL / min; The sample loading amount is 7-8.5 mg / mL of filler; After the sample loading is completed, the buffer E is used for balancing; Use the buffer E to equilibrate 1-2 column volumes; The elution is performed by isocratic elution using 100% buffer F, and the first impurity peak and the second target peak are collected; The buffer F is 50 mM NaAC-HAC, 1 M NaCl, pH 5.5 ± 0.1; Elute over 6-8 column volumes.

8. The method according to claim 1, characterized in that Step (1) further comprises the operation of flushing the column with a NaOH solution after the column regeneration is completed; Step (2) further comprises the step of using the buffer D to elute and collect the impurity peaks, and then using HAC to perform CIP cleaning to remove impurities on the chromatography column; Step (3) further includes the operation of performing CIP cleaning using NaOH after the collection is completed.

9. Use of the method for preparing recombinant human albumin from transgenic pig blood according to any one of claims 1 to 8 in preparing recombinant human albumin from transgenic pig blood.

10. Recombinant human albumin from transgenic pig blood prepared by the method according to any one of claims 1 to 8.

Images