Discussion On The Design Of Single Antigen Solution Workshop

Monoclonal antibodies, monoclonal antibodies, are produced by injecting antigens into host animals to initiate the body’s immune response. Monoclonal antibody drugs play a prominent role in the prevention and treatment of viral diseases, autoimmune diseases, malignant tumors and severe infectious diseases, and have also performed well in the prevention and treatment of COVID-19. Monoclonal antibodies in my country are expressed in mammalian cells, mainly Chinese hamster ovary cell line (CHO). In addition to considering the recovery rate and purity during the antibody purification process, it is also necessary to effectively inactivate and clear the virus. The original solution produced in the single antigen solution workshop is not suitable for thermal sterilization process, and the non-terminal sterilization production process of aseptic operation is usually selected, which has higher requirements on the control of workshop environment and microbial contamination level. This article will discuss the design method of the single antigen solution workshop from the production process and layout design of the single antigen solution workshop.

Part 1 About The Risk Of Monoclonal Antibody Virus

CHO cell lines are the workhorses for the manufacture of many therapeutic proteins. The biosafety level of CHO cells is level 1. It has been confirmed that CHO itself has endogenous retroviruses, but this virus is not pathogenic to humans. From the perspective of drug quality and safety, follow the “Guidelines for Quality Control Technology of Human Monoclonal Antibodies” published by SFDA, and add virus removal and inactivation methods.

In addition, based on the culture and fermentation of mammalian cells, product contamination by viruses is one of the main production risks to consider. Other potential sources of virus contamination may include: the use of contaminated master cell seed banks or working cell seed banks; operators may bring in viruses and contaminate the process; cell lines may contain viable viruses from the previous stage. Because mammalian cells provide suitable host cells for the viral contaminants introduced, viral loads may scale up with cell culture, which in turn may increase the overall risk of the production process.

Part 2 Design Concept Of Single Antigen Solution Workshop

In order to minimize the risk of virus contamination or reduce the risk of carrying pollutants to the final product, and minimize the microbial load in the production environment, all process steps with virus risks should be completed in their own independent production rooms with a one-way flow design. Unidirectional flow is achieved by dividing the workshop into supply-side corridors (shared supply corridors, the flow of people into the process rooms) and waste corridors (common corridors for collecting potentially contaminated materials between the various production processes). Materials and waste collected in waste corridors need to be adequately treated before they can be transferred to the supply side without carrying pollutants. The material can only enter the clean area from the return corridor after being decontaminated by the double-door sterilizer.

Part 3 Design Points Of The Core Content Of The Production Process

The production process of single antigen solution is mainly summarized into two stages: upstream cell culture, fermentation and downstream purification. The cell seeds are amplified through shake flasks, WAVE reactors, primary, secondary, and tertiary seed cultures, and then enter the fermenter for fermentation. After the fermentation is finished, it goes downstream through the centrifuge for purification. The feed liquid after centrifugation becomes the original liquid after chromatography and ultrafiltration. There are also auxiliary systems in the raw solution production process: culture medium preparation, buffer preparation and CIP and other systems, as shown in Figure 1 and Figure 2. specific

The specific process flow is described as follows:

(1) Cell Recovery

Cell resuscitation and cryopreservation pay attention to “slow freezing and quick thawing”. When resuscitating, the cell freezing solution frozen in liquid nitrogen or -80°C must be quickly thawed to 37°C, so that the ice crystals in extracellular cryopreservation can be melted quickly. Avoid ice crystals entering cells to form recrystallization when they melt slowly, causing damage to cells.

(2) Subculture Of Cells

Suspension cell culture, suspension culture refers to the culture technology in which cells grow and reproduce in a suspended state in the culture medium, and can be continuously cultured and harvested continuously. Usually, when the phenol red indicator in the culture medium turns yellow, suck out or pour out the cell suspension into a centrifuge tube on an ultra-clean bench after ultraviolet sterilization, discard the old culture medium, add new culture medium, and pipette fully to Scatter cells. Take an appropriate amount of cell suspension and transfer it to a cell culture bottle, or directly take an appropriate amount of cell suspension into a new culture bottle, and then add a certain amount of fresh complete culture medium for passage. For some fragile cells, use the natural sedimentation method to settle the cells. The old culture medium above was added to the new complete culture medium and blown off for subculture.

