Production process of intravenous human immunoglobulin (pH4)
Production process of intravenous human immunoglobulin (pH4) and related Quality Control. Human plasma contains about 20 kinds of proteins, most of which are albumin (35-40 mg/mL), and the concentration of immunoglobulin is about 8-12 mg/mL. The immunoglobulins in the blood are divided into IgG, IgA, IgM, IgD and IgE. The first three account for about 75%, 15%, and 10% respectively. IgG can be divided into IgG1, IgG2, IgG3 and IgG4 according to different subtypes. The half-life of IgG of different subtypes is different. The half-life of IgG1, IgG2 and IgG4 is 3 to 4 weeks, and the half-life of IgG3 is only about 2 weeks.
The human immunoglobulin products used in clinical practice are IgG mixtures obtained after separation and purification from raw plasma (at least 1000 people are mixed and put into production). It has a broad-spectrum anti-virus, bacteria or other pathogen function, and can quickly increase the blood of the recipient. IgG level, enhance the body’s anti-infection ability and immune regulation function, can be used for congenital gamma globulin deficiency, repeated infections, anti-infection after organ transplantation, etc. Studies have shown that immunoglobulin products can also stimulate certain T cell subgroups, thereby promoting T cell proliferation .
According to different injection methods, immunoglobulin products are divided into intramuscular immunoglobulin G (IMIG) and intravenous immunoglobulin G (IVIG). IVIG requires higher quality attributes such as purity than IMIG. In the past 30 years, with the improvement of IVIG production technology and management methods, the output and safety of IVIG have been greatly improved, but throm-boembolic events (TEEs) related to IVIG infusion still occur from time to time. In 2010, Octa-pharma urgently recalled 31 batches of products in the US market due to higher than conventional TEEs in 5% of IVIG products produced by Octa-pharma . Therefore, ensuring the quality of IVIG products is not only a guarantee for patient safety, but also a guarantee for the interests of blood product manufacturers.
1 Development of IVIG production process
The method of separating IgG from plasma began with the ethanol precipitation method in the 1940s . At first, the process did not include additional purification steps, resulting in the purity of the separated IgG monomer only reaching 70% ~ 80%, which contained a large amount of IgG aggregates and IgA, IgM. This type of product exhibits the biological function of body fluid antibodies when injected intramuscularly, and has a normal physiological half-life. However, the disadvantages of intramuscular injection are the small injection dose, strong local irritation at the injection site, and slow absorption. Administration can cause life-threatening allergic reactions, and the probability of occurrence is high, because immunoglobulin aggregates cause non-specific spontaneous complement activation reactions.
Enzyme digestion or chemical modification treatment can reduce the spontaneous complement activation caused by intravenous administration of immunoglobulin (for example, treatment with β-propiolactone will cause protein alkylation and acylation, and reduce the Fc fragment complement activation function) , So there are chemically modified or digested intravenous immunoglobulin products. However, the modified immunoglobulin products will be quickly removed from the circulation by the reticuloendothelial system, shortening the circulating half-life, thus affecting the use of immunoglobulin. The first-generation intact immunoglobulin product is produced by using component II in cold ethanol precipitation. After the component II is dissolved, filtered, and subjected to mild acid treatment (usually incubated at pH 4, 37 ℃ for 20 hours), It is treated with a trace amount of pepsin to ensure that the anti-complement effect caused by the product is eliminated without affecting the integrity of IgG. In the 1970s, people have been able to isolate complete immunoglobulin products with a purity greater than 99% .
As people’s understanding of immunoglobulin product technology and product quality continues to deepen, modern immunoglobulin production processes are becoming more and more diversified, and products are continuously improved in terms of raw plasma control, purification, preparation, and quality control [4 ].
2 IVIG modern production technology and quality control
2.1 Control raw materials of raw plasma
The quality of plasma is directly related to the quality of immunoglobulin. To ensure the safety of blood products, China’s existing laws and regulations have put forward requirements from different levels. First, plasma donors should be screened at the plasma station, and the donor review should be performed after the window; second, at the blood product manufacturer, each piece of plasma used for production should be reviewed and tested, and the retested samples should be quarantined. Production can only be put into production after the period, and samples of the slurry should be inspected.
