From lab to clinic: Large-scale recombinant protein production explained

Recombinant protein manufacturing has transformed biotechnology, enabling the production of therapeutic proteins, industrial enzymes, and bioengineered materials. The growing demand for recombinant proteins across regenerative medicine, disease diagnostics, and biopharmaceuticals has enhanced the importance of Contract Development and Manufacturing Organizations (CDMOs). These organizations bring essential expertise in process development, regulatory compliance, and large-scale production, ensuring the delivery of high-quality recombinant proteins. This review examines the critical role of CDMOs in optimizing recombinant protein manufacturing, exploring current challenges, innovations, and future advancements.

Bridging the gap between research and the patient

CDMOs serve as a crucial link between research institutions and commercial biomanufacturing by providing specialized services such as expression system optimization, purification strategies, and adherence to Good Manufacturing Practice (GMP) standards. CDMOs play a critical role in optimizing these expression platforms, ensuring high-yield, scalable, and regulatory-compliant production. These vital contributions in advancing recombinant protein production have a profound impact on the biopharmaceutical industry. As the demand for high-quality, scalable protein production escalates, CDMOs continue to be instrumental in providing dependable solutions.

Recombinant protein manufacturing

Recombinant protein expression is a cornerstone of scientific research and biopharmaceutical development, offering versatile solutions across various therapeutic applications. Bacterial and mammalian cell systems remain the most widely used platforms, each with distinct advantages.

Bacterial expression systems, particularly Escherichia coli, remain a cornerstone of recombinant protein production due to their rapid growth, ease of genetic manipulation, and cost-effectiveness. These systems offer high expression efficiency, making them ideal for large-scale manufacturing of simple proteins, enzymes, and non-glycosylated therapeutic proteins. However, bacterial hosts lack the intricate post-translational modification (PTM) machinery found in eukaryotic cells. This limitation can impact the correct folding, disulfide bond formation, and glycosylation of complex proteins, potentially affecting their functionality. Despite these challenges, bacterial systems continue to be a preferred choice for applications where process duration, scalability, and cost efficiency are paramount.

Mammalian cell systems, such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, are the gold standard for producing complex biopharmaceuticals. These platforms excel in generating proteins with native folding, proper post-translational modifications (PTMs), and human-like glycosylation—key factors in ensuring the efficacy and safety of monoclonal antibodies, recombinant vaccines, and other therapeutic proteins. With well-optimized expression technologies, mammalian systems offer a reliable and scalable solution for producing high-quality biologics, making them essential for advancing next-generation therapeutics.

A comprehensive approach to GMP-compliant large-scale recombinant protein production

Biopharmaceutical CDMO NorthX Biologics specializes in bacterial and mammalian cell culture processes for recombinant protein production. When embarking a new client project, a team of experts takes the gene sequence through gene cloning, cell bank generation, protein expression to final isolation and purification.

1. Expression system selection
   Choosing the appropriate expression system is a critical factor in recombinant protein production. CDMOs assess various elements such as yield, scalability, and post-translational modifications to select the optimal system. Commonly used expression hosts include:
   • Bacterial systems (e.g., Escherichia coli): Offer high expression efficiency but are limited in terms of post-translational modifications
   • Mammalian cells (e.g., CHO, HEK293): Preferred for producing complex biopharmaceuticals due to their ability to perform human-like modifications

2. Process development
   Co-funded by the Swedish Government, the Innovation Hub aims to advance infrastructure for advanced therapies and drive innovation in the biopharmaceutical sector. NorthX Biologics provides a clear distinction between non-GMP process development and GMP manufacturing, ensuring a seamless transition from early-stage development to full-scale, regulatory-compliant commercial production. An integrated approach across both upstream and downstream processes supports the production of high-quality recombinant proteins for advanced therapies.
   
   2.1 Upstream process development
   Efficient bacterial and mammalian cell culture processes are essential for maximizing recombinant protein production. Upstream process development focuses on optimizing media, fed-batch strategies, and bioreactor scale-up to improve protein yield and scalability. These processes are designed to ensure flexibility and efficiency in early-stage development.
   
   2.2 Downstream process development
   Downstream process development utilizes advanced chromatographic and filtration techniques to ensure high protein purity and yield. Chromatography resin screening, affinity chromatography, ion exchange, size exclusion chromatography and multimodal chromatography (MMC) are employed to refine and optimize protein purification for a variety of biopharmaceutical applications. This stage is critical in developing the optimal purification workflow before scaling up to GMP manufacturing.
   
