Cost Analysis and Economic Considerations in Large-Scale Protein Purification

1. Introduction

In the realm of biopharmaceutical production, the purification of proteins at a large scale is a critical yet challenging endeavor. The transition from laboratory-scale experiments to industrial-scale production necessitates meticulous planning and execution to maintain the quality, yield, and economic viability of the process. Downstream processing, which encompasses the purification stages, is often the most complex and costly phase of protein production. It involves several steps, including cell removal, protein capture, intermediate purification, and final polishing to ensure that the product meets the required purity and potency standards.

The importance of cost analysis and economic considerations in large-scale protein purification cannot be overstated. Biopharmaceutical companies are under constant pressure to reduce production costs while adhering to stringent regulatory requirements and ensuring the safety and efficacy of their products. The cost structure of downstream processing is influenced by various factors, including the choice of purification techniques, scalability of the process, equipment costs, consumables, labor, and compliance with Good Manufacturing Practices (GMP).

Understanding the economic landscape of protein purification is crucial for making informed decisions that balance cost and quality. This involves evaluating the cost-effectiveness of different purification strategies and implementing process improvements that enhance productivity while minimizing expenses. By addressing the economic challenges of downstream processing, biopharmaceutical companies can achieve a competitive edge in the market, ensuring the delivery of high-quality products at reduced costs. [1]

This article will analyze the various costs of large-scale expression in E. coli, yeast, insect and mammalian cell expression systems.

Fig.1 Recombinant proteins: useful reagents for many different applications. [21]

2. Cost Analysis in E. coli Expression Systems

Escherichia coli (E. coli) is one of the most widely used expression systems for the production of recombinant proteins. Its popularity stems from its well-understood genetics, ease of manipulation, rapid growth, and cost-effective cultivation. However, scaling up protein production in E. coli to an industrial level presents several economic challenges that must be addressed to maintain cost efficiency and product quality.

2.1 Production Costs:

Media and Fermentation: The cost of culture media and fermentation is a significant factor in the overall production costs. E. coli can be grown in relatively simple and inexpensive media, but optimizing media composition to enhance yield and reduce costs is crucial. High-density fermentation can maximize productivity but also increases the complexity of downstream processing. [2]

Induction Systems: The choice of induction system (e.g., IPTG induction) affects both the cost and efficiency of protein expression. While IPTG is a potent inducer, it is also relatively expensive. Alternatives such as auto-induction media can reduce costs and simplify the process.

Downstream Processing:

Cell Lysis: Efficient cell lysis methods are essential to release the target protein from E. coli cells. Mechanical methods (e.g., high-pressure homogenization) and chemical methods (e.g., detergents) have different cost implications. Mechanical lysis is effective but requires significant capital investment in equipment, while chemical lysis involves recurring costs for reagents. [3]

Protein Solubility and Inclusion Bodies: One of the major challenges in E. coli expression systems is the formation of inclusion bodies, where the protein aggregates and becomes insoluble. Solubilizing and refolding these proteins to their active forms adds to the downstream processing costs. Developing strategies to enhance protein solubility during expression can mitigate these costs.

2.2 Purification Techniques:

Affinity Chromatography: Affinity tags such as His-tags are commonly used to simplify purification. While affinity chromatography resins are relatively expensive, they provide high purity and yield, reducing the number of purification steps required. Optimizing the use of these resins and exploring cost-effective alternatives (e.g., mixed-mode chromatography) can reduce expenses.

Intein-Mediated Purification: The intein-mediated system, which allows for the tag-free purification of proteins, can be more cost-effective in the long run by reducing the need for additional purification steps and reagents. [4]

2.3 Scalability and Validation:

Process Scalability: Scaling up the purification process from laboratory to industrial scale requires careful consideration of equipment and process parameters. Pilot-scale studies are essential to identify potential issues and optimize conditions. The scalability of each step must be validated to ensure consistent performance and product quality. [5]

Regulatory Compliance: Compliance with GMP and other regulatory standards adds to the cost but is non-negotiable for biopharmaceutical products. Ensuring robust process validation and thorough documentation is essential to avoid costly regulatory setbacks.

