CN115466303A - a method of clarification - Google Patents

Abstract

This article provides a method for clarification of materials to be clarified, which includes repeating steps (a) to (c) during the process of clarification of materials to be clarified using a continuous flow centrifuge: (a) before slag discharge, feed The material is switched from the material to be clarified to the cleaning medium; (b) slagging; and (c) after the slagging, the feed material is switched from the cleaning medium to the material to be clarified; wherein, steps (a) to (c) are repeated at least two times Second-rate.

Description

a method of clarification

technical field

The present invention generally relates to a method of clarification; in particular, it relates to a method of clarification using a continuous flow centrifuge.

Background technique

In recent years, due to the rapid development of bioengineering pharmaceutical cell culture related technologies, the expression per unit volume of bioreactors has gradually increased. The increase of expression per unit volume is mainly achieved by increasing the expression of single cells and increasing the cell density. High cell density will lead to increased solid content and turbidity in the fermentation broth, which is a great challenge for the existing solid-liquid separation technology and will lead to a decrease in the recovery of products (eg, cell-expressed proteins). How to improve the product recovery of cell culture clarification methods has become a current concern.

The current common clarification treatment method is to combine continuous flow centrifugation and depth filtration, or to use two-stage depth filtration to remove cells and cell debris. For conventional cell culture fluids, these two operating modes can meet the clarification requirements of downstream processes. For the combined operation of continuous flow centrifugation and depth filtration, cells and large cell debris are generally removed by continuous flow centrifugation, and then small cell debris and some soluble impurities are removed by depth filtration. For the operation method of two-stage depth filtration, generally, the first-stage depth filter with a large pore size is used to remove cells and large cell debris, and then the second-stage depth filter with a smaller pore size connected in series is used to remove small cell debris. for the purpose of clarification. Compared with the two, the production cost of continuous flow centrifugation combined with depth filtration is lower than the latter, and it becomes the first choice for commercial production of biomacromolecules.

It was found that during continuous flow centrifugal clarification of material to be clarified with a high solids content (eg cell cultures), large product losses occur. There is a need for an improved method of treating materials to be clarified that further increases product (eg, protein product) recovery from preparative purification processes.

Contents of the invention

The inventors have discovered and proposed a method for clarification. Using the methods of the present invention, improved product (eg, protein product) recovery can be obtained during continuous flow centrifugal clarification of cell cultures.

On the one hand, this paper provides a kind of clarification treatment method for the material to be clarified, which comprises repeating steps (a) to (c) during the clarification process of the material to be clarified using a continuous flow centrifuge:

(a) Before slag discharge, switch the feed material from the material to be clarified to the cleaning medium;

(b) deslagging; and

(c) After slag discharge, switch the feed material from the cleaning medium to the material to be clarified;

Wherein, steps (a) to (c) are repeated at least twice.

In another aspect, this paper provides a method for separating and purifying cell cultures, comprising:

(i) clarifying the cell culture using the method for clarifying the material to be clarified as described herein to obtain a clarified cell culture supernatant; and

(ii) further separating and purifying the clarified cell culture supernatant obtained in step (i).

In another aspect, the present invention provides a system for clarifying materials to be clarified, comprising:

(A) a continuous flow centrifuge having:

The material to be clarified is fed into the interface, which is in fluid communication with the material to be clarified storage container;

one or more cleaning medium feed ports, each in fluid communication with the cleaning medium storage container;

(B) one or more processors; and

(C) memory having stored thereon instructions executable by said one or more processors;

Wherein, the instructions executable by the one or more processors are configured to perform the following operations: repeat steps (i) to (iii):

(i) Before slag discharge, stop the feeding of the material to be clarified, and feed the cleaning medium from the cleaning medium storage container to the continuous flow centrifuge, thereby switching the fed material from the material to be clarified to the cleaning medium;

(ii) deslagging; and

(iii) After the slag is discharged, stop the feeding of the cleaning medium, and feed the material to be clarified from the storage container of the material to be clarified to the continuous flow centrifuge, thereby switching the fed material from the cleaning medium to the material to be clarified;

Wherein, in the clarification process of the material to be clarified, steps (i) to (iii) are repeated at least twice.

In another aspect, provided herein is a computer readable medium having stored thereon instructions executable by a processor to perform the method described herein for clarifying a substance to be clarified in a continuous flow centrifuge.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings, wherein these illustrations are only for illustrating the embodiments of the present invention, and are not intended to limit the scope of the present invention.

Figure 1 is a schematic flow diagram according to one embodiment of the method described herein.

FIG. 2 is a trend diagram of depth filtering according to Embodiment 2.

FIG. 3 is a trend diagram of depth filtering according to Embodiment 2.

FIG. 4 is a trend diagram of depth filtering according to Embodiment 3.

FIG. 5 is a trend diagram of depth filtering according to Embodiment 3.

