Stability Problems And Solutions Of Biological Medicines in The Production Process
In recent years, biotechnology medicines, especially monoclonal medicines, have gradually become the main body of new medicine research and development. However, protein biologics generally have the problem of complex and unstable structure, especially a variety of unstable factors in the production process, which results in the degradation and inactivation of biologics. The preparation process of biologic medicines is very complex, often through biosynthesis (such as microbial fermentation/cell culture) stock purification and refining (such as chromatographic purification, virus removal) and preparation process (such as preparation configuration, aseptic filtration, filling, freeze-drying and lamp inspection) and other production, storage, transportation and other links. Therefore, solving these instability problems is the key to the successful application of biological medicines in clinical practice. In this article, the degradation ways in the production of biological medicines were summarized and the corresponding solutions were put forward.
As the biological technology (such as recombinant DNA technology, lymphocyte hybridoma technology, phage display technology) and the development of human genomics, biotech medicines (biological medicine, biotherapeutics, biolog-ics, biopharmaceuticals) especially monoclonal medicines, have gradually become the main body of new medicine research and development. In recent years, biological medicines accounted for 80% of the world's top 10 best-selling prescription medicines, and the proportion of the entire pharmaceutical field is also increasing year by year. Compared with traditional small molecule medicines based on chemical synthesis, biologic medicines are mainly prepared and produced by biotechnology methods, especially recombinant DNA technology, which have the characteristics of high activity, high specificity and low toxicity, and solve many medical problems that traditional small molecule medicines cannot solve, so they play an increasingly important role in saving lives and improving the quality of life of patients.
However, the development of biological medicines also faces many technical challenges. First, biopharmaceuticals are biomacromolecules (relative molecular mass is usually 5x103~2×105) with very complex structures and components. In addition to the primary structure, that is, the amino acid sequence, biological medicines usually have complex high-level structures (such as secondary, tertiary or even quaternary structures), which are the basis of their biological activity.
At the same time, due to factors such as post-translational modification, enzymatic hydrolysis and chemical degradation, common biological medicines are extremely complex mixtures containing millions or more molecules. Secondly, biological medicines are unstable and prone to chemical and physical degradation. Chemical degradation involves the breaking and formation of covalent bonds, while physical degradation is unique to biological medicines and does not involve covalent bond changes, but mainly changes in the high-level structure of proteins, including physical adsorption (to hydrophobic surfaces), denaturation, depolymerization, aggregation, and precipitation. These degradation will not only affect its biological activity, but also may cause many safety problems. Second, unlike small molecule medicines, almost all biologic medicines have potential immunogenicity, that is, the ability to stimulate the body to form specific antibodies or sensitize lymphocytes.
In addition to the structure of biologic medicines themselves, immunogenicity is also closely related to the stability of biologic medicines, especially polymers and protein particles, which are easy to stimulate the body to form corresponding antibodies to clear medicines, affecting the efficacy of medicines, and even due to cross reactivity can neutralize endogenous proteins in the human body. For example, the antibodies produced when using human erythropoietin (EprexR) treatment will not only neutralize protein medicines, but also bind human endogenous proteins to inactivate them, resulting in pure red blood cell regeneration disorders in patients. The immune response may also trigger hypersensitivity reactions, which can even endanger the patient's life in severe cases.
Some subtle changes (such as conformation) that occur during the production of biological medicines may be difficult to observe during the production process or short-term storage through existing analytical technologys, but may affect the stability of the long-term storage process, thus having a greater impact on the final quality of the product. The quality of production facilities, raw materials and packaging materials, as well as the training and operation of employees will also have a great impact on product quality. In this article, the common problems affecting the stability of biological medicines in the production process are summarized and the corresponding solutions are put forward.
01 Biological medicine preparation process
The preparation process of biologic medicines is very complicated. From biosynthesis to final packaging into clinical preparations, it is usually necessary to go through various production, storage and transportation steps including biosynthesis (such as microbial fermentation/cell culture), stock purification, refining (such as chromatographic purification, virus removal) and preparation process (such as preparation configuration, aseptic filtration, filling, freeze-drying and lamp inspection). Taking the most popular antibody biologic medicines as an example, a typical production procedure includes the following steps: First, the cell line is melted and gradually expanded in a reasonable growth environment to eventually meet the needs of production.