2.4 Technological Innovations:

Continuous Processing: Adopting continuous processing techniques can enhance efficiency and reduce costs by minimizing downtime and improving resource utilization. Continuous centrifugation and membrane filtration are examples of technologies that can streamline E. coli protein purification.

Single-Use Technologies: Implementing single-use systems can lower initial capital investment and reduce cleaning and validation costs. These systems offer flexibility and are particularly useful for multiproduct facilities. [6]

In conclusion, the economic considerations of using E. coli as an expression system for large-scale protein purification involve a careful balance of production, downstream processing, scalability, and regulatory compliance. By optimizing each stage of the process and embracing technological innovations, biopharmaceutical companies can achieve cost-effective production while maintaining high product quality.

3. Cost Analysis in Yeast Expression Systems

Yeast, particularly species like Saccharomyces cerevisiae and Pichia pastoris, is another widely used expression system for the production of recombinant proteins. Yeast expression systems offer several advantages, including the ability to perform post-translational modifications, high yield production, and relatively low-cost cultivation. However, scaling up protein production in yeast to an industrial level involves economic challenges that need to be addressed to optimize costs and maintain efficiency. [7]

3.1 Production Costs:

Media and Fermentation: The cultivation of yeast requires a nutrient-rich media, which can be more costly than bacterial media. Optimizing media composition to balance cost and productivity is crucial. High-density fermentation processes can improve yields but may also increase the complexity and cost of downstream processing.

Induction Systems: Pichia pastoris often uses methanol induction for protein expression, which can be costly and hazardous. Exploring alternative induction systems or auto-induction methods can help reduce costs and improve safety.

Downstream Processing:

Cell Lysis: Yeast cells have tougher cell walls compared to bacteria, making cell lysis more challenging and costly. Methods such as high-pressure homogenization, bead milling, or enzymatic lysis are commonly used. Each method has different cost implications, with mechanical methods requiring significant capital investment and enzymatic methods incurring recurring reagent costs. [8]

Glycosylation and Protein Heterogeneity: Yeast can perform post-translational modifications, such as glycosylation, which are crucial for the functionality of many therapeutic proteins. However, yeast-specific glycosylation patterns may differ from those in humans, potentially affecting product quality. This necessitates additional purification steps to ensure consistent glycosylation, adding to the costs.

3.2 Purification Techniques:

Affinity Chromatography: Affinity tags, such as His-tags or FLAG-tags, facilitate the initial capture of the target protein. While affinity chromatography is effective, the cost of resins can be high. Optimizing resin usage and exploring alternative purification methods, like mixed-mode chromatography, can help reduce expenses. [9]

Ion Exchange and Hydrophobic Interaction Chromatography: These techniques are often used for intermediate purification and polishing steps. Ion exchange and hydrophobic interaction chromatography can be cost-effective alternatives to affinity chromatography, but they require careful optimization to ensure high purity and yield.

3.3 Scalability and Validation:

Process Scalability: Scaling up the purification process from laboratory to industrial scale requires careful planning and validation. Pilot-scale studies are essential to identify potential issues and optimize conditions. Ensuring scalability of each step is crucial for maintaining performance and product quality at larger volumes. [10]

Regulatory Compliance: Compliance with GMP and other regulatory standards is essential for biopharmaceutical production. This involves rigorous process validation, thorough documentation, and ongoing quality control, all of which add to the cost. However, robust validation can prevent costly regulatory setbacks and ensure market access.

3.4 Technological Innovations:

Continuous Processing: Continuous processing techniques can enhance efficiency and reduce costs by minimizing downtime and improving resource utilization. Technologies like continuous centrifugation and membrane filtration are particularly beneficial for yeast protein purification.