Fig. 6 is a graph of the recovery amount of the rinsing experiment group 3 minutes before slag discharge in Example 1-3.

detailed description

The meanings of scientific and technical terms in this application are consistent with the common understanding of those skilled in the art, unless otherwise specified. In the present application, "a" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In the present application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically disclosing these numerical values, range end values, and numerical ranges formed by any combination thereof. In the present application, any numerical value, regardless of whether it has modifiers such as "about", covers an approximate range understandable by those skilled in the art, such as plus or minus 10%, 5%, etc. Herein, each "implementation" equally refers to and covers the implementation of each method and each system of the present application. In this application, one or more technical features in any implementation mode can be freely combined with one or more technical features in any one or more other implementation modes, and the resulting implementation mode also belongs to the content disclosed in this application .

Separation machinery is the main process equipment used in the field of biological product production. Continuous-flow centrifuges (for example, disc-type continuous-flow centrifuges) are widely used in high-volume biological product production processes due to their high centrifugation efficiency and good separation effects. It can effectively and continuously separate the solid particulate impurities from the liquid to obtain the desired material composition.

Disc type continuous flow centrifuge can quickly and continuously separate solid-liquid and liquid-liquid materials, it is a kind of vertical centrifuge. The disc type continuous flow centrifuge has a drum installed on the upper end of the vertical shaft, which is driven by a motor through a transmission device to rotate at a high speed. There is a group of disc-shaped parts nested together in the drum - discs. There is a small gap between the disc and the disc. Suspension (or emulsion) is fed into the drum through the feed pipe located in the center of the drum. When the suspension (or emulsion) flows through the gap between the discs, solid particles (or liquid droplets) can settle on the discs under the action of the centrifuge, forming a sediment (or liquid layer). The sediment slides along the surface of the disc and separates from the disc and accumulates in the largest diameter part of the drum, and the separated liquid is discharged from the drum through the liquid outlet. The role of the disc is to shorten the settling distance of solid particles (or liquid droplets) and expand the settling area of the drum. The production capacity of the separator is greatly improved due to the installation of the disc in the drum. The solids accumulated in the drum can be discharged from the drum at certain time intervals through the slag discharge device without stopping the machine.

The inventors have found through long-term research that during the operation of the disc-type continuous flow centrifuge, the frequency of slag discharge is positively correlated with the solid content of the material to be clarified, for example, cell culture (eg, cell harvesting liquid). The higher the solid content of the cell culture, the more slag removal times per unit time. In addition to solid impurities such as cells and cell debris, some target biological products (for example, target proteins) will also be discharged during each slag discharge, resulting in product loss. The higher the solids content of the cell culture, the greater the loss of product.

Herein, the term "solid content" of the material to be clarified (eg, cell culture) refers to the volume percentage of solids in the material to be clarified (eg, cell culture) to the material to be clarified (eg, cell culture).

In order to improve the product recovery in the process of preparation and purification of biological products, the inventor proposed an improved clarification treatment method after repeated research and practice, which can reduce or avoid the product loss caused by slag discharge in the continuous flow centrifugation process, and improve Increase product recovery and reduce cost per gram of product produced.

On the one hand, this paper provides a kind of clarification treatment method, it comprises in the process of using continuous flow centrifuge to clarify the material to be clarified, repeating steps (a) to (c):

(a) Before slag discharge, switch the feed material from the material to be clarified to the cleaning medium;

(b) deslagging; and

(c) After slag discharge, switch the feed material from the cleaning medium to the material to be clarified;

Wherein, steps (a) to (c) are repeated at least twice.

As used herein, the term "material to be clarified" generally refers to a material that is desired and suitable for a solid-liquid separation operation by a continuous flow centrifuge. The material to be clarified is generally a solid-liquid mixture, wherein the material desired to be collected is predominately present (eg, dissolved) in the liquid portion. In other words, the desired harvested material is expected to be obtained by collecting the liquid fraction after solid-liquid separation.

In some embodiments, the material to be clarified is cell culture. As used herein, the term "cell culture" refers to a culture suspension comprising cultured cell lines. In some cases, it is desirable to isolate and purify a biological product (eg, a cell-secreted protein) contained in a cell culture supernatant.

In some embodiments, the cell culture is a culture suspension of cells selected from the group consisting of eukaryotic cells, prokaryotic cells, and any combination thereof. In some embodiments, the cell culture is a culture suspension of cells selected from the group consisting of animal cells, plant cells, microbial cells, and any combination thereof. Suitable animal cells can include mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), avian cells (e.g., chicken, duck, and goose) or insect cells. Suitable mammalian cells may include CHO cells, HEK293 cells, lymphocytes, or myeloma cells (eg, NSO cells). Suitable microbial cells include bacterial cells or fungal cells (eg, yeast). Suitable yeast include Pichia methanolica, Pichia pastoris, Saccharomyces cerevisiae, or common baker's yeast. Suitable insect cells include Drosophila Schnieder S2 cells and Sf9 cells. In some exemplary embodiments, the cell culture may be selected from Culture suspensions of the following cells: CHO cells, NSO cells, cells of human origin (eg, primary human cells), insect cells, and other animal or microbial cells.