In the cell culture process, the environment of biologic medicines including cells, various proteolytic enzymes, nutrients and dissolved oxygen, etc., usually need to be maintained at a relatively high temperature (>30℃) and neutral pH conditions for≥10 days until sufficient protein synthesis and secretion to the extracellular so far. After the biologic medicine synthesis, the insoluble cell residue is removed by centrifugation or filtration, and then the supernatant containing the biologic medicine is purified by several chromatographic columns such as affinity protein A chromatography (protein A chromalography), cation exchange chromatography and anion-exchange chromatography, and the virus is removed and inactivated.
After purification, the biologics are replaced into the appropriate buffer by ultrafiltration or percolation, and stored in the medicine substance, or in the form of final bulk when added to the final preparation components. The finished product is obtained by filling into different inner packing materials (container-clo-sure), or further prepared into freeze-dried powder by freeze-drying treatment. Throughout the production process, proteins undergo a variety of destructive factors, such as low pH, high salt, freezing-thawing, light, oscillations, shearing, and various (hydrophobic) surfaces, which may cause structural changes or degradation of the protein, thereby affecting the quality of the biologic medicine, and each step can be optimized to avoid or reduce the resulting degradation.
02 Degradation and control of biologic medicines during microbial fermentation/cell culture
Microbial fermentation/cell culture process can affect the stability of protein medicines expressed by it, but there are few reports on the stability of biologic medicines in microbial fermentation/cell culture process, or this issue has not received enough attention. The main reason for this phenomenon may be that in the process of microbial fermentation L-cell culture, more attention is paid to the appropriate conditions for microbial/cell growth and expression quantity, and some degradation products can be removed by later purification, or the protein loss caused by degradation is considered to be caused by inadequate protein expression.
In accordance with the QbD principle of biological medicine development and the relevant guidelines such as FDA, product-related impurities are best suppressed at the forefront of production, followed by purification and other processes to remove. In the absence of an effective removal method, it is necessary to demonstrate that the impurity does not significantly affect the safety and efficacy of the medicine, but this will entail a lot of additional research, and there is a risk of some uncertainties due to poorly targeted studies. So the preferred strategy is to consider inhibiting these degradations at source.
There are many factors that cause protein degradation during microbial fermentation/cell culture, the first is environmental factors, such as high temperature, neutral pH, dissolved oxygen, salt ion strength, etc. The temperature of cell culture is much higher than the usual storage temperature (such as 2 to 8 ℃), and as with most chemical reactions, the higher the temperature, the faster the degradation of protein. At neutral pH, many proteins, including monoclonal antibodies, are more prone to aggregation and deamidation. Lower concentrations of dissolved oxygen may result in incomplete protein disulfide bond pairing.
In addition, the components of the medium such as metal ions (such as copper ions), amino acids (such as cysteine), etc., will also affect the quality of biological medicines, especially the formation and exchange of disulfide bonds. Optimized cell culture conditions can improve protein stability, but any process must be both effective and operable. Since the expression conditions of many proteins may conflict with the stability of proteins, changes in microbial fermentation/cell culture conditions in particular may affect the expression levels of target proteins, cell growth, process-related impurities and glycosylation levels. At this time, it is necessary to carry out comprehensive consideration and optimization.
03 Degradation and control of biochemical agents during purification and debacterialization/deviralization
3.1 Purification
The purification process is usually used to remove impurities and improve the purity of the medicine, but the conditions of some purification processes are relatively intense and the protein may be degraded. For example, protein A affinity chromatography used to purify monoclonal antibodies usually requires elution under acidic conditions (such as pH 3 to 4), however, some monoclonal antibodies are sensitive to acid, resulting in reduced or lost biologic activity. For example, the anti-CD52 monoclonal antibody alemtu-zumab (Campath) aggregated in >25% after purification by protein A chromatography. For these acid-sensitive proteins, the elution time needs to be minimized, and the elution should be neutralized in time after elution, or elution at lower temperatures. In addition, the use of optimized buffering systems (such as the addition of arginine) can significantly inhibit the generation of aggregation and improve the recovery of antibodies.
