April 9, 2024

Protein production and optimization strategies

Protein production is a fundamental process in biotechnology, enabling the creation of essential molecules for medical, industrial, and research purposes. As demands for diagnostic and therapeutic proteins continue to rise, the implementation of optimization strategies becomes imperative to enhance efficiency and yield.

In this article, we will be exploring the basics of protein production, along with considerations upon expression systems. After that, strategies will be presented that can improve biopharmaceutical processes across various phases in protein production.

Natural vs. recombinant proteins

Natural proteins, inherent to living organisms, are produced in vivo through gene expression processes regulated within the organism's cellular machinery. In contrast, recombinant proteins are engineered via molecular biology techniques like cloning, enabling their production in host organisms, often different from their native source. This heterologous expression broadens the spectrum of proteins that can be synthesized.

Natural protein synthesis occurs naturally within living organisms and does not necessitate interferences from outside. Recombinant proteins, on the other hand, necessitate the manipulation of host organisms to express specific target proteins.

Protein production process – step by step

In the pharmaceutical industry, the protein production process is characterized by a precise layout of predominantly in vitro steps. Initially, it begins with identifying the gene of interest, often encoding a specific antigen or functional protein. This gene is then integrated into a recombinant DNA construct (e.g. with a plasmid vector), serving as the blueprint for subsequent protein synthesis.

Both in small and large-scale high-throughput protein production, cell culture or fermentation techniques are utilized to cultivate host cells for optimized protein expression. Subsequently, the recombinant DNA construct finds its way into host cells via stable or transient transfection.

Once inside, the gene undergoes transcription by RNA polymerase (starting at the promoter sequence), producing mRNA strands containing codons, which dictate the sequence of amino acids in the eventual protein product.1 


These mRNA transcripts then journey to ribosomes where translation occurs. Ribosomes adeptly read the mRNA sequence and assemble amino acids brought in by tRNA molecules, forming a nascent polypeptide chain.

Post-translational modifications, including glycosylation or phosphorylation, may occur to confer functionality, particularly in membrane proteins or those intended for extracellular activity.

Throughout this process, chaperones and enzymes facilitate proper protein folding and assembly. However, challenges such as refolding issues or the presence of inhibitors may arise, requiring optimization strategies and the use of specialized reagents to obtain the protein of interest.

Following expression, protein purification and biochemical characterization steps are crucial, often involving techniques like mass spectrometry for thorough analysis. Ultimately, this process results in high-quality proteins suitable for diverse pharmaceutical applications.2 

Expression systems for in vitro protein production

In vitro protein production relies on various expression systems tailored to accommodate the diverse requirements of different proteins. Among the most commonly used expression systems are those based on bacterial, yeast, and mammalian cells, each offering distinctive advantages.

Bacterial Expression Systems: E. coli, a prokaryotic organism, is widely employed for its simplicity and cost-effectiveness in protein production. Expression vectors containing the gene of interest are introduced into E. coli cells, where they replicate alongside the bacterial genome. This system is particularly useful for producing simple proteins and peptides at high expression levels.

Yeast Expression Systems: Yeast, such as Saccharomyces cerevisiae, provides eukaryotic cellular machinery, allowing for the expression of complex proteins with post-translational modifications.

Mammalian Expression Systems: Mammalian cells, including Chinese Hamster Ovary (CHO) cells, are preferred for the production of complex proteins that require authentic post-translational modifications, such as glycosylation or disulfide bond formation. Expression vectors containing the gene of interest are transfected into mammalian systems, where they integrate into the genome of these eukaryotic cells and drive the expression of proteins. This system offers close resemblance to native mammalian protein synthesis, ensuring proper folding and functionality.

Insect Cell Expression Systems: Baculovirus expression systems utilize insect cells, such as those from the Spodoptera frugiperda (Sf9) cell line, for the production of recombinant proteins. Here, baculovirus vectors are used to insert a gene of interest into an insect cell expression system. This is particularly suitable for expressing complex proteins that require eukaryotic post-translational modifications.3 

eBook_Filling Gaps in Managing Large Volume of Biologics_Single Use Support

eBook: Filling Gaps in Managing Large Volumes of Biologics

Filesize: 3.99 MB – Mime-Type: application/pdf

Optimizing strategies – single-use technologies in protein production

From antibodies and peptides to diverse cell lines – challenges along aseptic processing, such as efficiency, safety, and cGMP compliance, need to be faced. This ensures that patients worldwide can rely on safe and high-quality protein products for various medical conditions. For that end, single-use systems offer unparalleled flexibility, scalability, and efficiency, resulting in high yields even at large scales.