Herein, the term "clarification operation" or "clarification treatment" as used generally refers to a liquid-solid separation operation.

In some embodiments, the centrifugal force of the clarification process is in the range of 4000G to 20000G, eg, in the range of 4000G to 13000G, 5000G to 12000G, 6000G to 11000G, or 7000G to 10000G.

In some embodiments, the centrifugal speed of the clarification treatment is in the range of 4000 rpm to 13000 rpm, for example, 5000 rpm to 10000 rpm.

In some embodiments, the continuous flow centrifuge is a disc continuous flow centrifuge.

Herein, the term "repeating steps (a) to (c)" as used means that during the entire clarification process (for example, from the start of the clarification process to the completion of the clarification process to obtain the desired amount of product), at least Two operations of steps (a) to (c). It should be understood that the start of the next step (a) to (c) is not immediately after the end of the previous step (a) to (c). At least a period of clarifying operation is included between the previous step (c) and the subsequent step (a). In some embodiments, the clarification operation for a period of time included between the previous step (c) and the subsequent step (a) starts from the end of the previous step (c) until the sediment accumulated in the drum reaches a level that needs to be discharged. When the amount is finished, the next steps (a) to (c) can be performed as required or only the slag discharge operation can be performed.

In some embodiments, steps (a) to (c) are performed in the following order: (a)→(b)→(c).

As used herein, the term "slagging" refers to the draining of the contents (eg, sediment) of the bowl bowl cavity of a continuous flow centrifuge. During the operation of a continuous flow centrifuge, the material to be clarified (eg, cell culture) flows into the bowl through the feed port and is clarified in the disc pack. Since the disc group is composed of a large number of conical discs superimposed on each other, the liquid is divided into thin liquid layers through the narrow disc spacer cavity, so that the settlement displacement is reduced to a minimum. The clarified liquid is squeezed towards the center of the bowl, then flows to the holder cavity and drains through it. The solid particles will be accelerated towards the underside of the disc and flow outward into the slag cavity. In order to prevent the slag cavity from being completely filled, the drum needs to discharge slag to the outside regularly. The separated solid material is regularly discharged through the solid material discharge port. During the slagging process, the feeding of materials can be interrupted or not.

In some embodiments, in step (a), at least 10 seconds before slagging, for example, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 1.5 minutes, at least 2 minutes before slagging Minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes or at least 8 minutes, the feed material is switched from the material to be clarified to the cleaning medium. In some embodiments, in step (a), at the moment of 10 seconds to 10 minutes before slag discharge, for example, 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes before slag discharge or 3 minutes, to 5 minutes, 8 minutes or 10 minutes, for example, 10 seconds to 8 minutes, 10 seconds to 5 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 20 seconds to 8 minutes, 20 seconds to 5 minutes, 20 seconds to 3 minutes, 20 seconds to 2 minutes, 20 seconds to 1 minute, 30 seconds to 8 minutes, 30 seconds to 5 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1.5 minutes, 30 seconds to 1 minute, 45 seconds to 8 minutes, 45 seconds to 5 minutes, 45 seconds to 3 minutes, 45 seconds to 2 minutes, 15 seconds to 1 minute, 1 minute At the moment of 8 minutes, 1 minute to 5 minutes, 1 minute to 3 minutes or 1 minute to 1.5 minutes, the feed material is switched from the material to be clarified to the cleaning medium.

In some embodiments, in step (a), the feed material is switched from the material to be clarified to the cleaning medium before the slag discharge, so that the sediment is rinsed with the cleaning medium. In some embodiments, the rinse lasts at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 1.5 minutes, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least A period of time of 6 minutes, at least 7 minutes, or at least 8 minutes. In some embodiments, the rinse lasts for a length of time of 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, or 3 minutes, to 5 minutes, 8 minutes, or 10 minutes, e.g., a rinse 10 seconds to 8 minutes, 10 seconds to 5 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 20 seconds to 8 minutes, 20 seconds to 5 minutes, 20 seconds to 3 minutes, 20 seconds to 2 minutes, 20 seconds to 1 minute, 30 seconds to 8 minutes, 30 seconds to 5 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1.5 minutes, 30 seconds to 1 minute, 45 seconds to 8 minutes, 45 seconds to 5 minutes, 45 seconds to 3 minutes, 45 seconds to 2 minutes, 15 seconds to 1 minute, 1 minute to 8 minutes, 1 minute to 5 minutes, 1 minute to 3 minutes or 1 minute to 1.5 minutes length of time.

