Development trend of monoclonal antibody production technology

Development trend of monoclonal antibody production technology. Monoclonal antibodies have established a leading position in therapeutic biological products. Establishing a sound production platform is the key to seamlessly transforming antibody drug discovery results into clinical and successful listing. Some leaders are influencing the design of monoclonal antibody production processes.

The emergence of biosimilars makes it possible to reduce the cost of medicines and the global production of biological products. Currently, monoclonal antibodies can easily achieve high expression in mammalian cell culture. These driving factors led to the discovery of significant improvements in platform technology.

In addition, in order to achieve these needs, there are also some new trends in biopharmaceutical production processes. Including the evaluation of different expression systems, continuous culture and non-chromatographic purification processes. This article discusses the changes these driving factors have produced in the monoclonal antibody production process.

1 Introduction of monoclonal antibody platform technology

Currently monoclonal antibodies are the most successful type of biological products. The number of monoclonal antibodies approved for marketing has exceeded 50, with sales expected to exceed US$125 billion by 2020. The high specificity and affinity that can bind to specific targets, and the easy humanization and fully humanization of the target protein sequence have led to the explosive growth of such biological products. These products have developed rapidly in the past 20 years, and more than 300 monoclonal antibodies are currently in clinical trials. Monoclonal antibodies have been approved for multiple indications such as malignant tumors, immunological diseases and rare diseases.

In addition to the highly specific binding ability to target cell surface targets, the ability to rapidly develop robust production processes to advance the monoclonal antibody candidate product into the clinical trial stage has also become a key factor in the smooth progress of the monoclonal antibody project. The convenience and speed of monoclonal antibody production enable these candidate products to quickly enter clinical trials. The scalability and robustness of these processes can greatly promote large-scale commercial production.

The development of protein production processes needs to consider many aspects, including impurity removal, robustness, scalability, and the use of readily available large quantities of raw materials. Process capability must consider not only the scale required to meet early clinical trials, but also the demand and scale to support long-term supply. Therefore, making full use of the platform method is the key to developing the production process. The level of robustness, scalability, and reproducibility means that these production processes appear to be significantly different from those used to purify small amounts of protein in the laboratory. Process development is a time-consuming task and requires extensive testing. Therefore, as long as circumstances permit, the industry will tend to adopt a platform approach.

From a business perspective, the platform approach has obvious advantages. The speed of entering the clinic is usually a key factor in determining the success or failure of a company. The monoclonal antibody platform can advance the project from the genetic design stage to the IND stage within one year, which is a huge improvement for molecular projects that usually take two years to develop. Reduced testing also means lower R&D costs. The predictability of the process platform enables organizations such as production and quality control to adopt a templated document set, which also reduces the time and resources spent on production and release inspections. The monoclonal antibody process platform makes the entire production process from initiation of clinical trials to product commercialization efficient and robust. The consistency and predictability of the platform approach has greatly promoted the development of such products.

Monoclonal antibodies are particularly suitable for the application of platform methods. Using mature mammalian cell culture systems, stable cell lines can be developed quickly and templated. There are several optimized expression vectors dedicated to monoclonal antibody production. There are also robust fed-batch culture techniques suitable for monoclonal antibodies. Some of these cell lines and culture processes have been used in large-scale production, and the operating parameters that affect these processes have been deeply characterized.

Cell line development and upstream cell culture processes are very suitable for templated methods. However, for most proteins, the biggest difference is the downstream purification process. A personalized purification process should be designed for each protein according to protein characteristics and key impurities. The Fc region of the monoclonal antibody can bind highly specifically to protein A, which is a component of the cell wall of Staphylococcus aureus. Protein affinity chromatography has been widely used in the production of monoclonal antibodies, and the chromatography method requires almost no optimization to achieve a purity of over 95%.

