Monoclonal antibodies (mAbs) are an important part of biopharmaceuticals, which are used to treat diseases such as cancers, autoimmune conditions, and infectious diseases. Production of these antibodies involves complex biological processes to ensure high quality, efficiency, and consistency. The demand for mAbs continues to rise due to their therapeutic efficacy, driving advancements in production and process optimization.
Cell Line Development
The production of mAbs starts with the development of suitable cell lines. Chinese Hamster Ovary (CHO) cells are known to be the preferred host system due to their ability to perform complex human-like glycosylation, which is critical for the efficacy of mAbs. In addition, CHO cells are very stable, easy to work with, and possess high productivity. These CHO cells are well-accepted by regulatory agencies worldwide and are therefore the gold standard for biomanufacturing.
The transfer of the gene of the antibody of interest into Chinese Hamster Ovary (CHO) cells is the first step in mAb production. This is done using plasmid vectors that are short pieces of DNA carrying the gene of the antibody and selection markers. Selection markers are employed in the selection of the cells that have taken in the gene by making only such cells grow in specific conditions.
After transfection, the cells are subjected to a selection process to isolate the cells that have incorporated the antibody gene. The isolated cells are cloned, i.e., individual cells are separated and grown into new cell lines. The cloned cells are screened very rigorously to choose the cells that produce the most antibodies and are genetically stable in the long term.
Advanced technologies such as fluorescence-activated cell sorting (FACS) and automated imaging platforms speed up the detection of the most productive cells. They allow researchers to screen and identify the most productive and stable cell lines at high speed.
It is important that the final cell line is genetically stable and produces antibodies consistently over many generations. This ensures that the manufacturing process is uniform with strict regulatory requirements to produce safe and effective mAbs.
Sophisticated genetic technologies such as CRISPR/Cas9 have revolutionized cell line development efficiency. These technologies enable gene integration of antibodies into a desired genomic location that increases yield as well as consistency. Multiplex gene editing is being used by companies nowadays to engineer CHO cells with improved metabolic pathways to improve cell viability and reduce production bottlenecks.
Upstream Processing
The production process in bioreactors begins by warming frozen cells from a working cell bank (WCB). The cells are gradually expanded through a series of shake flasks and small bioreactors until they’re ready to inoculate a large production bioreactor. This stage aims to generate enough viable cells to produce significant quantities of monoclonal antibodies. The scale-up process is carefully monitored to maintain cell health and to ensure optimal growth conditions.
The mAb production mainly uses two types of bioreactor operations: fed-batch and perfusion. In a fed-batch system, the cells grow in a controlled environment while receiving regular nutrient feeds. This method helps achieve higher cell density and increases antibody yield. The process usually takes 10–20 days to complete.
In contrast, perfusion bioreactors work by continuously adding fresh media while removing waste products. This allows the cell culture to stay active for a long time—up to 60 days—and supports even greater productivity. However, perfusion systems are more complex and need advanced monitoring and control systems to maintain stable conditions throughout the process.
Media formulation is very important in upstream processing. Chemically defined media are now commonly used because they’re more consistent and do not contain animal-based ingredients. Improving the media and how nutrients are fed helps cells grow better and produce higher quality products. New methods like dynamic flux balance analysis (DFBA) study cell metabolism to find the best nutrient mix, increasing productivity.
Downstream Processing
After mAbs are produced, they’re harvested from the culture media by separating the cells from the product using centrifugation and depth filtration. The mAbs then go through a multi-step purification process to ensure they’re safe and effective. The most important step is Protein A chromatography, where the antibodies bind to special Protein A resin, allowing them to be selectively captured. Additional purification steps, like ion-exchange chromatography and hydrophobic interaction chromatography, remove unwanted substances such as host cell proteins (HCP) and DNA. To ensure the product is safe for patients, viral inactivation and filtration are performed to eliminate any potential viral contaminants.
New advanced techniques are being developed to make the purification process faster and more cost-effective. Some alternatives to Protein A chromatography, such as multimodal chromatography and precipitation techniques, are being tested. Multi-column continuous chromatography, like periodic counter-current chromatography, allows for quicker and more efficient purification while using less resin, which helps reduce costs. Ensuring that all viruses are removed is also a key step, usually done through low pH inactivation and virus filtration to maintain product safety.
Challenges and Technological Innovations
One of the biggest challenges in mAb production is ensuring consistent product quality across different batches. Important product characteristics, known as critical quality attributes (CQAs), such as glycosylation profiles and aggregation levels, must be carefully monitored and controlled. To achieve this, real-time monitoring tools like Raman spectroscopy and multi-analyte technology (PAT) frameworks also help by enabling real-time decision-making and adaptive control of the process.
Process variability is another concern because small changes in cell culture conditions can affect the quality of the final product. To address this, Quality by Design (QbD) methods are used to set acceptable variability limits, which improves process understanding and ensures compliance with regulatory standards. Advanced technologies like digital twins (virtual models of the production process) and hybrid models (which combine data-driven and physical approaches) are now being used to optimize and predict production outcomes in real time.
Recent innovations focus on process intensification, which aims to increase productivity while reducing costs and shortening production times. For example, N-1 perfusion is a method where cells are expanded before entering the main bioreactor, allowing for faster growth and higher antibody yields. Another advanced method, dynamic flux balance analysis (DFBA), helps optimize media composition and feeding strategies, improving cell viability and overall production efficiency.
Future Directions
The future of mAb production is moving towards continuous manufacturing, where upstream and downstream processes are combined in a single, uninterrupted operation. This method can reduce production times and lower costs while ensuring high product quality. Advances in automation, machine learning, and single-use technologies (SUT) are also transforming the industry. Automated control systems, combined with real-time analytics, allow manufacturers to adjust bioreactor conditions in real time, improving efficiency and consistency.
Single-use technology (SUT) is becoming more popular for both upstream and downstream processes because it is flexible and reduces the risk of cross-contamination. Although current SUT bioreactors are limited to about 2000 liters, future improvements in materials and design may allow for larger-scale production. Hybrid facilities, which combine single-use and stainless-steel systems, provide flexibility to produce multiple products within the same facility.
New purification techniques are also emerging to replace traditional Protein A chromatography, which is expensive and requires thorough validation. Methods like multimodal chromatography and membrane-based technologies offer promising alternatives that are more cost-effective and efficient. As genetic engineering and bioprocess optimization continue to advance, the industry is moving towards scalable, flexible, and cost-efficient manufacturing methods to meet the increasing demand for mAbs.
In the future, the integration of new technologies, real-time monitoring, and advanced process control systems will lead to faster, more efficient mAb production. This will ensure high-quality treatments are produced and delivered affordably to patients worldwide.
References
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Author
Mr. Akshay Yalameli
Second Year, B.Tech. Biotechnology Student
Dr. D. Y. Patil Biotechnology and Bioinformatics Institute,
Dr. D. Y. Patil Vidyapeeth, Tathawade, Pune