In some embodiments, (b) deslagging is performed when the sediment accumulated in the drum reaches the amount to be discharged. For the specific material to be clarified, in some embodiments, the "amount to be discharged" of the sediment accumulated in the drum can be set as needed, and/or, the time length of the slagging cycle can be set as needed, so as to The rinsing effect of the method described in this paper is further optimized for specific materials to be clarified.

In some embodiments, step (c) is performed when (b) deslagging is complete, ie, switching the feed material from the cleaning medium to the material to be clarified.

In some embodiments, steps (a) to (c) are repeated at least two times, e.g., at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times Second-rate. In some embodiments, steps (a) through (c) are repeated until the desired clarification is complete. In some embodiments, the steps (a) to (c) are repeated from the beginning of the clarification treatment, that is, the first slagging during the clarification treatment is to perform the operations of the steps (a) to (c). In some embodiments, steps (a) to (c) are repeated throughout the clarification process, that is, the operations of steps (a) to (c) are performed every time the slag is discharged during the clarification process. In some embodiments, steps (a) to (c) can be set to repeat one or more start times and one or more end times in the entire clarification process as needed, that is, the steps (a) to (c) can be repeated as needed during the clarification process. The operations of steps (a) to (c) are carried out during two or more slag discharges.

In some embodiments, the washing medium can be selected from the group consisting of water (e.g., distilled water, purified water, water for injection, etc.), cell culture medium (e.g., fresh medium or spent medium), buffer (e.g., PBS), and any combination thereof.

In some embodiments, the feed rate of the material is in the range of 10 to 12000L/h, for example, 20 to 10000L/h, 30 to 8000L/h, 40 to 5000L/h, 50 to 2000L/h, 50 to 1000L/h h, 50 to 200L/h, 60 to 180L/h, 70 to 150L/h, 80 to 120L/h or 90 to 120L/h.

In some embodiments where the material to be clarified is a cell culture, the cell culture contains viable cells at a density in the range of 5×10 ⁵ to 1×10 ⁸ cells/mL, for example, 1×10 ⁶ to 2.5× 10 ⁷ cells/mL or in the range of 1×10 ⁶ to 2×10 ⁷ cells/mL.

In some embodiments where the material to be clarified is a cell culture, the cell culture contains cells with a viable rate in the range of 5% to 95%, for example, 5% to 90%, 5% to 80%, 5% to In the range of 70%, 5% to 60%, 5% to 50%, 5% to 40% or 5% to 30%, based on 100% of the total number of cells contained in the cell culture.

In some embodiments where the material to be clarified is a cell culture, the turbidity of the cell culture is greater than 1000 NTU, for example, greater than 1200 NTU, greater than 1500 NTU, greater than 1800 NTU, greater than 2000 NTU, greater than 2500 NTU, greater than 3000 NTU, greater than 3500 NTU, greater than 5000NTU, greater than 6000NTU, greater than 7000NTU, greater than 8000NTU, greater than 9000NTU, greater than 10000NTU, greater than 12000NTU, greater than 14000NTU, greater than 16000NTU, greater than 18000NTU, or greater than 20000NTU.

In some embodiments, repeating steps (a) to (c) is performed manually or automatically. For example, operation automation can be achieved by improving the continuous flow centrifuge equipment (eg, adding a cleaning medium feed interface) and operating system (eg, setting a program to repeat steps (a) to (c) as needed).

In some embodiments, repeating steps (a) to (c) can be performed manually.

The methods provided herein for treating material to be clarified (e.g., cell cultures) can significantly reduce or avoid product loss due to slagging (especially frequent slagging due to high solids content), increase product recovery, and reduce the yield per gram of product production cost.

In another aspect, this paper provides a method for separating and purifying cell cultures, comprising:

(i) clarifying the cell culture using the method for clarifying the material to be clarified as described herein to obtain a clarified cell culture supernatant; and

(ii) further separating and purifying the clarified cell culture supernatant obtained in step (i).

In some embodiments, the further separation and purification includes performing one or more deep filtration on the clarified cell culture supernatant.

As used herein, the term "depth filtration" refers to the operation of trapping impurity particles (rather than surface trapping) from the mobile phase using porous media. Depth filtration generally uses a filter bed with a thicker medium layer (such as sand layer, diatomaceous earth, etc.) as the filter medium. The impurity particles whose size is smaller than the pore diameter of the medium cannot form a filter cake on the surface of the filter medium. These impurity particles can enter the interior of the medium, approach the wall of the pore by inertia and diffusion, and deposit under the action of static electricity and surface force, thus interacting with the fluid separate.

In another aspect, provided herein is a continuous flow centrifuge for clarifying materials to be clarified, comprising:

a material to be clarified feed interface, which is in fluid communication with the material to be clarified storage container; and

One or more cleaning medium feed ports, each in fluid communication with the cleaning medium storage container.