In ion exchange chromatography, it is often necessary to use a higher concentration of salts (such as sodium chloride and sodium acetate), and to adjust the pH of the solution to be suitable for anion or cation exchange chromatography, while ensuring that these conditions do not affect the quality of the protein. Some monoclonal antibodies are more sensitive to high salt and tend to form protein aggregates such as opalescence and particles. We found that elution with histidine as a buffer instead of high salt could effectively inhibit such aggregation reactions (data not published).
In hydrophobic exchange chromatography, proteins are separated by the affinity between the hydrophobic group and the mobile phase, and are easily adsorbed on the hydrophobic surface to denature. However, it is much milder than reverse-phase chromatography, which requires the elution of proteins using organic solvents. The method of adding arginine to the sample solution or mobile phase can also be used to improve the recovery of protein.
3.2 Sterilization/removal of viruses
Since biologic medicines need to be administered by injection route, sterilization and viral clearance is also a necessary process for biopharmaceuticals, mainly including physical removal and chemical inactivation. Physical removal is the separation of bacteria or viruses from biological medicines by physical means, the main methods are membrane filtration/nanofiltration and chromatography. Chemical inactivation is the inactivation of bacteria or viruses by chemical methods, mainly including the use of surfactants, heating, acid treatment and UV/ Y-ray treatment.
Sterilization by heat treatment means that the solution is heated to 60 ℃ for 10 h. When sterilizing by heat treatment, it is necessary to pay attention to whether the target protein can withstand the conditions. If the melting temperature (Tm) of human blood albumin is close to 60 ℃, it is generally necessary to add some protective agents, such as sodium caprylate and acetyltryptophan, to raise the Tm to >70 ℃ before heat treatment sterilization. At the same time, attention should be paid to the impact of some miscellaneous proteins, especially trace amounts of miscellaneous proteins with low melting temperatures, and the particles formed after the degradation of these impurities will become nucleation sites for protein aggregation, accelerating the aggregation of target proteins. If the solution contains sucrose, it should also be considered that sucrose is prone to hydrolysis to form glucose and fructose under high temperature conditions, and these two reduced sugars will have a Maillard reaction with the free amino group of proteins, resulting in the degradation of biological medicines.
To sterilize by radiation, it is necessary to pay attention to the chemical and physical degradation of proteins caused by free radicals, and it is usually necessary to add some free radical scavengers to protect proteins.
3.3 Freeze-Thaw
Freeze-thaw is a necessary process in the production of biological medicines, such as the waiting process in different steps of the production process, or the change of site/transfer, and is also a common method for long-term storage of the stock solution. In addition, accidental freeze-thaw may also be caused when the finished product is transported or the patient uses it at home. Some proteins are very sensitive to freeze-thaw, especially in the absence of suitable protective agents, which can easily cause protein inactivation. Therefore, freeze-thaw experiment is also an essential part of formulation prescription screening.
The mechanisms of protein freeze-thaw destruction are as follows: First, the ice water surface formed during freezing is an important cause of protein denaturation, and proteins tend to be adsorbed to these surfaces for denaturation and aggregation; Secondly, after a large amount of water becomes ice during the freezing process, the concentration of the remaining solute and the protein itself will increase sharply, and the higher the protein concentration, the more chances of intermolecular collision occur, and the more serious the formation of aggregation.
According to the reaction mechanism of protein degradation, there are different ways to inhibit protein degradation caused by freeze-thaw. For example, the eighth in the ice water agent (such as polysorbate 20, polysorbate 80) to inhibit the degradation caused by the surface of ice water. Thermodynamic stability (keeping the protein in its natural state) is increased by adjusting the pH and ionic strength of the solution and by adding excipients/protectants.
For long-term storage of biologic medicine stock, it is usually necessary to keep the protein below the glass transition temperalure (T') of the maximum frozen concentrate to ensure very low motility (kinetic stability). For example, a protein solution containing sucrose as a protective agent, since its T' is about -30 ℃, it needs to be kept at a temperature of -40 ℃ or even lower.