The main challenge after protein A chromatography is the ability to remove residual host cell proteins, high molecular weight polymerization, DNA and virus contamination. Leading biopharmaceutical companies have developed some downstream process platforms for monoclonal antibody production. Compared with other protein projects that cannot include related steps in the downstream process, the ability to use a common starting method from the template can reduce the number of corresponding experiments.

These downstream process platforms have successfully enabled a large number of monoclonal antibody products to enter the clinical and commercial fields. However, there are still some emerging trends that continue to shape the biopharmaceutical industry. The next section will discuss the changing trends of the leader’s monoclonal antibody production process.

2 Driving changes in biological products

Many factors are driving changes in the production of traditional biopharmaceuticals. Because the selling price of drugs is determined by the value of the patient’s life and health, production costs are not considered an important factor. As a result, production costs have become the focus of increasing attention. In addition, organizations with production capabilities are seeking to use existing factories more effectively to reduce the need for new factory construction. In the context of promoting process innovation, many factors have been tested.

2.1 Biosimilars

The emergence of biosimilars (generally referred to as biosimilars) is a key change in traditional biopharmaceutical methods.
Although due to its own complexity, the price drop of biosimilar drugs is not as significant as that of small molecule drugs, the focus of biosimilar drugs is still on production costs. On October 30, 2005, the European Union promulgated the “Guiding Principles for Biosimilar Drugs”. Since then, many large biotechnology companies and large pharmaceutical companies have announced the establishment of biosimilar pharmaceutical companies, including Sandoz, Amgen, Biogen (belonging to Samsung), Pfizer and Merck Serono. It is expected that biosimilars will be launched by 2020. The market size is nearly 20 billion U.S. dollars. The US FDA is more cautious in accepting the application of biosimilar drugs. Currently, two products have been approved, Zarxio (Neupogen biosimilar drug) and Inflectra (Remicade biosimilar drug). As of May 2016, EMA has approved 22 biosimilar drugs. As this trend continues to expand globally, biosimilars will significantly reduce the cost of goods (COGS).

2.2 Global biopharmaceuticals

With the rise of biosimilar drugs, people’s interest in global biopharmaceuticals has become increasingly strong. This is also related to the preferential treatment of local biopharmaceutical companies in several markets. The situation in China is particularly obvious. Pfizer and General Electric jointly launched a biosimilar production plant called KU Bio. Other biopharmaceutical companies have also entered the Chinese market, including Wuxi. In December 2015, the China Food and Drug Administration (cFDA) announced that China will speed up the approval process for drug marketing applications. In Latin America and South Africa, the localized production trend of biosimilar drugs has also begun to rise. A series of Indian companies engaged in the production of biopharmaceuticals has also increased significantly.

2.3. One-off production technology

The growth of one-off production technology has promoted global production, and this technology has significantly reduced infrastructure investment. The one-time production technology makes it possible to quickly use the disposable method for the production of clinical samples throughout the entire process. One-time production of biopharmaceuticals is a trend, and it has now developed into the commercial manufacturing field. Several manufacturers have used multiple 2000L disposable bioreactors to produce biopharmaceuticals.

For example, Amgen’s manufacturing plant in Singapore uses 6×2000L disposable bioreactors for cell culture. Compared with the large stainless steel equipment that is still in a dominant position, these technologies make the cGMP production of biopharmaceuticals easier to achieve. Combined with a modular structure that can be assembled quickly, one-off production becomes a technology that can expand biopharmaceuticals worldwide.

2.4 Increase in expression

In recent decades, the expression level of cell culture has also increased significantly. Now, after 14 days of batch culture, monoclonal antibodies can usually reach an expression level of 5 g/L. The continuous feeding of the fed bioreactor can also obtain higher expression levels. These advances are achieved by the development of cell line expression vectors, clone screening, and changes in cell culture media. This increase in product expression has in turn enabled small bioreactors (such as 2000L disposable bioreactors) to be used in commercial production.

