Optimizing the Production of Recombinant Proteins & Antibody Fragments

The production of recombinant proteins and antibody fragments is an essential step in advancing biopharmaceuticals and developing new therapeutics. However, manufacturing these can be challenging and time-consuming, so you may be wondering – how can I streamline my production processes?

This blog post will explore various techniques for optimizing the production of recombinant proteins, and antibody fragments, and explain how you can maximize yield and efficiency.

Understanding Recombinant Proteins & Antibody Fragments

Recombinant proteins and antibody fragments have an abundance of applications in the biopharmaceutical industry. For example, recombinant proteins have been used to produce human insulin to manage diabetes, while antibody fragments are used for anti-cancer treatments, as their small size allows more effective targeting of tumors.

Regardless of which option is most appropriate for your project, they can both be optimized to help you reach your project’s end goal faster and more efficiently. Here are some key considerations for enhancing their production…

The First Step: Choosing Your Expression System

Choosing the right expression system is an important first step for your project. Keep in mind that different expression systems all have their respective advantages and optimal applications – for example, E. coli is a popular choice for recombinant proteins due to its ability to achieve high yield and rapid growth.

However, there are a number of other factors to consider too – your project’s budget, for instance. Although E. coli was the first host for producing antibody fragments and has many advantages, Saccharomyces cerevisiae is an efficient, cost-effective alternative that is capable of post-translational modifications. Pichia pastoris is also a widely used, cost-effective option.

Want more advice on expression systems and how to choose your Contract Development and Manufacturing Organization (CDMO)? Check out our post on Microbial vs mammalian systems: Choosing the right CDMO partner for your biopharmaceutical development for further guidance.

The finer details: Vectors, codons, promoters, and affinity tags

Vector design is another crucial step for efficient protein and antibody fragment production. This can be enhanced by incorporating elements to improve protein stability, folding, secretion, and purification, which will also facilitate production processes. Other essential vector elements include the coding sequence, promoter, and affinity tag, which we’ve outlined below:

• Codon optimization: This is the process of optimizing the codon sequence of the inserted gene to enhance expression in the selected host. It is commonly used in recombinant protein production, but also across a range of other biopharmaceuticals, including gene therapy and DNA vaccines. It is an important step to make sure you can maximize the yield of your desired product in the expression organism
  
  
• Promoter selection: A promoter is the RNA polymerase DNA binding site which enables transcription of your inserted gene. Selecting a strong promoter is essential to achieve high-level protein expression. Inducing expression during the fermentation process can be beneficial, particularly for recombinant proteins toxic to the host. For example, the T7 lac promoter is commonly used as part of the vector design for expression of cloned proteins in e.coli. This promoter can be essentially ‘switched on’ with a chemical reagent Isopropyl β-D-1-thiogalactopyranoside (IPTG) to efficiently produce recombinant proteins once the biomass has reached a certain point. Your CDMO should also make sure the chosen promoter is compatible with your chosen expression system. Bacterial promoters are less complex and may be used in closely related species, but mammalian promoters have little to no crossover with one another
  
  
• Affinity tags: These are widely used for streamlining protein purification. Incorporating an affinity tag, such as a
  His-tag, can enable high purity with fewer purification steps. The tag can also be removed by introducing a protease site between the target protein and affinity tag, if necessary

Once these finer details have been established, it’s time to move on to the final steps of establishing culture conditions and undertaking downstream purification.

Process development: The DOE approach and downstream purification

Regardless of which expression system you choose, it is important to understand the importance of process development when selecting your CDMO partner. Process development is the refinement of production procedures, ensuring efficient and consistent manufacturing outcomes. By identifying and addressing potential challenges and bottlenecks early on, process development helps minimize risks, reduce costs, and enhance scalability, leading to improved product quality and regulatory compliance during large-scale manufacture.

At SEKISUI, we recommend application of Design of Experiments (DOE) to optimize fermentation. We use Ambr® 250HT, a valuable bioreactor system which is fully automated to run many experiments in parallel, saving time and money. This screening tool offers high throughput capabilities while mimicking larger-scale bioreactor conditions.

Enhancing culture conditions, such as temperature, oxygen, pH, and nutrients ahead of the downstream purification stages is also important. Efficient downstream purification techniques are essential for purifying recombinant proteins and antibody fragments due to their intricate structures and the need for high purity levels. These techniques enable the separation and removal of impurities such as host cell proteins, DNA, and aggregates, ensuring the final product meets quality standards for safety, efficacy, and regulatory compliance.

Your CDMO should be able to advise and optimize appropriate downstream purification techniques, such as filtration and chromatography, for your product. Ensure they have appropriate monitoring and control technologies to allow for any adjustments to maintain conditions throughout production.

Conclusion

Optimizing the construct for recombinant protein expression is essential for maximizing yield, stability, and functionality. Strategically design your construct with the correct codon usage and promoters to enhance your recombinant protein and antibody fragment expression.

Improving the production of recombinant proteins and antibody fragments is vital for your project’s efficiency. Be sure your CDMO has the end goal in mind and has process development capabilities. Optimizing the production process, from DOEs for upstream culture conditions to downstream purification techniques, is instrumental in the development of a robust, scalable and cost-effective process.

BioProduction by SEKISUI

Now you understand more about how to optimize recombinant protein and antibody fragment production for your project, why not explore what BioProduction by SEKISUI has to offer?

BioProduction by SEKISUI is a CDMO service with expertise in microbial fermentation and downstream purification. We have deep experience with E. coli but also yeast, and a variety of other bacteria, so you can be assured our team know the ins and outs of whichever microorganism you use. SEKISUI’s Quality by Design (QbD) approach ensures efficient development to large manufacturing scale with a robust process.

With more than 100 products manufactured across plasmids, enzymes, antibody fragments, and protein complexes, and over 40 years of manufacturing expertise in the UK, why not have the SEKISUI advantage? Download our services brochure here.