The entire process from the collection of raw plasma to the application of the product to the clinic requires a good communication and feedback mechanism with plasma collection stations, production companies and regulatory agencies to ensure that safety events can be properly identified and handled. As the main body responsible for product safety, blood product manufacturers have the responsibility to effectively supervise their plasma collection stations to ensure that the collection, inspection, storage and transportation of raw plasma comply with China’s laws and regulations, and conduct raw plasma processing in accordance with current laws and regulations. Re-examination and sample retention are the prerequisites for the production of safe blood products.
2.2 Stock solution production process
The traditional IVIG production process is: raw plasma is cryoprecipitated under controlled conditions at 2 to 3 ℃, and then the cryoprecipitated supernatant is taken, and the prothrombin complex, antithrombin or C1-inhibitor is separated by chromatographic adsorption After 3~4 ethanol precipitation steps, component II is separated, and the precipitate is treated with low pH/low concentration pepsin, or combined with cation exchange/anion exchange chromatography steps to reduce or remove impurities. In this process, the content of IVIG produced in component II is 3~4g/L. In order to increase production, some enterprises have improved the process. One of the methods is to reduce the loss caused by the precipitation of component III and component II, and adopt improved low pH inactivation and or S/D inactivation, ion exchange and other process steps, the content of IgG can reach 4~4.5g/L after the process is improved .
Regardless of the process used for the separation and purification of components, blood product manufacturers should conduct full research on the process, understand the impact of each production process on product quality, safety, and effectiveness, and identify key process parameters and their control ranges. Strengthen the process control of the production process by carrying out necessary tests on intermediates. For example, during the chromatography process, in addition to the research on the removal ability of the target impurities and the impact of product quality, consideration should also be given to whether there is any impact on the product virus safety. If there is any impact, the virus removal should be carried out on a reduced representative scale. Capability research, in addition, the research should also consider the influence of different cycles of fillers.
2.3 Preparation production process
The initial formulation of IVIG formulations was to freeze-dry the formulations under neutral pH conditions. This formulation can be stored at 2~8°C for 2~3 years, but this formulation often contains IgG polymer complexes after reconstitution. IgG is more stable in liquid conditions with a pH of 4.25, so the most common IVIG formulations currently use sorbitol, sugars (fucose, glucose) or amino acids (glycine, proline, isoleucine) As a stabilizer, the pH range is 4.5 to 5.5. The use of sucrose as an IgG stabilizer may cause fatal renal failure in some patients, so it is replaced in the formulation of the prescription. For the research of formulation formulation, it is recommended that applicants carry out research under different forced degradation conditions in order to more sensitively find whether the formulation formulation is stable. In addition, the formulation formulation research should also consider the compatibility with direct contact packaging materials.
2.4 Virus inactivation/removal process
Compared with plasma-derived blood products such as fibrinogen, coagulation factor Ⅷ, and coagulation factor IX isolated from the upstream of cold ethanol precipitation, IgG obtained by multi-step cold ethanol precipitation has higher virus safety. On the one hand, it may be due to The product itself has the presence of antibodies that neutralize the virus. On the other hand, the multi-step cold ethanol precipitation in the IVIG production process will play a certain role in the elimination of the virus.
However, in actual clinical applications, there are still examples of hepatitis C virus (HCV) transmission due to immunoglobulin infusion , so the virus safety of IVIG should be highly valued. At least one step of effective virus inactivation/removal process should be included in the production of immunoglobulin (virus titer drop ≥4log), blood product producers should carry out small-scale virus inactivation/removal verification based on actual process conditions, and select representative viruses , And carry out production within the scope of verification. In order to improve the virus safety of products, it is recommended that blood product producers incorporate two different principles of virus inactivation/removal processes in the production process.