2.5 New downstream process technology

The increase in upstream process productivity has caused downstream processes to become production bottlenecks. Although some people believe that the currently used fixed chromatography process can meet the production requirements of several tons of monoclonal antibodies, people’s interest in purification technology is still increasing, hoping that it will significantly increase productivity. Recent hotspots include continuous biological treatment (usually used for continuous feed culture and downstream operations), and non-chromatographic techniques that can be used on a large scale. Another area that has regained attention is non-chromatographic separation methods, in which separation methods can completely get rid of the dependence on chromatography columns.

2.6 New generation antibody structure

Another major factor leading to the evolution of platform methods is that specific monoclonal antibody-like structures are being developed as potential treatments. The biotechnology industry is rapidly surpassing traditional monoclonal antibodies and is involved in various structures including Fc fusion proteins, bispecific antibodies (bsAbs) and fusion proteins. These new structures need to adjust the original monoclonal antibody platform technology to support their production.

The Fc fusion protein is expressed by linking the coding sequence of the Fc region of the monoclonal antibody with the coding sequence of another protein. The Fc region has several advantages as a fusion site. Many biologically active peptides and proteins show rapid clearance in the kidneys and a short plasma half-life. The Fc region can bind to the newly born Fc receptor, prolonging the half-life of the antibody, and has the same effect on the fusion part.

Seven Fc fusion proteins have been approved for marketing, of which at least two (Enbrel and Orencia) have annual sales of more than $1 billion, reaching blockbuster level. Amgen’s monoclonal antibody original platform includes the purification of Fc fusion proteins. However, for Fc fusion proteins, there are several key downstream differences, including the sensitivity to proteolysis and the possibility of higher high molecular weight aggregation (HMW) levels than conventional monoclonal antibodies. The typical monoclonal antibody downstream platform method is usually feasible after appropriate adjustment of the elution conditions to ensure the stability of the molecule and the effective removal of multimers.

Bispecific antibodies (bsAbs) are designed to simultaneously bind and neutralize two different antigens (ligands, cell receptors or cytokines) or two different target proteins. Because of this feature, bispecific antibodies can be used as modulators to transfer immune effectors and cytotoxic factors, such as T cells, to tumors or infected bacteria. The two arms of the antibody are different, which leads to several challenges in the production process of bispecific antibodies.

For example, if the two units of an antibody are expressed separately, the downstream process needs to be split into two halves and then reassembled to form a heterodimer. Unless a recombination process is used, even if the formation of homodimers is suppressed (by using a hole-cavity method or similar technology to promote the formation of heterodimers), a small amount of homodimers will still be formed, which requires Remove in downstream processing. Therefore, the construction of this bispecific antibody requires a more complicated downstream process.

However, the use of a common light chain and eyelet technology (KiH) allows the expression of bispecific antibodies in cell culture. Due to the structure of KiH, the formation of homopolymers is suppressed. If the selected sequences are biochemically different, the small amount of homodimers formed can be removed. This can be done even in the absence of a common light chain. For example, in the X monoclonal antibody technology derived from Xencor, homodimers and heterodimers have slight differences in their designed Fc regions, allowing them to be separated in CEX. Another purpose of this engineering is to improve the functional effects of bispecific antibodies.

Antibody-conjugated drug (ADC) is a drug in which an antibody is coupled to a cytotoxic agent, which is mainly used for cancer treatment. At the end of the downstream process of ADCs, except for the chemical bonding step, the remaining downstream processes are the same as traditional monoclonal antibodies. This usually requires an ultrafiltration/filtration process to remove uncoupled small molecules. The main difference with monoclonal antibodies is that due to the toxicity of the linking molecule, stricter control and personnel protection are required.

Other next-generation monoclonal antibody structures include fusion of antibodies at the C or N-terminus to further enhance the ability to simultaneously bind to multiple targets. Such as anti-lytic protein fused with antibody structure. As shown in Figure 1, it includes engineered scFvs, double antibodies and tertiary antibodies, as well as Fab couplers in the form of dimers or trimers. If these treatment modes become a treatment mode, it will lead to the development of platform technology methods.