Advantages: It is easier to expand the culture, occupies a small area, the culture process is simple, and the cell proliferation is fast.

Disadvantages: Only a few cells are suitable for suspension culture.

Adhesive cell culture, culture of anchorage-dependent cells is initially cultured in a spinner bottle system, which is generally used for the transitional stage from small-scale culture to large-scale culture. When the cell coverage rate in the bottom of the bottle reaches 70%~80%, discard the old cell culture medium on the ultra-clean bench sterilized by ultraviolet light, add preheated fresh complete culture medium to stop the digestion, and blow gently to make the cells If it falls off and disperses evenly, directly transfer an appropriate amount of cell suspension to a new culture bottle or centrifuge to remove the supernatant, add an appropriate amount of fresh complete culture medium to blow and resuspend the cells, then transfer an appropriate amount of cell suspension to a new culture bottle, and then add a certain amount of Measure the complete culture solution, shake the bottom of the bottle and culture the cell solution.

Advantages: simple structure, low investment, mature technology, good repeatability, enlargement only needs to simply increase the number of spinner bottles.

Disadvantages: high labor intensity, large floor space, small surface area per unit volume for cell growth, low cell growth density, and limited monitoring and monitoring of environmental conditions during culture.

(3) Cell Culture

Most animal cell culture methods in my country adopt microcarrier culture. Microcarrier culture is to add microcarriers to suspend them in the growth solution, so that cells that cannot be suspended can be attached to the surface of microcarriers for growth and reproduction. This culture method takes into account the advantages of monolayer culture and suspension culture, and is usually divided into continuous perfusion and fed-batch culture.

Perfusion culture, during the cell culture process, the culture medium is continuously perfused into the culture vessel, and at the same time flows out at the same flow rate. The advantage of the perfusion process is to maintain a low concentration of the culture solution, which is conducive to the formation of the target product. At the same time, the continuous outflow of the culture solution can bring out cell debris and by-products, reduce various enzymes released by cell metabolism, and improve equipment utilization and efficiency. output per unit time.

Fed-batch culture, in the process of cell culture, a stirring system is used for continuous suspension culture. The volume of the culture medium for cell inoculation is generally about 1/2 of the final volume. During the process, add nutrients or culture fluid to the incubator according to the consumption and demand of the cells for nutrients, supplement carbon sources, nitrogen sources, trace Elements, etc., to promote the continuous growth of cells and improve the yield and quality of target products.

(4) Monoclonal Antibody Fermentation Broth Separation Process

The clarification of cell harvesting fluid is the first step in starting the downstream process in biopharmaceuticals. It provides impurity-free feed liquid for downstream purification. The active ingredients of biological products are mostly biologically active macromolecules such as proteins and polysaccharides. Therefore, commonly used in production Separation methods mainly include cell crushing technology, precipitation technology, centrifugation technology, filtration technology and chromatographic separation technology, usually using a gentle multi-stage separation method. If only one filtration technology is used, two-stage filtration is usually used: first use a filter medium with a larger pore size to remove cells or cell debris; then use a filter medium with a tight pore size to remove colloidal impurities. For high-density cell harvesting fluid, flocculation and depth filtration can be selected. To avoid premature clogging of the downstream chromatographic purification column and reduce bioburden, the clarification process will be combined with 0.45 μm and 0.2 μm terminal sterilization filtration at the end.

(5) Downstream Purification Of Monoclonal Antibody

The production of monoclonal antibody drugs usually includes four modules: Upstream process, Downstream process, Analysis and Formulation [3]. The downstream is purification. After the downstream obtains the cell culture clarified liquid from the upstream, it is purified and produced to obtain the drug stock solution. The downstream purification process relies on three types of chromatography columns and two types of membrane filters. The three kinds of chromatography columns generally refer to rProtein A (recombinant protein A) affinity chromatography column, cation exchange chromatography column (cation exchange chromatography, CEX) and anion exchange chromatography column (anion exchange chromatography, AEX). The two membranes refer to virus removal membrane (viral filter) and ultrafiltration membrane (ultrafiltration/diafiltration, UF/DF).