Single-Use Technologies: Single-use systems offer flexibility and reduce cleaning and validation costs, making them ideal for multiproduct facilities. Implementing single-use bioreactors, tubing, and filters can lower initial capital investment and operational costs.

3.5 Energy Efficiency:

Energy Consumption: Yeast fermentation processes can be energy-intensive, especially at large scales. Implementing energy-efficient practices, such as optimizing fermentation conditions and using energy-saving equipment, can lead to substantial cost savings. [11]

Sustainable Practices: Adopting sustainable practices, like recycling heat and using renewable energy sources, can further reduce energy costs and contribute to environmental sustainability.

In conclusion, the economic considerations of using yeast as an expression system for large-scale protein purification involve a careful balance of production costs, downstream processing efficiency, scalability, and regulatory compliance. By optimizing each stage of the process and embracing technological innovations, biopharmaceutical companies can achieve cost-effective production while maintaining high product quality.

4. Cost Analysis in Insect Expression Systems

Insect cell expression systems, particularly the Baculovirus Expression Vector System (BEVS) using Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tn) cells, are valuable for producing complex recombinant proteins. These systems offer several advantages, such as high protein yields and the ability to perform complex post-translational modifications. However, they also present unique economic challenges that need to be addressed for cost-effective large-scale production.

4.1 Production Costs:

Media and Cultivation: Insect cells require specific media formulations that are more expensive than bacterial or yeast media. The optimization of media composition to enhance cell growth and protein expression while reducing costs is essential. High-density cell culture techniques can improve yields but may increase the complexity and cost of downstream processing. [12]

Baculovirus Production: Producing and maintaining baculovirus stocks for infection can be costly. Ensuring high-quality and high-titer baculovirus stocks is critical for consistent protein production. Strategies to optimize baculovirus production and reduce costs include improving viral stability and infection efficiency.

4.2 Downstream Processing:

Cell Lysis and Clarification: Efficient cell lysis and clarification are necessary to release recombinant proteins from insect cells. Methods such as mechanical disruption or detergent lysis can be used, each with different cost implications. Mechanical lysis requires significant capital investment in equipment, while detergents add to recurring costs.

Protein Glycosylation: Insect cells can perform post-translational modifications, including glycosylation, which are essential for the functionality of many therapeutic proteins. However, the glycosylation patterns in insect cells differ from those in mammalian cells, potentially affecting product quality. Additional purification steps may be required to ensure consistent glycosylation, increasing costs. [13]

4.3 Purification Techniques:

Affinity Chromatography: Affinity tags, such as His-tags or FLAG-tags, are often used to facilitate the initial capture of the target protein. Affinity chromatography resins are effective but expensive. Optimizing resin usage and exploring alternative purification methods, such as mixed-mode chromatography, can help reduce costs.

Size Exclusion and Ion Exchange Chromatography: These techniques are commonly used for intermediate purification and polishing steps. They are generally more cost-effective than affinity chromatography but require careful optimization to ensure high purity and yield. [14]

4.4 Scalability and Validation:

Process Scalability: Scaling up insect cell culture and downstream processing from laboratory to industrial scale requires careful planning and validation. Pilot-scale studies are essential to identify potential issues and optimize conditions. Ensuring scalability of each step is crucial for maintaining performance and product quality at larger volumes.

Regulatory Compliance: Compliance with GMP and other regulatory standards is crucial for biopharmaceutical production. This involves rigorous process validation, thorough documentation, and ongoing quality control, all of which add to the cost. Robust validation processes can prevent costly regulatory setbacks and ensure market access.

4.5 Technological Innovations:

Continuous Processing: Adopting continuous processing techniques can enhance efficiency and reduce costs by minimizing downtime and improving resource utilization. Continuous centrifugation and membrane filtration are examples of technologies that can streamline insect cell protein purification. [15]

Single-Use Technologies: Single-use systems offer flexibility and reduce cleaning and validation costs, making them ideal for multiproduct facilities. Implementing single-use bioreactors, tubing, and filters can lower initial capital investment and operational costs.