In some embodiments, a continuous flow centrifuge described herein is used in the methods of clarification described herein.

In another aspect, the present invention provides a system for clarifying materials to be clarified, comprising:

(A) a continuous flow centrifuge having:

The material to be clarified is fed into the interface, which is in fluid communication with the material to be clarified storage container;

one or more cleaning medium feed ports, each in fluid communication with the cleaning medium storage container;

(B) one or more processors; and

(C) memory having stored thereon instructions executable by said one or more processors;

Wherein, the instructions executable by the one or more processors are configured to perform the following operations: repeat steps (i) to (iii):

(i) Before slag discharge, stop the feeding of the material to be clarified, and feed the cleaning medium from the cleaning medium storage container to the continuous flow centrifuge, thereby switching the fed material from the material to be clarified to the cleaning medium;

(ii) deslagging; and

(iii) After the slag is discharged, stop the feeding of the cleaning medium, and feed the material to be clarified from the storage container of the material to be clarified to the continuous flow centrifuge, thereby switching the fed material from the cleaning medium to the material to be clarified;

Wherein, in the clarification process of the material to be clarified, steps (i) to (iii) are repeated at least twice.

In some embodiments, the material to be clarified is a solid-liquid mixture.

In some embodiments, the material to be clarified is cell culture.

In some embodiments, the cell culture is a culture suspension of cells selected from the group consisting of eukaryotic cells, prokaryotic cells, and any combination thereof. In some embodiments, the cell culture is a culture suspension of cells selected from the group consisting of animal cells, plant cells, microbial cells, and any combination thereof. Suitable animal cells can include mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), avian cells (e.g., chicken, duck, and goose) or insect cells. Suitable mammalian cells may include CHO cells, HEK293 cells, lymphocytes, or myeloma cells (eg, NSO cells). Suitable microbial cells include bacterial cells or fungal cells (eg, yeast). Suitable yeast include Pichia methanolica, Pichia pastoris, Saccharomyces cerevisiae, or common baker's yeast. Suitable insect cells include Drosophila Schnieder S2 cells and Sf9 cells. In some exemplary embodiments, the cell culture may be selected from Culture suspensions of the following cells: CHO cells, NSO cells, cells of human origin (eg, primary human cells), insect cells, and other animal or microbial cells.

In some embodiments, the centrifugal force of the clarification process is in the range of 4000G to 20000G, eg, in the range of 4000G to 13000G, 5000G to 12000G, 6000G to 11000G, or 7000G to 10000G.

In some embodiments, the centrifugal speed of the clarification treatment is in the range of 4000 rpm to 13000 rpm, for example, 5000 rpm to 10000 rpm.

In some embodiments, the continuous flow centrifuge is a disc continuous flow centrifuge.

Herein, the term "repeating steps (i) to (iii)" as used means that during the whole clarification process (for example, from the clarification process to the clarification process is completed to obtain the desired amount of product), at least Two operations of steps (i) to (iii). It should be understood that the start of the next step (i) to (iii) is not immediately after the end of the operation of the previous step (i) to (iii). At least a period of clarifying operation is included between the previous step (iii) and the subsequent step (i). In some embodiments, the clarification operation for a period of time included between the previous step (iii) and the subsequent step (i) starts from the end of the previous step (iii) until the sediment accumulated in the drum reaches a level that needs to be discharged. When the amount is finished, the subsequent steps (i) to (iii) can be performed as required or only the slag discharge operation can be performed.

In some embodiments, steps (i) to (iii) are performed in the following order: (i)→(ii)→(iii).

In some embodiments, in step (i), at least 10 seconds before slagging, for example, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 1.5 minutes, at least 2 seconds before slagging Minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes or at least 8 minutes, the feed material is switched from the material to be clarified to the cleaning medium. In some embodiments, in step (i), at the moment of 10 seconds to 10 minutes before slag discharge, for example, 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes before slag discharge or 3 minutes, to 5 minutes, 8 minutes or 10 minutes, for example, 10 seconds to 8 minutes, 10 seconds to 5 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 20 seconds to 8 minutes, 20 seconds to 5 minutes, 20 seconds to 3 minutes, 20 seconds to 2 minutes, 20 seconds to 1 minute, 30 seconds to 8 minutes, 30 seconds to 5 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1.5 minutes, 30 seconds to 1 minute, 45 seconds to 8 minutes, 45 seconds to 5 minutes, 45 seconds to 3 minutes, 45 seconds to 2 minutes, 15 seconds to 1 minute, 1 minute Between 8 minutes, 1 minute to 5 minutes, 1 minute to 3 minutes or 1 minute to 1.5 minutes, the feed material is switched from the material to be clarified to the cleaning medium.