 
Figure 1 Possible structures of next-generation antibodies

3 Current status and progress of monoclonal antibody upstream and downstream processes

3.1 Development drive of monoclonal antibody platform

This section outlines the downstream processes that several large biopharmaceutical companies have developed and successfully used for mass production of monoclonal antibodies. Several aspects of these processes are common. In the process of mammalian cell culture, the monoclonal antibody is secreted into the medium outside the cell. Harvesting and recovery procedures usually use centrifugation, followed by deep filtration and a series of membrane filters. If the scale is small, centrifugal separation can be used, using a series of depth filters, which can usually be reused. The platform process is almost always protein A affinity chromatography. In some cases, a selective elution method can be added to the protein A chromatography to further enhance the clearance of host cell proteins (HCP). Non-protein A solutions have been developed and used in the commercial production of certain therapeutic proteins, but due to the lack of general methods and robustness of the treatment process, they have not yet been widely used. According to WHO guidelines, the process should include two different principles of virus removal procedures. Low pH virus activity and nanofiltration are widely used in monoclonal antibody production. The classic method of low pH is to immediately displace the low pH buffer solution after the protein is eluted from the protein A chromatography. After protein A affinity chromatography and virus inactivation, one or two refined chromatography methods are used to remove high molecular weight polymers, host cell proteins, DNA, and remove potential viruses. The main difference of each platform method lies in the order of purification chromatography steps of different principles. In view of the increasingly common high-expressing cell culture process (5-10g/L) in the industry, a high column load is required in the refined chromatography process.

Each company will eventually customize the method of the monoclonal antibody downstream platform according to its own expression system and cell culture process, as well as the types and subclasses of monoclonal antibodies they mainly use in research and development. The main criterion is that the platform method needs to remain robust and applicable in a wide range of IgG molecules without significant modification. Another key criterion is that the platform can adapt to the production schedule and minimize the time spent in downstream processing. Therefore, another key driving factor is the capacity of each refining process.

3.2 Current status of monoclonal antibody downstream platforms

Amgen is one of the first companies to disclose its downstream process platform. The fully templated method proved to be infeasible, but a small amount of development experiments can be performed to obtain the required process parameters. Examples of these process parameters include: the elution pH of the protein and the selection of the elution chromatography step, which depends on the main components of the impurities that need to be removed. Figure 2 shows the downstream platform solution used in Amgen at that time.

A typical refining process usually adopts first cation exchange chromatography (CEX) adsorption mode, and then uses hydrophobic chromatography (HIC) flow-through mode to remove polymers, or anion exchange chromatography (AEX) to remove HCP as needed. In a few cases, hydroxyapatite is used to remove specific product-related impurities. After the first step of refining the CEX process to make HCP and HMW meet the requirements, AEX membrane chromatography was used as the second step of virus inactivation process.

Genentech is another company that pioneered the development of a monoclonal antibody process using a platform method. Genentech historically used CEX and AEX chromatography as part of its downstream platform (Figure 3). This trend was initially to use the binding and elution mode of CEX as the first refining step, and then the AEX flow-through mode. Generally, the pI of monoclonal antibody is alkaline, so it is easy to bind tightly with CEX packing, and it is easy to flow through AEX packing. The CEX step cleared HMW and HCP. However, the AEX procedure requires low conductivity to successfully remove HCP. AEX requires a large volume of tank load before sample loading, which may cause bottlenecks during operation. Using mixed mode Captoadhere packing instead of traditional AEX packing helps reduce the need for high dilution.

A major disadvantage of using AEX is that it can only remove HCPs but not HMW polymers. The AEX process can be optimized and the weak separation mode can be used to reduce this defect, but in this case a part of the product will remain on the filler. In this mode of operation, the AEX process can remove part of HMW while removing HCP. We speculate from the structure that under conditions of very low conductivity, some hydrophobic interactions of the filler framework can remove part of the HMW.