4.6 Energy Efficiency:

Energy Consumption: Insect cell culture processes can be energy-intensive, especially at large scales. Implementing energy-efficient practices, such as optimizing cultivation conditions and using energy-saving equipment, can lead to substantial cost savings.

Sustainable Practices: Adopting sustainable practices, such as recycling heat and using renewable energy sources, can further reduce energy costs and contribute to environmental sustainability. [16]

In conclusion, the economic considerations of using insect cells as an expression system for large-scale protein purification involve a careful balance of production costs, downstream processing efficiency, scalability, and regulatory compliance. By optimizing each stage of the process and embracing technological innovations, biopharmaceutical companies can achieve cost-effective production while maintaining high product quality.

5. Cost Analysis in Mammalian Cell Expression Systems

Mammalian cell expression systems, particularly Chinese hamster ovary (CHO) cells, are the gold standard for producing complex therapeutic proteins, including monoclonal antibodies and other glycoproteins. These systems offer several advantages, such as the ability to perform human-like post-translational modifications, but they also present significant economic challenges that must be managed effectively for large-scale production.

5.1 Production Costs:

Media and Culture Conditions: Mammalian cells require complex and nutrient-rich media, which are more expensive than those used for bacterial or yeast systems. Additionally, the culture conditions (e.g., controlled temperature, CO2 levels) add to the operational costs. Optimizing media formulations and culture conditions can enhance cell growth and protein yield while reducing costs.

Cell Line Development: Developing a stable, high-yielding cell line is time-consuming and expensive. This process involves multiple stages, including transfection, selection, and cloning. Investing in robust cell line development processes can reduce variability and improve long-term productivity, ultimately lowering production costs. [17]

5.2 Downstream Processing:

Harvest and Clarification: The large size and fragility of mammalian cells require gentle but effective methods for cell lysis and clarification. Centrifugation and depth filtration are commonly used, but they need to be optimized to balance efficiency and cost. These steps can be expensive due to the need for specialized equipment and consumables.

Protein Glycosylation and Heterogeneity: One of the main advantages of mammalian cells is their ability to perform human-like glycosylation. However, this also introduces variability, requiring additional purification steps to ensure product consistency. Managing glycosylation heterogeneity is crucial for maintaining product quality, but it adds to the purification costs.

5.3 Purification Techniques:

Affinity Chromatography: Protein A affinity chromatography is widely used for the purification of monoclonal antibodies due to its high specificity and yield. However, Protein A resins are expensive, and their cost needs to be managed through optimization and recycling strategies. Alternative purification methods, such as mixed-mode chromatography, can be explored to reduce costs.

Polishing Steps: Additional purification steps, such as ion exchange and size exclusion chromatography, are necessary to remove impurities and achieve the desired product quality. These steps can be costly, and their efficiency must be maximized to reduce overall expenses.

5.4 Scalability and Validation:

Process Scalability: Scaling up mammalian cell culture and downstream processing requires careful planning and validation. Pilot-scale studies are essential to identify potential issues and optimize conditions. Ensuring scalability of each step is crucial for maintaining performance and product quality at larger volumes. [18]

Regulatory Compliance: Compliance with GMP and other regulatory standards is essential for biopharmaceutical production. This involves rigorous process validation, thorough documentation, and ongoing quality control, all of which add to the cost. Robust validation processes can prevent costly regulatory setbacks and ensure market access.

5.5 Technological Innovations:

Continuous Processing: Adopting continuous processing techniques can enhance efficiency and reduce costs by minimizing downtime and improving resource utilization. Technologies such as continuous centrifugation and perfusion culture systems are particularly beneficial for mammalian cell protein purification.

Single-Use Technologies: Single-use systems offer flexibility and reduce cleaning and validation costs, making them ideal for multiproduct facilities. Implementing single-use bioreactors, tubing, and filters can lower initial capital investment and operational costs.