In some embodiments, in step (i), the feed material is switched from the material to be clarified to the cleaning medium before the slag discharge, so that the sediment is rinsed with the cleaning medium. In some embodiments, the rinse lasts at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 1.5 minutes, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least A period of time of 6 minutes, at least 7 minutes, or at least 8 minutes. In some embodiments, the rinse lasts for a length of time of 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, or 3 minutes, to 5 minutes, 8 minutes, or 10 minutes, e.g., a rinse 10 seconds to 8 minutes, 10 seconds to 5 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 20 seconds to 8 minutes, 20 seconds to 5 minutes, 20 seconds to 3 minutes, 20 seconds to 2 minutes, 20 seconds to 1 minute, 30 seconds to 8 minutes, 30 seconds to 5 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1.5 minutes, 30 seconds to 1 minute, 45 seconds to 8 minutes, 45 seconds to 5 minutes, 45 seconds to 3 minutes, 45 seconds to 2 minutes, 15 seconds to 1 minute, 1 minute to 8 minutes, 1 minute to 5 minutes, 1 minute to 3 minutes or 1 minute to 1.5 minutes length of time.

In some embodiments, (ii) deslagging is performed when the sediment accumulated in the drum reaches the amount to be discharged. For the specific material to be clarified, in some embodiments, the "amount to be discharged" of the sediment accumulated in the drum can be set as needed, and/or, the time length of the slagging cycle can be set as needed, so as to The rinsing effect of the method described in this paper is further optimized for specific materials to be clarified.

In some embodiments, step (iii) is performed upon completion of (ii) deslagging, switching the feed material from the cleaning medium to the material to be clarified.

In some embodiments, steps (i) to (iii) are repeated at least two times, e.g., at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times Second-rate. In some embodiments, steps (i) through (iii) are repeated until the desired clarification is complete. In some embodiments, the steps (i) to (iii) are repeated from the beginning of the clarification treatment, that is, the first slagging during the clarification treatment is to perform the operations of the steps (i) to (iii). In some embodiments, steps (i) to (iii) are repeated throughout the clarification process, that is, the operations of steps (i) to (iii) are performed every time slagging is discharged during the clarification process. In some embodiments, steps (i) to (iii) can be set to repeat one or more start moments and one or more end moments in the entire clarification process as needed, that is, one or more end moments can be set during the clarification process as needed The operations of steps (i) to (iii) are carried out during two or more slag discharges.

In some embodiments, the washing medium can be selected from the group consisting of water (e.g., distilled water, purified water, water for injection, etc.), cell culture medium (e.g., fresh medium or spent medium), buffer (e.g., PBS), and any combination thereof.

In some embodiments, the feed rate of the material is in the range of 10 to 12000L/h, for example, 20 to 10000L/h, 30 to 8000L/h, 40 to 5000L/h, 50 to 2000L/h, 50 to 1000L/h h, 50 to 200L/h, 60 to 180L/h, 70 to 150L/h, 80 to 120L/h or 90 to 120L/h.

In some embodiments where the material to be clarified is a cell culture, the cell culture contains viable cells at a density in the range of 5×10 ⁵ to 1×10 ⁸ cells/mL, for example, 1×10 ⁶ to 2.5× 10 ⁷ cells/mL or in the range of 1×10 ⁶ to 2×10 ⁷ cells/mL.

In some embodiments where the material to be clarified is a cell culture, the cell culture contains cells with a viable rate in the range of 5% to 95%, for example, 5% to 90%, 5% to 80%, 5% to In the range of 70%, 5% to 60%, 5% to 50%, 5% to 40% or 5% to 30%, based on 100% of the total number of cells contained in the cell culture.

In some embodiments where the material to be clarified is a cell culture, the turbidity of the cell culture is greater than 1000 NTU, for example, greater than 1200 NTU, greater than 1500 NTU, greater than 1800 NTU, greater than 2000 NTU, greater than 2500 NTU, greater than 3000 NTU, greater than 3500 NTU, greater than 5000NTU, greater than 6000NTU, greater than 7000NTU, greater than 8000NTU, greater than 9000NTU, greater than 10000NTU, greater than 12000NTU, greater than 14000NTU, greater than 16000NTU, greater than 18000NTU, or greater than 20000NTU.

In another aspect, provided herein is a computer readable medium having stored thereon instructions executable by a processor to perform the method described herein for clarifying a substance to be clarified in a continuous flow centrifuge.

In some embodiments, the computer readable medium has stored thereon instructions executable by a processor to automate the method of clarifying a substance to be clarified as described herein in a continuous flow centrifuge.

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are all within the scope of the present invention.

The following examples all use an instrument: a disc-type continuous flow centrifuge, model MBPX 404-SGP-31, from AlfaLaval Company.

Example 1

The parameters of the continuous flow centrifugation used in this embodiment are: the feed rate is 90 L/h, and the centrifugal force is set at 9500 g. The feed liquid used is the culture liquid of CHO cells, the viable cell density is 14.01×10 ⁶ cells/mL, the cell viability is 73.3%, the solid content is 7.9%, and the turbidity is 2940 NTU. The cell culture expression of the target protein was 1.022g/L.