3. GMP Manufacturing
   3.1 Microbial protein expression
   NorthX Biologics specializes in microbial development and GMP-compliant protein expression, offering tailored solutions for the development and production of microbial proteins across a wide range of applications, including therapeutics, vaccines, and industrial uses. With advanced facilities and extensive expertise, flexible manufacturing options for both GMP and non-GMP applications are provided, ranging from fully closed single-use systems to cost-effective large-scale stainless-steel plants, supporting batch sizes from milligrams to kilograms. A long experience in producing extracellular, intracellular and periplasmic proteins ensures high-quality, soluble protein expression or inclusion body production, all while meeting the specific requirements of GMP manufacturing.
   
   3.2 Mammalian protein expression
   NorthX Biologics has deep expertise in the development and GMP-compliant manufacturing of proteins expressed in mammalian systems, with a strong focus on optimizing both upstream and downstream processes to ensure high yields and product quality. A comprehensive range of GMP-compliant mammalian protein production services is offered, including adherent and suspension-based manufacturing systems. Adherent manufacturing capabilities, using cell lines such as HEK293, are fully optimized for effective GMP translation, supporting scales from T-flasks to 500m² fixed-bed reactors for seamless scale-up. For suspension cell lines like CHO, HEK293, and PerC6, a variety of GMP-compliant options, including shake flasks, wave-mixing bioreactors, and single-use stirred tank bioreactors, are provided, all operated in batch, fed-batch, or perfusion modes.

4. GMP compliance and quality control
   NorthX Biologics offers comprehensive biopharmaceutical testing services with customized solutions designed to meet the unique needs of each product, including analytics services. Batches produced meet stringent regulatory standards for clinical or commercial applications through quality control measures, including:
   • Process validation to ensure consistency in protein expression and purification
   • Analytical characterization using techniques such as ELISA and HPLC for functional validation
   • Adherence to GMP standards (Ph. Eur./USP), including the implementation of standard operating procedures (SOPs) for traceability

5. Quality assurance and regulatory compliance
   NorthX Biologics ensures that all recombinant protein manufacturing adheres to regulatory requirements, ensuring compliance with FDA, EMA, and ICH guidelines. This can be achieved through:
   • Process validation
   • Analytical characterization and rigorous quality testing
   • Full GMP compliance to meet regulatory expectations

Selective challenges in recombinant protein manufacturing

• While significant progress has been made, CDMOs continue to face several challenges, including:
  • Process scalability: Managing the transition from lab-scale to commercial-scale production while maintaining consistency and quality
  • Protein aggregation: Overcoming stability issues that may affect protein formulation and storage
  • Regulatory variability: Adapting to the evolving landscape of global regulatory standards

Closing thoughts: The strategic value of CDMOs in protein production

Selecting the appropriate recombinant protein expression system is crucial for driving both scientific research and biopharmaceutical advancements. Whether utilizing bacterial or mammalian platforms, each offers unique benefits aligned with specific project needs. For CDMOs like NorthX Biologics, effectively applying their expertise in expression systems and advanced bioprocessing technologies is essential to deliver high-quality, scalable, and GMP-compliant solutions.

By collaborating with researchers and companies, NorthX Biologics plays a pivotal role in optimizing the development of therapeutic proteins, monoclonal antibodies, and other biologics, ensuring successful commercialization. Through their commitment to regulatory compliance and technical expertise, NorthX Biologics is a key partner in advancing next-generation therapies that improve patient outcomes globally.

Naresh Thatikonda, PhD
Scientist
NorthX Biologics

Naresh, a Scientist at NorthX Biologics and SME at our Matfors facility, holds a PhD in Biotechnology and a MBA in Industrial Management and Economics. He joined NorthX Biologics in 2019, where he has been providing process and CMC support for the GMP manufacturing of drug substance (DS) and drug product (DP), contributing to the production of biological drugs for clinical trials.

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References

Thatikonda, N. (2018). Functionalization of spider silk with affinity and bioactive domains via genetic engineering for in vitro disease diagnosis and tissue engineering (Doctoral dissertation, KTH Royal Institute of Technology).

U.S. Food and Drug Administration. (2023). A Quick-Start Guide to Biologics Manufacturing. Retrieved from https://www.fda.gov/media/170955/download 

BiologicsCorp. (2013). A Guide to the Production of Recombinant Proteins. Retrieved from https://biologicscorp.com/wp-content/uploads/2013/05/A-Guide-to-the-Production-of-Recombinant-Protein.pdf