It should be noted that although some process steps are not effective virus inactivation/removal processes, they have an effect on virus inactivation/removal (4log＞virus titer reduction ≥ 1log). Changes in these process steps may affect product virus safety Bring hidden dangers. The processes used for immunoglobulin virus inactivation/clearance include: low pH virus inactivation (mainly inactivating enveloped viruses), pasteurization (inactivating enveloped viruses and to a certain extent, inactivating non-enveloped viruses) Virus), S/D inactivation (mainly inactivating lipid-enveloped viruses), caprylic acid treatment (mainly inactivating lipid-enveloped viruses) and nanofiltration (including 15, 20, 35 nm pore filters, which can remove envelopes and Non-enveloped virus) [5-8].
2.5 Quality control
At present, the “Chinese Pharmacopoeia” (2015 edition)  stipulates that the quality attributes that should be controlled for IVIG products include: identification test, physical inspection (appearance, osmolality, visible foreign matter, insoluble particles, loading, thermal Stability), chemical test (pH, protein content, purity, sugar and sugar alcohol content), molecular size distribution (exclusion chromatography), antibody titer (anti-HBs, diphtheria antibody), kininogen activator, Antibody complement activity, anti-A and anti-B hemagglutinin, sterility test, abnormal toxicity test, heat source test. At the same time, producers should conduct more quality research in actual research, which is conducive to the management of the entire life cycle such as product development and post-marketing process changes.
2.5.1 Distribution of IgG subtypes
Because the biological activities of different subtypes of IgG in antitoxin, antiviral, and antibacterial aspects are not completely the same , the distribution of different IgG subtypes may cause certain differences in product titer. The normal human serum IgG distribution has a certain range. By detecting the IgG subtype distribution in the product, the blood product manufacturer can understand the impact of the production process on the IgG subtype distribution on the one hand, and on the other hand, it is easier to update when the process changes. Comprehensive comparison of product changes before and after production process changes.
2.5.2 Detection of Fc segment function
There are receptors expressing a variety of Fc fragments in the human body, including activating receptors (such as FcγRI, FcγRⅡA, FcγRⅡC and FcγRⅢA) and inhibitory receptors (such as FcγRⅡB). IVIG uses its Fc fragment to interact with different receptors, thereby exerting different Physiological function . The 2015 edition of the Chinese Pharmacopoeia requires the detection of antibody complement activity. Not all IgG subtypes have complement activity. Therefore, complement activity can only reflect the Fc segment activity of a specific type of IgG. Blood product producers can Combining the characteristics of the IgG Fc fragment to carry out a variety of detection methods, such as the detection of the binding ability of IVIG products to different Fc receptors.
2.5.3 FⅪ Residual detection
Studies have reported that coagulation factors II, VII, IX, and X can be removed in the process of ethanol precipitation, but the isoelectric point of coagulation factor FXIa is high (pI8.9~9.1), and it is difficult to completely remove FXIa from FXI by ethanol precipitation. It is completely separated in IVIG, and it needs to be removed by a suitable chromatography process . Foreign researchers found that among 29 IVIG products from 8 different manufacturers, 26 products can shorten the coagulation time of FXI-deficient plasma. Among them, 14 products have higher procoagulant activity than raw plasma. Therefore, it is recommended that manufacturers conduct blood coagulation testing for intermediate products and final products of key processes in order to understand the process’s ability to remove thrombin. The prothrombin content in the intermediate product can be detected by Western blot or ELISA, and its activity can be detected by the thrombin generation assay (TGA), and the thrombin activity in the final product can be detected by TGA and non-activated thrombin The original time test (non-activated partial thromboplastin time, NaPTT) and other methods to detect .
2.5.4 Detection of IgA and IgM content
As product-related substances, IgA and IgM may exist in immunoglobulin products to varying degrees. Unlike coagulation factors, IgA and IgM are related to IgG in their physiological functions. Therefore, it cannot be simply considered that the content of IgA and IgM in the product is as small as possible. Some immunoglobulin products containing high IgA and IgM are more conventional Immunoglobulin products have a stronger antibacterial effect . But IgM exists in aggregate form, so it has a higher complement activation effect than IgG. The use of IgA-containing IVIG in patients with IgA deficiency may produce IgA antibodies, which can cause severe anaphylactic shock. “European Pharmacopoeia” 9.0  requires that the content of IgA in IVIG should be approved by regulatory agencies. For the safety and effectiveness of the product, it is recommended that the applicant pay attention to the content of IgM and IgA in the product during process development or process changes. For the detection of IgA and IgM, immunological methods such as immunoturbidimetric method and ELISA method can be used for detection .