Figure 2 Amgen’s monoclonal antibody downstream process platform method

Both of the above solutions have a disadvantage, that is, a large amount of dilution is required for the AEX step. In addition, CEX and AEX usually cannot achieve sufficient HMW clearance.
The HMW levels of different monoclonal antibodies are not consistent, but the HMW concentration after protein A chromatography generally does not exceed 5%. Such a level often requires the use of HIC technology as mentioned earlier. Another innovative method is to carry out HIC process operation under high sample load (200g/L) without adding salt.

This method requires the use of a highly hydrophobic HIC filler without adding additional hypertonic salts. Adjust the pH value before loading to remove HMW under this high overload condition. Using this ultra-high-capacity step, a large number of monoclonal antibodies can be processed and used as part of Biogen’s (Figure 4) monoclonal antibody platform with the AEX flow-through mode. The ultra-high sample loading refining process capability reduces the number of chromatography cycles and also reduces the loading time of batch production.

   
Figure 3 Genentech monoclonal antibody downstream process platform

Millipore-Sigma has discussed a solution that is almost completely refined in flow-through mode. In this proposal, CEX uses a high overload mode to load samples. When doing this, CEX has a certain ability to clear HCP. We also speculate that this is due to the hydrophobic interaction with the chromatography medium. The refining process finally completes the entire process in AEX flow-through mode.

Figure 4 Biogen’s monoclonal antibody downstream process platform

Contract development and manufacturing (CMDO) requires the integration of many different cell lines and cell culture processes into downstream platform methods. Because HCP and HMW need to be removed at the same time, this is a major challenge for downstream platform solutions. The various requirements for downstream monoclonal antibody platforms are shown in Figure 5. There are many variability in different cell lines and media types, which are processed by the downstream process platform. Therefore, the two-step refining process needs to be able to remove HMW and HCP at the same time.

Figure 5 The range of factors considered to determine the KBI biopharmaceutical platform method

3.3 The widely used monoclonal antibody downstream platform

The traditional craftsmanship of CEX and AEX cannot fully meet this demand. A key modification to achieve this goal is to use mixed mode chromatography as part of the monoclonal antibody downstream platform. Mixed mode chromatography adds hydrophobic groups to the backbone structure of AEX or CEX. The enhanced hydrophobicity of the chromatography filler improves the ability to remove polymers in both CEX and AEX modes, making it more suitable for monoclonal antibody technology.

In addition, these two chromatographic methods can simultaneously remove HCP and DNA. Different monoclonal antibodies have different hydrophilicities. Therefore, KBI’s flat downstream process is defined as anion exchange chromatography (such as Q-dextran FF or Capto Q or Fractogel SO3 and other hydrophobic media to Captoadhere or Nuvia cPrime mixed mode media). This adjustment of hydrophobicity can tailor the best conditions for each monoclonal antibody. Similarly, depending on the type of filler selected, the CEX binding and elution process will also slightly or moderately adjust the hydrophobicity.

Although this method requires a certain degree of experimental work, if it is suitable for a certain monoclonal antibody, the preferred method uses mixed mode chromatography including AEX and CEX, so that a smaller amount of hydrophobic stationary phase can be used. The process shown in Figure 6 has been widely used in KBI company including monoclonal antibody structure, cell line and cell culture process.

Figure 7 shows that a large number of monoclonal antibody HMW polymers and HCPs cleared map files were carried out through this platform process. It can be seen from the figure that after adopting this platform, the total level of HMW is less than 1%, and HCP is less than 50 ppm. Especially for CMDOs like KBI, the ability to cover a wide range of monoclonal antibodies with different structures is the key to the platform.

  
Figure 6 The monoclonal antibody downstream process used by KBI Biopharmaceuticals for FIH manufacturing

Figure 7 KBI biological harma platform DSP method performance. (a) HMW result, (b) HCP result

4 Emerging process technologies that may have an impact on monoclonal antibody production

Various downstream platform processes can meet the processing needs of the most advanced monoclonal antibody production process for cell batch culture. However, the increase in productivity in the cell culture process requires the development of alternative methods to improve downstream production efficiency. Due to the desire to make fuller use of existing production facilities and to reduce the production cost of biopharmaceuticals, these technologies have aroused great interest in the field of biopharmaceuticals. The ultimate goal is to reduce the cost of biosimilars produced to $10 per gram.