5.6 Energy Efficiency:

Energy Consumption: Mammalian cell culture processes are energy-intensive, especially at large scales. Implementing energy-efficient practices, such as optimizing culture conditions and using energy-saving equipment, can lead to substantial cost savings.

Sustainable Practices: Adopting sustainable practices, such as recycling heat and using renewable energy sources, can further reduce energy costs and contribute to environmental sustainability.

In conclusion, the economic considerations of using mammalian cells as an expression system for large-scale protein purification involve a careful balance of production costs, downstream processing efficiency, scalability, and regulatory compliance. By optimizing each stage of the process and embracing technological innovations, biopharmaceutical companies can achieve cost-effective production while maintaining high product quality.

6. Other consumption

In addition to the purification system, there are some unavoidable consumable costs.

6.1 Chromatography media and resins:

Chromatography is the cornerstone of protein purification and is used to capture and refine the target protein. The choice of chromatography media and resin greatly affects the efficiency and cost of the process. Although more expensive, high-performance resins generally have excellent binding capacity and selectivity, which can improve yield and purity. However, balancing the cost and performance of these resins is critical. [19]

To manage costs, companies can explore bulk purchasing agreements, resin regeneration and reuse strategies, and alternative chromatography methods such as mixed-mode chromatography, which can reduce reliance on expensive affinity resins.

6.2 Filtration systems:

Filtration is another critical step in downstream processing and is used for cell removal, concentration, and buffer exchange. The scalability of filtration systems poses challenges, especially in maintaining filtration efficiency and reducing fouling. Costs associated with filtration include the purchase of filter membranes, equipment, and ongoing consumables.

Strategies to optimize filtration costs include selecting high-capacity filters that require fewer changes, implementing prefiltration steps to reduce the load on the final filter, and optimizing process conditions to extend the life of the filter.

6.3 Labor and operating costs:

The labor involved in operating and maintaining a purification system has a large impact on total costs. Skilled technicians and operators are essential to ensure the smooth operation of complex purification processes, and their expertise is invaluable. In addition, operating costs include utilities, maintenance, and process monitoring.

Automation of the purification process can reduce labor costs and improve consistency. Investing in automated chromatography systems, online monitoring, and control technologies can streamline operations and reduce the need for human intervention. [20]

6.4 Buffer preparation and consumption:

The preparation and use of buffers account for a considerable cost in protein purification. Buffers need to be used in large quantities, and the cost of raw materials, preparation, and handling can quickly increase.

Strategies to reduce costs include optimizing buffer formulations to minimize waste, recycling buffers whenever possible, and implementing continuous buffer exchange systems to reduce the total amount of buffer required.

6.5 Equipment and facility costs:

The capital investment in purification equipment and facilities is significant. Large-scale bioprocessing requires specialized equipment, including bioreactors, chromatography columns, filtration units, and ancillary systems. GMP-compliant facility design and maintenance further increase costs.

Single-use technologies are becoming increasingly popular because they reduce the need for cleaning and sterilization between batches, thereby reducing equipment and facility costs. Single-use systems also offer the flexibility to scale up operations and are particularly cost-effective for smaller production runs.

6.6 Regulatory Compliance:

In biopharmaceutical manufacturing, adhering to regulatory requirements is non-negotiable. Costs associated with compliance include process validation, quality control testing, and documentation. Ensuring that purification processes meet regulatory standards is critical for product approval and market access.

Efficient process validation and a strong quality management system can streamline compliance efforts and reduce associated costs. Investing in comprehensive validation protocols and regular audits can prevent costly regulatory setbacks.

In summary, managing the costs of large-scale protein purification involves a multifaceted approach to address the various cost components of the process. Biopharmaceutical companies can achieve significant cost savings by optimizing the use of chromatography resins, filtration systems, labor, buffers, equipment, and ensuring regulatory compliance. Employing technological innovations and strategic process improvements can further improve the economic viability of protein purification, making high-quality therapeutic proteins more accessible and affordable.