The rate of freeze-thaw also affects the stability of biological medicines. If the freezing is too slow, the protein will be degraded more easily in a higher concentration state for a long time. On the contrary, under very fast conditions (such as -80℃), a large amount of ice water surface may be formed, which also causes degradation due to the surface. The rate of melting is also very important, with slow melting (e.g. 4℃) causing further damage by recrystallization of the water that has melted on the surface of the ice water. Therefore, in the production process, it is generally recommended to melt frozen products at a faster speed as far as possible, such as using flowing water to accelerate melting.
In addition, during the freezing process, some solutes will crystallize out due to the formation of ice and the reduction of solubility. The most typical is sodium phosphate buffer, compared with sodium dihydrogen phosphate, the solubility of sodium dihydrogen phosphate is very sensitive to temperature, at low temperature conditions will be the first precipitation, resulting in a decrease in the pH of the solution up to 3 to 4 units, at this time the acid-sensitive protein is prone to degradation. Some proteins with multiple subunit structure, such as aponeocarzinostatin and staphylococcal nuclease, have low temperature denaturation due to the decrease of hydrophobic action of linking subunits with decreasing temperature.
3.4 Filtration/ultrafiltration
There are three main types of membrane filtration for protein solutions, namely sterile filtration, nano-filtra-tion and ultrafiltration/percolation. Bactericidal filtration is mainly used to remove insoluble particles and bacteria, usually used before the final product filling; Nanofiltration is mainly used to remove viruses; Ultrafiltration/percolation is mainly used to replace the purified sample into the buffer of the final preparation and concentrate it, while avoiding the direct addition of strong alkali or strong acid to the protein solution to adjust the pH of the solution, and the addition of other solid excipients may cause local heat release and affect the stability of the protein.
However, membrane filtration itself will have some effects on proteins, and the interaction between proteins and filtration membranes may reduce the concentration of proteins in the stock solution and denature the proteins, which has a more significant impact on medicines with low protein concentration. Generally, the interaction between protein and filter membrane, and between protein and protein can be reduced by adding surfactants. In addition, some poor quality filters themselves will shed some particles and become nucleation points for protein aggregation, accelerating protein aggregation. Selecting a high quality filter membrane is critical.
The Donnan effect also needs to be considered in the ultrafiltration process. The Donnan effect means that during the membrane filtration process, the polymer (such as protein macromolecules) is trapped in the membrane, and the electrolyte with opposite charge in the solution gathers more around the polymer due to the mutual attraction of charge, so that the filtration membrane cannot be completely permeated during the ultrafiltration process, resulting in an increase in concentration. Conventional antibodies are positively charged in the ultrafiltrate, so the anionic electrolyte will be enriched with the antibody and the concentration will increase.
Generally, the lower the initial buffer concentration and the higher the protein concentration after ultrafiltration, the more obvious the Daunan effect and the more significant the impact on the pH of the buffer. If the buffer containing histidine, the pH value will rise when the ultrafiltration concentrates the antibody medicine, and even the pH value of the preparation will exceed the quality control standard and make the product unqualified.
04 Degradation and control of biological medicines in the process of finished product preparation
4.1 Configuration and Mixing
In the production process, due to the large size of the biologic medicines involved, preparation configuration and mixing operations become very important, such as local protein or excipient concentration is too high, or the change of solution pH and ionic strength may lead to protein denaturation or precipitation. The type, size, mixing speed and time of the mechanical agitator during production may affect the stability of the biologic medicine, for example, the mixing rate is too high, resulting in accelerated protein aggregation. Therefore, it is necessary to optimize these parameters as much as possible under the premise of achieving uniform mixing.
4.2 Filling
Biologic medicines are prone to denaturation and aggregation during the filling process, mainly due to mechanical forces such as shear forces generated by the pumping process and degradation caused by some precipitates. It has been reported that the stainless steel of the piston pump will precipitate some nanoparticles and become nucleation points for antibody aggregation. The small bubbles generated during the filling process can denature the protein on the gas-liquid surface, and the small bubbles will produce free radicals and/or local heat changes when broken, which may cause protein denature.