4.1 Continuous production

Small molecule drugs usually adopt continuous production methods to maximize the production efficiency of production equipment. However, traditional biopharmaceutical production can only use discrete batch production methods, including upstream cell culture processes and downstream chromatography processes. In this regard, people are increasingly interested in realizing the value of continuous processing in biopharmaceutical production.

The fed-through cell culture process is to add fresh medium to the original medium and continuously extract the medium containing the product, which is mainly used for unstable or low expression products. The traditional fed-batch culture process is only used to cultivate products that are inhibited or unstable in batch culture. However, for the sake of production efficiency rather than the product itself, the fed-batch culture process is becoming more and more common recently.Because there is no need to wait for the expansion of cells in a low inoculation volume, but a continuous continuous medium is used to maintain a high cell density production stage, the fed-batch culture process can significantly improve the productivity of the bioreactor.

The disadvantage of requiring a large amount of cell culture medium can be appropriately solved in logistics. This system proves that it is cost-effective to spend a higher cost to obtain higher productivity. In addition, improvements in cell separation technology (such as Repligen’s tangential flow separation technology) have also promoted the wide application of flow-added cell culture.

In continuous fed culture, product purification can be processed through multiple cycles of a batch chromatography system, which has also caused renewed interest in continuous upstream and downstream processes. Traditional chromatography is a batch process. The steps range from the sanitation, equilibration, and loading of the chromatography column to washing, elution, column removal and column regeneration, and storage.

The throughput and productivity of batch processes are limited. First, due to the limitation of flow distribution, the maximum diameter of the chromatography column can only reach 2 meters. Due to the limitation of pressure drop, the height of the packed bed can only be filled up to 30 cm (usually 20 cm). This fundamentally limits the number of products processed in a single cycle. When multiple cycles are required, intermediate products need to be stored for a longer period of time. The intermediate sample storage step also requires the use of large-volume storage tanks, which is another limitation on productivity.

Continuous chromatographic separation can turn this batch process into a continuous or semi-continuous process, as shown in Figure 8. Combined with a continuous upstream fed-batch culture process, the current design mode of biopharmaceutical production processes can be changed. Recent economic analysis of the continuous production process shows that the continuous production process can be compared with the traditional batch production process, and the production cost of a greatly reduced bioreactor is basically the same. This can significantly reduce the capital expenditure required to build a commercial manufacturing facility. Consistent with what was discussed in Sections 2.1 and 2.2, this law is one of the driving factors for low-cost production of biosimilars. Some analysis shows that even at the scale of a 500L bioreactor, the continuous production process can reach a low cost of $17/g.

Continuous chromatographic separation can take many forms. Periodic reflux chromatography is a form (from GE Healthcare) that uses multiple chromatography columns to perform continuous operations at different stages of the operating cycle. Other forms include multi-column countercurrent solvent gradient purification, using ChromaCon’s ContiChrom® system, Tarpon Biosystems (now Pall)’s BioSMB technology, and Semba Bio’s Octave chromatography system to recover the front and Tail to increase yield and purity.

A recent debate in this field is whether and how to implement an end-to-end continuous process in order to maximize productivity. To achieve this goal, multiple process steps, including virus inactivation, ultrafiltration/dialysis and virus filtration processes need to be integrated into a continuous process. Over time, continuous progress will appear, but whether a completely continuous process is required is still controversial. Combining continuous cell culture with continuous adsorption chromatography, and then completing the remaining mAb downstream processes through the high-load refining process described in section 3.2 may be sufficient.