7. Conclusion

The economic considerations of large-scale protein purification are multifaceted and require a strategic approach to ensure cost-effectiveness while maintaining high product quality. From the choice of expression systems to downstream processing techniques, each step in the production chain must be carefully optimized to balance costs and efficiency.

1. Expression Systems:

Each expression system, whether it be E. coli, yeast, insect, or mammalian cells, has unique advantages and economic challenges. E. coli offers cost-effective cultivation but poses challenges in protein solubility and inclusion bodies. Yeast systems can perform post-translational modifications but require more expensive media and present difficulties in cell lysis. Insect cells excel in producing complex proteins with proper folding but involve higher costs in media and baculovirus production. Mammalian cells, particularly CHO cells, are the gold standard for producing therapeutic proteins but require nutrient-rich media and careful control of culture conditions to manage costs.

2. Downstream Processing:

Downstream processing is often the most expensive phase in protein production. It includes cell lysis, protein capture, and various purification steps. Techniques such as affinity chromatography, ion exchange, and size exclusion chromatography must be optimized for cost-efficiency and yield. The choice of purification methods and the scalability of these processes play a crucial role in determining the overall production costs. Continuous processing techniques and single-use technologies can significantly reduce costs by improving efficiency and reducing downtime.

3. Technological Innovations:

Adopting technological innovations such as continuous processing and single-use systems can enhance the economic viability of protein purification. Continuous processing minimizes downtime and improves resource utilization, leading to significant cost savings. Single-use technologies reduce the need for cleaning and sterilization, lower initial capital investments, and provide flexibility for multiproduct facilities.

4. Regulatory Compliance:

Ensuring regulatory compliance is essential for market approval and involves rigorous process validation, thorough documentation, and ongoing quality control. These steps add to the overall costs but are non-negotiable for maintaining product safety and efficacy. Efficient process validation and robust quality management systems can streamline compliance efforts and prevent costly regulatory setbacks.

5. Energy Efficiency and Sustainability:

Implementing energy-efficient practices and sustainable processes can further reduce costs and contribute to environmental sustainability. Optimizing culture conditions, using energy-saving equipment, recycling heat, and utilizing renewable energy sources can significantly lower energy consumption in large-scale protein purification processes.

In summary, managing the costs of large-scale protein purification requires a holistic approach that addresses production, downstream processing, scalability, regulatory compliance, and energy efficiency. By optimizing each stage of the process and embracing technological innovations, biopharmaceutical companies can achieve cost-effective production while maintaining high product quality. This strategic approach not only ensures the economic viability of protein purification but also enhances the accessibility and affordability of high-quality therapeutic proteins.

FAQ

1. What are the primary cost drivers in large-scale protein purification?

The main cost drivers include the choice of expression system, media and culture conditions, downstream processing techniques, equipment costs, labor, and regulatory compliance. Each of these factors needs careful optimization to balance cost and quality.

2. How do single-use technologies help in reducing costs in protein purification?

Single-use technologies reduce initial capital investment and operational costs by eliminating the need for cleaning and sterilization between batches. They offer flexibility and are particularly beneficial for multiproduct facilities, allowing for quicker turnaround and reduced contamination risks.

3. What are the benefits of using continuous processing in protein purification?

Continuous processing enhances efficiency by minimizing downtime and improving resource utilization. It can lead to significant cost savings by reducing the footprint of manufacturing facilities, lowering labor costs, and ensuring consistent product quality with fewer deviations.

4. Why is regulatory compliance a significant cost factor in protein purification?

Regulatory compliance involves rigorous process validation, thorough documentation, and ongoing quality control to meet Good Manufacturing Practices (GMP) and other standards. These steps are essential to ensure product safety and efficacy but add to the overall costs of biopharmaceutical production.

5. How can energy-efficient practices contribute to cost savings in protein purification?

Implementing energy-efficient practices, such as optimizing culture conditions and using energy-saving equipment, can reduce the energy consumption of large-scale protein purification processes. Sustainable practices, like recycling heat and using renewable energy sources, further contribute to cost savings and environmental sustainability.