Three equal-volume cell cultures were taken, one was used as a control experiment, and the remaining two were subjected to feed material switching operations before continuous flow centrifugation (see Figure 1). The specific operation is: close the pipe clamp on the cleaning medium pipeline, open the pipe clamp connected to the fermentation liquid storage tank, after centrifuging for a period of time, close the pipe clamp connected to the fermentation liquid storage tank, and open the cleaning medium (buffer) Tube clamps on the tubing to allow the cleaning medium to pass through the centrifuge in continuous flow. Rinse for 3 minutes before deslagging for the first part, and 8 minutes for 8 minutes before deslagging for the second part.

The turbidity, lactate dehydrogenase (LDH) and protein content of the product after continuous flow centrifugation were measured respectively. The results are shown in Table 1. Compared with the control, under the same loading conditions, the amount of protein collected increased from 56.3g to 61.9g (3 minutes, compared with the control recovery, an increase of 9.95%) and 64.1g (8 minutes, compared with Compared with the recovery ratio, it has increased by 13.85%). The systematic error is about 3%.

Using the methods described herein for processing cell cultures increases protein recovery from the centrate. Because this method increases the centrifugate volume appropriately, the turbidity and LDH concentration of the centrifugate are reduced.

Table 1. Embodiment 1 handles product detection result

Example 2

The parameters of the continuous flow centrifugation used in this embodiment are: the feed rate is 120 L/h, and the centrifugal force is set to 9000 g. The feed liquid used is the culture liquid of CHO cells, the viable cell density is 1.35×10 ⁶ cells/mL, the cell viability is 3.5%, the solid content is 12.5%, and the turbidity is 1508 NTU. The cell culture expression level of the target protein was 1.14g/L.

Three equal-volume cell cultures were taken, one was used as a control experiment, and the remaining two were subjected to feed material switching before continuous flow centrifugation. The specific operation is: close the pipe clamp on the cleaning medium pipeline, open the pipe clamp connected to the fermentation liquid storage tank, after centrifuging for a period of time, close the pipe clamp connected to the fermentation liquid storage tank, and open the cleaning medium (buffer) Tube clamps on the tubing to allow the cleaning medium to pass through the centrifuge in continuous flow. Rinse for 3 minutes before deslagging of the first part, and 5 minutes of rinsing before deslagging of the second part.

The turbidity, LDH and protein content of the product after continuous flow centrifugation were measured respectively. See Table 2 for specific data. Compared with the control, under the same loading conditions, the amount of protein collected increased from 14.7g to 19.4g (3 minutes, compared with the control recovery, an increase of 31.97%) and 18.7g (5 minutes, compared with Compared with the recovery ratio, it has increased by 27.21%). The systematic error is about 3%.

Using the methods described herein for processing cell cultures increases protein recovery from the centrate. Because this method increases the centrifugate volume appropriately, the turbidity and LDH concentration of the centrifugate are reduced.

In addition, the effect of the method of processing cell culture described in this paper on the subsequent depth filtration was also investigated, and the results are shown in Figure 2 and Figure 3 . The results show that the methods of processing cell cultures described herein have no negative impact on the capacity of the depth filter.

Table 2. Embodiment 2 handles product detection result

Example 3

The parameters of the continuous flow centrifugation used in this embodiment are: the feed rate is 100 L/h, and the centrifugal force is set to 9500 g. The culture medium of CHO cells was used, the viable cell density was 19.21×10 ⁶ cells/mL, the cell viability was 37.7%, the solid content was 30%, and the turbidity was 183750 NTU. The cell culture expression of the target protein was 0.239g/L.

Three equal-volume cell cultures were taken, one was used as a control experiment, and the remaining three were subjected to feed material switching before continuous flow centrifugation. The specific operation is: close the pipe clamp on the cleaning medium pipeline, open the pipe clamp connected to the fermentation liquid storage tank, after centrifuging for a period of time, close the pipe clamp connected to the fermentation liquid storage tank, and open the cleaning medium (buffer) Tube clamps on the tubing to allow the cleaning medium to pass through the centrifuge in continuous flow. Rinse for 1 minute before deslagging of the first part, rinse for 2 minutes before deslagging of the second part, and rinse for 3 minutes before deslagging of the third part.

The turbidity, LDH and protein content of the product after continuous flow centrifugation were measured respectively. See Table 3 for specific data. Compared with the control, under the same loading conditions, the amount of protein collected increased from 0.86g to 1.30g (1 minute, compared with the control recovery, increased by 51.16%), 1.30g (2 minutes, compared with Compared with the control recovery, it increased by 51.16%) and 1.38g (3 minutes, compared with the control recovery, it increased by 60.47%). The systematic error is about 3%.