(6) Virus Removal

Generally, the most effective virus removal/inactivation methods in the monoclonal antibody production process are: low pH value (pH value = 3.2, keep for 30-60 min), heating (55-65°C for 1 h), S/D (solvent/ detergent), membrane filtration (such as nanofiltration), etc. In antibody process design, a typical design is to add virus inactivation process after Protien A affinity chromatography. Incubate the combined eluate after affinity chromatography at low pH, use strong acid buffer to lower the pH value of the eluate (pH = 3.2 for 30-60 min), and then quickly use strong alkali buffer to lower the pH value. The value was adjusted to 5.5 to facilitate the next step of purification. Minimize the time the protein is exposed to low pH environments. (There is also the use of phosphate buffer instead of strong acid/strong alkali HCL/NaOH for pH adjustment, but the disadvantage of this method is that it will lead to a larger volume of the next purification process, and it may be considered to increase the ultrafiltration concentration process. less; strong acids and bases may increase the risk of protein precipitation during the process).

For low pH incubation, a disposable mixing system (50-1 000 L) should be used, which has low shear force mixing, online pH value detection, and does not need to be equipped with CIP\SIP, which is conducive to quality assurance and verification. One drive unit can be used with multiple One-time mixing system greatly reduces the investment cost of fixed equipment and saves energy. The affinity chromatography and ion exchange chromatography used for the purification of fermentation broth also have a certain ability to remove viruses. In the monoclonal antibody process, nanofiltration membrane filtration can be used as the final step of virus removal, and then the single antigen solution (immunoglobulin) can be obtained by sterile filtration.

Part 4 Engineering Example

According to the characteristics of single antigen solution production and key points of engineering design, combined with engineering examples, the plane design and air-conditioning purification system of the workshop are described and analyzed. Figure 3 is the floor plan of a single antigen solution workshop.

1 Graphic Design

The single antigen solution production workshop needs to be designed and partitioned according to the production process and preparation process, and is divided into two main production areas, the upstream area and the downstream area. Upstream zone: cell recovery, cell expansion, cell culture, harvesting; downstream zone: stock solution purification, ultrafiltration and filtration to remove viruses. According to production needs, two auxiliary functional areas need to be set up: medium preparation area and storage area, buffer preparation and storage area. Separate the entry and exit passages for personnel to avoid pollution to the clean area when personnel exit. The production personnel in the workshop enter the production purification area through the clean changing facilities, and enter each functional room through the supply side corridor [3]. The inlets for clean materials and non-clean materials are set up, and raw and auxiliary materials enter the production purification area through logistics channels and air locks. The used utensils and waste generated in the upstream and downstream areas enter the cleaning area through the waste treatment corridor for cleaning and sterilization. Set up an independent waste outlet to ensure that waste will not cause secondary pollution to the production purification area. The plan layout adheres to the principle of one-way flow, dividing the workshop into the supply side and the waste disposal corridor. The overall space layout is carried out according to the concept of one-way flow of people and goods, so as to reduce the risk of microbial contamination and effectively control biological load and virus contamination.

2 Air Conditioning Purification System

The air-conditioning system is divided according to the zoning principle of the production area and the preparation area to prevent cross-contamination. As an auxiliary post, the two areas of medium preparation and buffer preparation are relatively independent from the upstream and downstream production areas. Separate air-conditioning systems are set up for human purification facilities, supply-side corridors, cell banks, upstream areas, downstream areas, and waste disposal corridors. In the downstream area, the purification process, the ultrafiltration of the raw liquid and the filtration of virus removal are all set in an air-conditioned partition.

According to GMP requirements, the cleanliness level of the upstream area and the downstream area are both Class C. Considering the process characteristics and product added value of monoclonal antibody products, the air-conditioning system chooses the form of dual-machine hot standby to ensure the production environment of the original solution. The purification air-conditioning system is equipped with HEPA at the terminal of the air supply system, and adopts the air-conditioning mode of fixed supply and variable discharge. The number of air changes should not be less than 40 times/h. Control the pressure gradient to ensure that the air flow flows from the low pollution risk area to the high pollution risk area, that is, from the upstream area to the downstream area. Highly polluted rooms such as virus removal and inactivation rooms should be kept in a relatively negative pressure state.