Reference

[1] Stevens RC. Design of high-throughput methods of protein production for structural biology. Structure. 2000;8(9):R177-R185. doi:10.1016/s0969-2126(00)00193-3
[2] Gupta SK, Shukla P. Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review. Front Pharmacol. 2017;8:419. Published 2017 Jul 4. doi:10.3389/fphar.2017.00419
[3] Aricescu AR, Lu W, Jones EY. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr. 2006;62(Pt 10):1243-1250. doi:10.1107/S0907444906029799
[4] Zhao Y, Bishop B, Clay JE, et al. Automation of large scale transient protein expression in mammalian cells. J Struct Biol. 2011;175(2):209-215. doi:10.1016/j.jsb.2011.04.017
[5] Tian L, Sun SS. A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform. PLoS One. 2011;6(8):e24183. doi:10.1371/journal.pone.0024183
[6] Milne JJ. Scale-Up of Protein Purification: Downstream Processing Issues. Methods Mol Biol. 2017;1485:71-84. doi:10.1007/978-1-4939-6412-3_5
[7] Sharma SS, Chong S, Harcum SW. Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system. Appl Biochem Biotechnol. 2005;126(2):93-118. doi:10.1385/abab:126:2:093
[8] Vicente T, Roldão A, Peixoto C, Carrondo MJ, Alves PM. Large-scale production and purification of VLP-based vaccines. J Invertebr Pathol. 2011;107 Suppl:S42-S48. doi:10.1016/j.jip.2011.05.004
[9] Zhang C, Long AM, Swalm B, et al. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography. Protein Expr Purif. 2016;128:29-35. doi:10.1016/j.pep.2016.08.005
[10] Kelley B. Very large scale monoclonal antibody purification: the case for conventional unit operations. Biotechnol Prog. 2007;23(5):995-1008. doi:10.1021/bp070117s
[11] Peti W, Page R. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif. 2007;51(1):1-10. doi:10.1016/j.pep.2006.06.024
[12] Beshay U, Miksch G, Friehs K, Flaschel E. Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli [published correction appears in Bioprocess Biosyst Eng. 2009 Feb;32(2):159]. Bioprocess Biosyst Eng. 2009;32(2):149-158. doi:10.1007/s00449-008-0227-3
[13] Hunt I. From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expr Purif. 2005;40(1):1-22. doi:10.1016/j.pep.2004.10.018
[14] Schwarz D, Klammt C, Koglin A, et al. Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods. 2007;41(4):355-369. doi:10.1016/j.ymeth.2006.07.001
[15] Shukla AA, Thömmes J. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol. 2010;28(5):253-261. doi:10.1016/j.tibtech.2010.02.001
[16] Bommarius B, Jenssen H, Elliott M, et al. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides. 2010;31(11):1957-1965. doi:10.1016/j.peptides.2010.08.008
[17] Sullivan CJ, Pendleton ED, Sasmor HH, et al. A cell-free expression and purification process for rapid production of protein biologics. Biotechnol J. 2016;11(2):238-248. doi:10.1002/biot.201500214
[18] Sarramegna V, Talmont F, Demange P, Milon A. Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification. Cell Mol Life Sci. 2003;60(8):1529-1546. doi:10.1007/s00018-003-3168-7
[19] Korn J, Schäckermann D, Kirmann T, et al. Baculovirus-free insect cell expression system for high yield antibody and antigen production. Sci Rep. 2020;10(1):21393. Published 2020 Dec 7. doi:10.1038/s41598-020-78425-9
[20] Kittler S, Ebner J, Besleaga M, et al. Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand. J Biotechnol. 2022;359:108-115. doi:10.1016/j.jbiotec.2022.10.002
[21] Remans K, Lebendiker M, Abreu C, et al. Protein purification strategies must consider downstream applications and individual biological characteristics. Microb Cell Fact. 2022;21(1):52. Published 2022 Apr 7. doi:10.1186/s12934-022-01778-5