4.3 Freeze-drying
Biologic medicines tend to use liquid formulations because liquid formulations have significant advantages over lyophilized formulations from the point of view of cost, process simplicity, and patient convenience. However, some proteins are very unstable in aqueous solutions, and if sufficient stability has not been achieved after preparation optimization, then the use of lyophilized preparations should be considered. The lyophilization process will form many destructive factors, the first is the destructive factors in the freezing process, which have been detailed previously.
In addition, proteins may also encounter degradation factors under dry conditions. For example, the hydration layer on the surface of proteins is very important for the stability of proteins. Hageman proposed that the surface of proteins contains about 7% water, which is very important for maintaining the structure of proteins, and the water content after lyophilization is generally between 1% and 2%, so other substances are needed to replace the role of water during dehydration. Therefore, it is very important to choose the right prescription and lyophilization process. It is generally believed that disaccharides such as sucrose and trehalose can play a relatively effective role as hydrogen bond donors, while polymer compounds can not effectively play the role of water substitutes due to steric effect.
In addition, under the premise of controlling the freeze-dried water content (such as 1% to 2%), sucrose and trehalose can form an amorphous powder with a high T, so that the whole system can be maintained in a solid state and inhibit physical and chemical degradation during long-term storage. However, for polypeptide biological medicines (such as glucagon), because they do not have a relatively fixed high-level structure, polymer sugars such as hydroxyethyl starch that cannot play a hydrogen bond role can also play a high protective effect like seaweed sugars. Recently, it has been reported that the use of amino acids as a new biological medicine freeze-drying protectants, especially arginine, can be used alone or mixed with sucrose very effectively to protect the stability of proteins under freezing and freeze-drying conditions.
05 Degradation and control of biological medicines during storage, transport and use
In the process of storage, transportation and use, proteins will also experience various degradation conditions, such as short-term temperature changes during storage and transportation, transportation oscillations, or light damage during transportation and use, which may have a greater impact on protein quality. For biopharmaceuticals and vaccines, cold chain transportation is a key factor in ensuring product quality. In recent years, there have been several vaccine safety incidents in China, such as the Shanxi vaccine case in 2010 and the Shandong illegal vaccine case in 2016. These cases all involved improper storage and transportation of vaccines, and the potential medicine safety risks caused by them have aroused great concern of the whole society. Therefore, strengthening the management and control in the storage, transportation and use process is an important link to ensure the safe application of biological medicines.
06 Conclusion
Biologic medicines are highly fragile molecules, and the quality of their products is closely related to the production process. In the production process, various chemical and physical degradation is easy to occur, especially the physical degradation of biological medicine macromolecules, which can occur under various physical or mechanical conditions, so the experience of small molecule medicines can not be directly applied to biological medicines.
Extreme conditions should be avoided in the production process, such as mixing the biological medicine solution with a mixer with too high stirring rate, directly using strong acids or alkali to adjust the pH of the solution, or directly adding solid excipients to the protein solution to dissolve. Although it may not cause detectable effects in the short term, it may have affected the local normal fine structure of the biologic medicine, and these structural changes will be amplified during long-term storage, ultimately affecting the quality of the product.
If necessary, the comparative evaluation of different production processes or protectants can be accelerated by using accelerated and forced degradation stability tests on the stock or finished product. Particular attention should be paid to these degradation products during purification and filling, as they will remain in the finished product and eventually be used in patients, raising safety, efficacy and immunogenicity issues.
In a sense, the production process of biopharmaceuticals determines their quality, which requires the analysis of the degradation mechanism of these molecules and the inhibition of their possible degradation throughout the production process to ensure that the final product can be safely and effectively applied to patients.
About Guidling
Guidling Technology is a national high-tech enterprise focusing on biopharmaceuticals, cell culture, purification and concentration of biomedicine, diagnosis and industrial fluids. We have successfully developed centrifugal filter devices, ultrafiltration & microfiltration cassettes, virus filter, TFF system, depth filter, hollow fiber, etc. Which fully meet the application scenarios of biopharmaceuticals, cell culture, and so on. Our membranes and membrane filters are widely used in concentration, extraction and separation of pre-filtration, microfiltration, ultrafiltration and nanofiltration. Our many product lines, from small, single-use laboratory filtration to production filtration systems, sterility testing, fermentation, cell culture and more, meet the needs of testing and production. Guidling Technology is looking forward to cooperating with you!