By implementing optimization strategies at different levels of protein production, biopharmaceutical companies can enhance protein yields and accelerate production timelines. And with the platform solutions by Single Use Support, manufacturers have future-proof protein production solutions at hand.

Filling & homogenization of proteins

Efficient filling and homogenization processes pose significant challenges in protein production workflows. Multi-use methods may encounter issues such as cross-contamination, inefficient mixing, and time-consuming setups. Additionally, maintaining sterility and ensuring uniform distribution of components can be complex and labor-intensive.

The single-use filling system RoSS.FILL provides a sterile and efficient way to fill biologics and biosimilars with outstanding precision and consistency – even larger volumes. By eliminating the need for complex cleaning and sterilization procedures associated with traditional filling methods, RoSS.FILL streamlines the process and minimizes the risk of contamination.

Designed for homogenization, RoSS.PADL offers a scalable platform for gently kneading single-use bags to ensure homogeneous mixing of solutions. With multiple cooling and massaging mechanisms, RoSS.PADL facilitates optimal product quality while maintaining the integrity of sensitive biologics and biosimilars.

RoSS.FILL - single use filling system for drug substances

RoSS.FILL | Fill-Filtration

RoSS.FILL is a fully automated single-use bag filling system. This allows maximum flexibility and scalability of both the filling and draining process. It is possible to fill unlimited volumes per batch with a speed of up to 300 liters per hour. The entire filling process is fully disposable, thus warranting an absolutely sterile fill & drain process. For further productivity improvements check out our pinch valve innovation.

Primary and secondary packaging

Primary packaging often struggles to ensure compatibility with the product, maintain sterility, or provide adequate protection during storage and transportation. Secondary packaging must meet regulatory requirements (just like the remaining process components, by that matter), facilitate efficient handling, and provide additional protection against environmental factors.

Single Use Support provides IRIS single-use bioprocess containers designed to meet the diverse needs of protein production. These containers offer excellent compatibility with various biopharmaceutical products, ensuring sterility and integrity throughout the manufacturing process.

The RoSS® Shell, a protective secondary packaging is designed to safeguard IRIS single-use bioprocess containers during storage and transportation. These solutions offer robust protection against environmental stressors and facilitate efficient handling and distribution of biopharmaceuticals.

Controlled plate freezing of proteins

Controlled plate freezing has become a favorable approach in preserving the quality and stability of proteins. After all, freezing biologics on a large scale presents several challenges. Maintaining precise temperature control, ensuring uniform freezing rates, and minimizing ice crystal formation are crucial for preserving protein stability. Additionally, traditional freezing methods can be time-consuming and labor-intensive, leading to inefficiencies in production.

The plate freezing platform RoSS.pFTU provides elevated temperature control and customizable freezing rates, ensuring optimal conditions for preserving protein integrity. The platform caters solutions for both small and large-scale applications, allowing biopharmaceutical companies to efficiently freeze specific proteins of different volumes.

Freeze-thaw technology for handlings mAbs on another quality level

Freeze & Thaw platform

The Single Use Support freeze-thaw platforms provide insular solutions for the freeze/thaw processes of each clinical phase. Our new freeze-thaw units are fully scalable and compatible with all batch sizes and bags from all established manufacturers – you will only require one single system from the lab to blockbuster production.

Temperature-controlled storage

Proper temperature control is paramount in preserving stability and integrity during protein storage. Fluctuations in temperature can lead to structural changes in proteins, compromising their efficacy and safety. To address this critical need, Single Use Support offers the RoSS.FRDG ultra cold storage freezer.

This ULT freezer is designed to maintain proteins at a consistent temperature of, going down as far as -75°C to ensure long-term stability and prevent degradation. It provides a reliable environment for storing proteins, safeguarding their structural integrity and biological activity with state-of-the-art manufacturing execution systems (MES).

By utilizing the RoSS.FRDG freezer, biopharmaceutical companies can confidently store their proteins, knowing that they are protected from temperature fluctuations that could compromise their quality. This is yet another puzzle piece that aids in ensuring the continued success of protein-based therapies.

  1. The Art of Protein Synthesis, https://humanbiology.pressbooks.tru.ca/chapter/5-6-protein-synthesis/ , Published 2020
  2. The Art of Protein Synthesis, https://humanbiology.pressbooks.tru.ca/chapter/5-6-protein-synthesis/, Published 2020
  3. A concise guide to choosing suitable gene expression systems for recombinant protein production, http://dx.doi.org/10.1016/j.xpro.2023.102572, Published 2023-11-01
Optimizing pDNA production using advanced single-use technologies

Khalil Essani

Product Line Management

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