Further development of the continuous production process is inevitable and will result in changes in the number of suppliers’ production and marketing systems. Some key technical obstacles have been overcome in this field, and the current focus is on large-scale verification of these systems and the use of rapid process control techniques to improve the control of these systems. In the emerging technology fields listed in this article, because it still uses traditional chromatography media, continuous production seems to be the first system that will be realized on a large scale.

Figure 8 Principle of continuous chromatography

4.2 Non-chromatographic separation

Another way to increase productivity is to switch from chromatographic processes to non-chromatographic processes. A key reason for the reliance on chromatography is that it can handle a large number of host cell protein impurities while separating components that are highly similar to the product. However, the chromatography step is an obvious rate-limiting step, especially when it comes to mass-produced products, such as high-expression culture in a large-volume bioreactor.

The chromatography process that biological separation relies on is limited by the bed diameter and bed height of the chromatography column. Most chromatography media are compressible, which means that low-pressure pumps and the systems used in cGMP biopharmaceutical systems cannot be used to increase the height of the column. In addition, the largest diameter chromatography column currently has a diameter of 2 meters. Therefore, even with continuous chromatography, the capacity is still limited. The idea of ​​non-chromatographic separation is to find an alternative to chromatographic method to realize biological separation by processing the entire batch of cell culture harvest liquid at one time. This may greatly increase the production capacity of biopharmaceuticals.

The selective precipitation scheme using polymers can capture the product in the entire bioreactor in one operation, rather than relying on multiple chromatography cycles. If these types of unit operations are highly selective and general, they will be widely used in large-scale biological treatment. There are a variety of polymers that can be used for the precipitation of monoclonal antibodies, such as anionic polymer precipitation products and caprylic acid hydrolyzed host protein impurities.

The combination of polymers with different mechanisms can create better selectivity. For example, salt (precipitation caused by high ionic strength) can be combined with a charged polymer, and separation can be achieved by neutralization of the charge or by polyethylene glycol (polyethylene glycol) that repels protein molecules. Appropriate combination may produce highly selective separation, which can reduce the dependence on multiple continuous chromatographic processes in the downstream process, and it is also possible to design polymers with multiple mechanisms to achieve the control of host cell protein impurities or products. Selective precipitation.

Another extension of selective precipitation is the flocculation of cells in the supernatant of the bioreactor. Flocculants such as low pH (< pH 5.0) and polymerization agents, such as PDDA (polydiallyldimethylammonium chloride) can not only be used to precipitate cells and cell debris, but also host cell proteins and DNA and other impurities. Therefore, the flocculant can also be used to remove impurities on a large scale while having other functions in harvesting. In some cases, large amounts of host cell proteins can be removed, which can reduce downstream process steps.

Aqueous Two-Phase Extraction (ATPS) announces the addition of a mixed polymer and salt or two polymers to the solution to produce two separate aqueous phases. Such as adding PEG-salt and dextro-PEG APTS. There have been several reports using ATPS for highly selective separation. However, because the partitioning mechanism is difficult to develop and usually does not have enough specificity to support the separation of proteins such as monoclonal antibodies, its large-scale application is limited.

A PEG/phosphate ATPS system has been used to achieve the purpose of monoclonal antibodies from transgenic plant extracts. Recently, multi-step ATPS has been developed, trying to create a unified platform for a variety of different types of monoclonal antibodies. Taking into account the needs of the separation field, it is expected that ATPS will have greater development in the future.

All of these separation technologies have the potential to attract attention and can greatly increase the processing capacity of existing mainstream production equipment. However, all these technologies require further development to make themselves a scalable technology that can be universally applied to the purification process of various monoclonal antibodies without the need for optimization.

4.3 Screening expression system

In addition to mammalian cell culture, monoclonal antibodies can also be produced in other expression systems. A key area for further development is the study of alternative expression systems that can produce higher productivity while retaining glycosylation patterns compatible with the human immune system. These developments may make this field surpass the most advanced CHO cell culture.
A key alternative expression system is transgenic plants.