Using the methods described herein for processing cell cultures increases protein recovery from the centrate. Because this method increases the centrifugate volume appropriately, the turbidity and LDH concentration of the centrifugate are reduced.

In addition, the effect of the method for treating cell culture described herein on the subsequent depth filtration was also investigated, and the results are shown in Fig. 4 and Fig. 5 . The results show that the methods of processing cell cultures described herein have no negative impact on the capacity of the depth filter.

Table 3. Embodiment 3 handles product detection result

As can be seen from the foregoing examples, the method for treating cell cultures described herein can significantly reduce or avoid product loss caused by slagging (especially frequent slagging due to high solid content), improves protein recovery, and reduces Production cost per gram of product. Also, treatment of cell cultures using the methods described herein has no negative impact on subsequent depth filtration mass loading.

Further, the higher the solid content of the cell culture treated by the method described herein, the greater the increase in recovery can be obtained. As shown in Figure 6, in the 3 minutes before slagging, compared with the recovery obtained in Example 1 (solid content 7.9%), the recovery of 9.95% was improved with respect to the contrast, and embodiment 2 (solid content 12.5% %), the amount of recovery obtained has increased by 31.97% relative to the contrast, while the amount of recovery obtained in Example 3 (30% solid content) has increased the recovery by 60.47% relative to the contrast.

The research results on the length of leaching before slag discharge show that when the approximately time of leaching before slag discharge in Example 1 is increased from 3 to 8 minutes, and the approximately time of leaching before slag discharge in Example 2 is increased from 3 to 5 minutes, In Example 3, when the rinsing time before slag discharge was increased from 1 to 3 minutes, the recovery amount did not increase significantly, indicating that the 1-minute rinsing time was basically sufficient and could be further reduced, for example, to half a minute or 15 seconds for rinsing time.

The above embodiments are only preferred exemplary embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A method for clarifying material to be clarified, which comprises repeating steps (a) to (c) in the process of using a continuous flow centrifuge to clarify material to be clarified: (a) Before slag discharge, switch the feed material from the material to be clarified to the cleaning medium; (b) deslagging; and (c) After slag discharge, switch the feed material from the cleaning medium to the material to be clarified; Wherein, steps (a) to (c) are repeated at least twice. 2. The method according to claim 1, wherein the material to be clarified is a solid-liquid mixture; specifically, the material to be clarified is a cell culture; more specifically, the cell culture is selected from A culture suspension of cells from the group: animal cells, plant cells, microbial cells, and any combination thereof; more specifically, the animal cells include insect cells. 3. The method according to claim 1, wherein the continuous flow centrifuge is a disc type continuous flow centrifuge; optionally, repeating steps (a) to (c) is performed manually or automatically. 4. The method of claim 1, wherein the washing medium is selected from the group consisting of water, cell culture medium, buffer, and any combination thereof. 5. The method according to claim 1, wherein in step (a), at least 10 seconds before slag discharge, the feed material is switched from the material to be clarified to the cleaning medium. 6. The method of claim 1, wherein the centrifugal force of the clarification treatment is in the range of 4000G to 20000G. 7. The method as claimed in claim 1, wherein the centrifugal rotation speed of the clarification treatment is in the range of 4000rpm to 13000rpm. 8. The method as claimed in claim 1, wherein the feed rate of the material is in the range of 10 to 12000 L/h. 9. A method for separating and purifying cell cultures, comprising: (i) using the method as claimed in claim 1 to carry out clarification treatment to the cell culture to obtain a clarified cell culture supernatant; and (ii) further separating and purifying the clarified cell culture supernatant obtained in step (i); Specifically, the further separation and purification includes performing one or more deep filtrations on the clarified cell culture supernatant obtained in step (i). 10. A system for clarifying materials to be clarified, comprising: (A) a continuous flow centrifuge having: The material to be clarified is fed into the interface, which is in fluid communication with the material to be clarified storage container; one or more cleaning medium feed ports, each in fluid communication with the cleaning medium storage container; (B) one or more processors; and (C) memory having stored thereon instructions executable by said one or more processors; Wherein, the instructions executable by the one or more processors are configured to perform the following operations: repeat steps (i) to (iii): (i) Before slag discharge, stop the feeding of the material to be clarified, and feed the cleaning medium from the cleaning medium storage container to the continuous flow centrifuge, thereby switching the fed material from the material to be clarified to the cleaning medium; (ii) deslagging; and (iii) After the slag is discharged, stop the feeding of the cleaning medium, and feed the material to be clarified from the storage container of the material to be clarified to the continuous flow centrifuge, thereby switching the fed material from the cleaning medium to the material to be clarified; Wherein, in the process of clarifying the material to be clarified, steps (i) to (iii) are repeated at least twice; Specifically, the material to be clarified is a solid-liquid mixture; more specifically, the material to be clarified is cell culture.

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