Recombinant proteins are proteins that are produced using recombinant DNA technology, which is a genetic engineering technique of combining genetic material from multiple sources. This technology enables the creation of genetic combinations that do not naturally occur and allows for production of several biotherapeutics, such as monoclonal antibodies (mAbs) and genetically modified organisms (GMOs).

The Science Behind Recombinant DNA

All organisms use the same genetic code and this allows for transferability of functional genes between organisms and species – recombining the DNA. Using tools, such as the restriction endonucleases or CRISPR (scissors), DNA ligase (glue), and vectors (carriers), we can cut, insert, and express foreign genes in a host organism to produce desired proteins or traits.

Applications of Recombinant Proteins in Therapeutics

Recombinant proteins are pivotal in the development and manufacturing of biotherapeutics. Insulin, for instance, was one of the first recombinant proteins to be approved for therapeutic use, revolutionizing diabetes treatment. Before the first recombinant human insulin (Humulin) was produced using a bacterial expression system, insulin was extracted from the pancreases of pigs and cows, often causing immune reactions in patients and had a limited supply. Similarly, monoclonal antibodies, which are engineered to target specific antigens, have become crucial in treating diseases like cancer and autoimmune disorders. The ability to produce these proteins in large quantities has transformed how diseases are treated, offering targeted and effective solutions that were previously unavailable.

Manufacturing of Recombinant Protein Biotherapeutics

Producing recombinant proteins (rProteins) involves inserting a gene encoding the protein of interest into an expression system, such as bacteria, yeast, mammalian, or insect cells, and then culturing the host organism in a controlled environment to produce the protein in large quantities. The desired gene is first identified and then isolated from a donor organism. Restriction enzymes are used to cut the DNA at a specific sequence followed by insertion of the gene into a vector such as a plasmid or a virus. The vector is used to introduce the gene into the host cells using transfection or transduction techniques and often contain promoters, which are sequences that signal the start of transcription, as well as selection markers, which help identify cells that have successfully incorporated the vector. The choice of vector can influence the level of protein expression and the ease of purification. Selecting the appropriate host cell is also crucial for the successful production of recombinant proteins.

Monitoring and Control of Protein Production

Continuous monitoring of cultivation conditions allows for real-time adjustments to ensure optimal cell expansion and efficient protein expression. Process analytical technologies and bioprocessing automation systems work together to maintain optimal conditions while also detecting and mitigating process deviations early. By doing so, manufacturers can prevent issues that could compromise protein quality and yield.

Advances in biotechnology have led to several innovations aimed at optimizing recombinant protein production. These solutions help biopharma scientists develop, optimize, and implement manufacturing processes that deliver higher yields, improved efficiency, and greater robustness.

The Purification of Recombinant Proteins

The purification of recombinant proteins is a critical step that ensures their functionality and safety for various applications. Recent advancements in purification techniques have focused on enhancing efficiency, yield, and purity while minimizing costs. Process chromatography remains the gold standard for protein purification. PAT (Process Analytical Technology) is starting to be utilized for optimization of purification with immediate feedback on key attributes like protein concentration, purity, and aggregation levels. By integrating PAT, manufacturers can transition from fixed-recipe batch processes to dynamic, data-driven workflows that enhance product quality and yield. Additionally, advancements in automation and single-use technologies have streamlined the purification process, reducing the time and labor required, and reducing contamination risks. These disposable systems eliminate the need for extensive cleaning and validation procedures, thereby reducing cross-contamination risks and operational costs. Single-use chromatography columns, membranes, and filtration consumables are now widely adopted in biopharmaceutical manufacturing, offering flexibility and scalability.

Recombinant Protein Analysis

Mass spectrometry (MS) is a powerful analytical tool for characterizing recombinant proteins, providing detailed information on molecular weight, composition, and structural modifications. By ionizing protein samples and measuring their mass-to-charge ratio, MS can identify post-translational modifications and sequence variations with high precision. Integrating Capillary Zone Electrophoresis (CZE) sample separation solutions, such as ZipChip, with MS enhances this analytical capability by offering high-resolution separation of protein isoforms and variants based on their charge-to-size ratios. In CZE, an electric field propels proteins through a capillary, effectively resolving complex mixtures. When coupled with MS, CZE is used for distinguishing protein isoforms, sequence confirmation, and detection of post-translational modifications (PTMs). CZE-MS has been successfully applied to the analysis of highly glycosylated proteins, facilitating in-depth characterization of glycoforms and their distribution. The synergy of CZE’s separation efficiency with MS’s detailed molecular analysis provides a comprehensive platform for recombinant protein characterization.

Challenges in Recombinant Protein Manufacturing

Despite the many advances in recombinant protein production, several challenges remain. Addressing these challenges is crucial for optimizing the manufacturing process. Protein aggregation is a common issue that can reduce yield and complicate purification. Protein aggregation occurs when proteins clump together, often due to improper folding or environmental stresses. Several strategies can help prevent protein aggregation. These include optimizing expression conditions, such as temperature and pH, to promote proper folding. Additionally, monitoring amino acids in cell culture media, with at-line analyzers such as REBEL, can help identify optimal concentrations of amino acids, which are crucial to proper protein folding and stability. Furthermore, maintaining the right concentrations of glucose and lactate during cell culture, with the help of real-time analytical devices such as MAVEN and MAVERICK, helps reduce cellular stress than can lead to protein aggregation.

Conclusion

The production of recombinant proteins is a complex yet essential process in biotechnology and medicine. By understanding the key steps, challenges, and optimization strategies involved, manufacturers can achieve high yields, ensure quality, and maintain regulatory compliance. As the field continues to evolve, ongoing research and collaboration between analytical technology providers, bioprocessing systems manufacturers and biopharma developers will be crucial for driving further advancements. The future of recombinant protein manufacturing lies in the ability to integrate emerging technologies and innovative approaches, ensuring that these valuable biomolecules continue to play a pivotal role in healthcare and industrial applications.