The purification air-conditioning system of the production workshop adopts a constant air volume and fixed fresh air ratio primary return air system. The air is sent to the room through three-stage filtration of primary effect, medium effect and high efficiency filters. Monitor the key environmental parameters related to the clean area, such as temperature and humidity, pressure difference, wind speed, etc. When the ambient temperature, humidity and pressure difference exceed the set range, the system will sound and light alarm and record, and the system will archive the data at the same time [4]. The pressure gradient is reasonably determined for each room in the clean area, and the airflow flows from the clean corridor to the dirt corridor.

Part 5 Selection Of Equipment Required For Single Antigen Solution Purification Process

1 Centrifuge

The centrifuge uses the high-speed rotation of the centrifuge rotor to generate a strong centrifugal force, forcing the suspended particles in the liquid to overcome the diffusion effect and accelerate their sedimentation speed. Suspended particles here often refer to suspended cells, organelles, viruses, and biological macromolecules. Most industries use Disk-stack centrifugation to remove cells and their debris. Disk-stack centrifugation enables continuous centrifugation, increasing throughput and reducing processing time. During the development of the centrifuge process, there are many parameters that need to be optimized, such as feeding speed, centrifuge speed, frequency of filter cake layer removal, etc. These parameters will affect the stability of cells, processing time, and antibody yield. By optimizing these parameters, various demands can be reasonably balanced.

2 Filters

Using porous material as the medium, with the help of the sieving properties of the filter medium, and generally the surface of the medium is under the action of external force, the technology of separating, purifying and enriching different components through the channels of the medium. Compared with other separation technologies, filtration separation technology is widely used, simple process, high efficiency and energy saving. When the particle content in the stock solution is high, depth filtration is often selected. Depth filter media typically consist of a bed of cellulose or polypropylene fibers, a filter medium such as diatomaceous earth, and a binder to create a smooth interface. Because centrifugation cannot completely remove cells and their debris, and the cost of tangential flow is too high, depth filtration is usually used after centrifugation, and can also be used alone for clarification of cell fermentation broth. Since the pore size of the depth filter medium is not uniform, a sterile filtration process will follow after the depth filter to facilitate the purification of the chromatographic column.

3 Resin Column

Affinity chromatography column rProtein A, cation exchange chromatography column (CEX), anion exchange chromatography column (AEX). The first chromatography column is an affinity chromatography column rProtein A. Chromatographic methods for biomacromolecules are mainly divided into affinity, ion exchange, hydrophobic interaction and size exclusion. Affinity chromatography uses the ligand on the medium to specifically bind to the target protein molecule, but does not adsorb other proteins and impurities, so the impurities flow out from the chromatography column, and the adsorbed target biomolecules are separated by changing the eluent. Under certain conditions, the separated substance and the ligand can be desorbed to achieve the purpose of separation and purification. It can remove more than 90% of HCP and DNA, and even one step can obtain antibody purity greater than 98%, and it also has obvious advantages in recovery rate, the recovery rate of affinity capture can reach 95%.

The second column is a cation exchange chromatography column (CEX) [5]. In the antibody purification process, CEX generally uses the adsorption mode. Cation exchange can remove impurities such as HCP, substances shed from affinity chromatography columns, and some high molecular weight polymers. Usually, the cation exchange chromatography column does not undertake the task of removing viruses, but it can assist in the removal of viruses.

The third column is an anion exchange chromatography column (AEX). Anion exchange can remove deoxyribonucleic acid, viruses and endotoxins, shedding substances, partial aggregates and acidic HCP in the process of affinity chromatography in the antibody purification process, and establish a guarantee for the further purification of the original solution.

4 Filter Membrane

In addition to virus nanofiltration membrane (Viral Filter) and ultrafiltration membrane (Ultrafiltration/Diafiltration, UF/DF).

Part 6 Conclusion

As an intermediate product of biological products, single antigen solution has high requirements for the design of the workshop. Based on the research on the production process of single antigen solution, this article comprehensively considers the biological safety, process characteristics and human flow organization in the production process, and proposes the design concept and process design points of one-way flow to discuss with you the single antigen solution workshop. design.