Some of these systems have been used in clinical production, such as genetically modified tobacco. Use Agrobacterium (Agrobacterium) to obtain transgenic tobacco in a short time. This method is widely used to produce antibodies that neutralize the HIV virus. Many companies have begun to use tobacco as the preferred expression system (Medicago, Kentucky Bioprocessing). However, the challenges of transgenic plant expression still include high levels of endotoxin and low expression levels. Another concern is the secretion of proteases, which shortens the validity period of plant extracts.

In addition, current concerns about the growth of genetically modified plants limit the scalability of this technology, and the implementation of large-scale production depends on public approval. The requirement to separate genetically modified plants from the general ecosystem means that their cultivation can only be limited to large, automated greenhouses. This limits the rapid expansion of this technology. A lot of research has been done in this field, and plant expression may be a technology for large-scale commercial production in the future.

E. coli can produce non-glycosylated monoclonal antibodies and antibody fragments both intracellularly and extracellularly. It is very attractive that E. coli can be cultured quickly and reach high expression levels. However, E. coli does not have a glycosylation mechanism, so if glycosylation is important for activity, this may be a major limitation that limits its application. At present, E. coli is mainly used as a supplement to the monoclonal antibody production platform and is only used for the production of clinical samples of antibody fragments.

Yeast expression system has been used in clinical production. In particular, Saccharomyces cerevisiae has been used to express a variety of commercial biological therapies. However, a key limitation is the production of excessive non-mammalian glycosylation patterns in S. cerevisiae. In addition, due to misexpression and folding in the endoplasmic reticulum, the expression level of full-length monoclonal antibodies in S. cerevisiae is limited. Pichia pastoris is a better recombinant protein expression system. This is a methylotrophic yeast that can be cultured at a very high cell density. The promoters used in the Pichia pastoris system are very powerful and will produce significant expression levels (up to 20 g/L) in the case of extracellular secretion. The glycosylation of Pichia pastoris is less than that of beer yeast. The engineered strain of Pichia pastoris eliminates the problem of protease expression and at the same time inhibits the production of high mannose. Another challenge facing this system is the lack of proper protein folding partners in this expression system. As a result, the product can exist in many forms. However, with the development of engineered strains of Pichia pastoris, this obstacle can be overcome. The high production capacity of Pichia pastoris makes it a candidate expression system for monoclonal antibodies in the future.

Another emerging platform for biopharmaceutical production is the microalgae production system. Microalgae are photosynthetic microorganisms that can be cultivated in a large number of fermenters. Microalgae have been used in the production of industrial biotechnology products. The output of the current microalgae fermentation system is still relatively low. Before this expression system can accept biopharmaceutical production, other obstacles need to be overcome, including glycosylation and other post-transcriptional modifications.

5 Conclusion

This article discusses the monoclonal antibody platform approach and its need to accelerate the clinical and market development of many different therapies. The use of a platform approach enables many biopharmaceutical companies to successfully succeed from genes in a year or less. Based on their internal antibody structure, cell lines and cell culture process, each biopharmaceutical organization has developed its most suitable platform method. The latest trends include the use of multimodal tomography as part of the process platform and the use of a two-step refining process in one flow mode. These process improvements make the monoclonal antibody platform more widely applicable, as well as meaningful to the throughput bottleneck of downstream processing.

With the continuous improvement of cell culture capabilities, other alternative forms that can increase the productivity of downstream processes are also constantly being developed. These include the operation of the protein in a continuous mode, rather than a batch mode. It is conceivable that continuous processing can be developed in the entire downstream process in the future. The non-chromatographic separation process using precipitation or ATPS is another possible future direction for the downstream process of monoclonal antibodies. The next decade will see further development of the monoclonal antibody downstream process platform, which is driven by productivity and new molecular models.

references:
Shukla AA, Wolfe LS, Mostafa SS, Norman C. Evolving trends in mAb production processes.Bioeng Transl Med. 2017 Apr 3;2(1):58-69.

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