2.5.5 Detection of hemagglutinin IVIG
During use, hemolysis may occur due to the presence of hemagglutinin, including anti-A, Anti-B and Anti-D . Currently, the Chinese Pharmacopoeia (2015 edition)  ] Only anti-A and Anti-B are required to be tested. Anti-D is not required for the time being. The European Pharmacopoeia 9. 0  requires the detection of anti-D in human immunoglobulins. The reason may be due to the negative RhD in China Blood types are relatively rare, and blood product manufacturers may consider anti-D testing in extended quality studies.
2.5.6 IVIG for impurity residue detection
Process-related impurities in the production process may include ethanol, caprylic acid, sedimentation aids, etc. The residues of these process impurities may cause side reactions in the use of the product. In order to ensure the safety of the product, the applicant should test the relevant residual impurities , It should be proved that the relevant impurities can be removed during the process, or that the residual impurities in the process will not affect the safety of users.
3 Considerations about IVIG process changes
The production history of IVIG in China is long. After the market, blood product companies often improve the production process to increase the recovery rate and utilization rate of IVIG products. In the process of process improvement or scale expansion, the product may undergo unexpected changes. It has been reported that the low pH inactivation process used for virus inactivation can reduce the coagulation factor activity of IVIG products to a certain extent. Therefore, changing the low pH inactivation process may not only affect the virus safety of the product, but may also cause blood coagulation after the change. Unexpected changes in factor activity. In addition, some experimental studies have shown that the viral load of human immunodeficiency virus (HIV) can be significantly reduced after cold ethanol precipitation. In the IVIG case that caused the transmission of HCV, it may be that the manufacturer used chromatography instead of cold ethanol precipitation in order to increase the yield, which reduced the removal effect of the virus and caused the product to contain HCV.
For the safety of patients, blood product manufacturers have the responsibility to deepen the research on the process. It is recommended that manufacturers use commercial scale for verification. In the process of process verification, if it is not possible to prove that the changed process steps have no effect on the downstream process, they should also consider verifying the downstream process steps. In the process of process verification, attention should be paid to whether the original detection method is still suitable for the modified process and whether it can detect new impurities that may be introduced.
Although it is not necessary to conduct a comprehensive quantitative detection of all impurities in routine production, in order to ensure that the changes will not cause adverse effects such as the increase of impurities such as coagulation factors, it is recommended to carry out related impurities in the starting material plasma, intermediate products and final products Quantitative detection. In order to determine the impact of process changes on product quality, necessary quality comparability studies should be carried out. Representative batches of products are used to conduct quality comparability studies before and after the change. The research content is in addition to the provisions of the Chinese Pharmacopoeia (2015 edition)  In addition to the verification items, necessary quality expansion research should be carried out according to the specific conditions of the process change.
In addition, due to the product storage process, some unnoticeable changes may be gradually amplified over time, such as some IVIG storage process FⅪ will change to FⅪa, which will lead to the increase of FⅪa activity in the product, so it should be carried out. The necessary stability comparative study. Through the comprehensive results of the process, quality and stability comparative study, it is finally determined whether the process change will affect the product.
According to reports, the global sales of IVIG in 2014 were 8 million U.S. dollars , and the estimated sales of IVIG in 2019 will reach 12.5 million U.S. dollars. I believe that as China’s economy continues to develop, the demand for IVIG will also increase. Bigger. Therefore, on the one hand, manufacturers should strengthen the comprehensive utilization of raw plasma and increase the production capacity of unit raw plasma to ensure clinical supply; on the other hand, they should always put patient safety in the first place to ensure product quality. While satisfying the supply and ensuring product quality, the primary task of blood product manufacturers is to strengthen research, deepen their understanding of processes and products, and introduce quality for design (Quality by Design, QbD) and overall quality in the development and production of blood products. Life cycle management and other concepts ensure safe and effective products with robust processes.