Fusion proteins, which consist of two or more naturally unconnected proteins linked by a peptide chain, are a popular and highly successful class of recombinant protein drugs with a variety of functions and therapeutic applications. Fusion proteins are engineered from two or more genes that encode separate proteins, which are genetically linked to create a single polypeptide that possesses the functional properties of both parent proteins. These recombinant proteins combine unrelated domains that do not occur in nature. Therapeutic fusion proteins represent a new generation of biopharmaceuticals, as they are entirely designed through human ingenuity rather than evolving from natural processes. They often exhibit enhanced molecular functionality, such as an extended half-life.
These new proteins can be classified based on the functions of their integrated domains. Typically, one fragment is responsible for molecular recognition or binding, while the other fragment contributes a specific function, such as an extended half-life, enhanced stability, cytotoxicity, or a novel targeting or delivery pathway. Most fusion proteins aim to achieve three primary objectives: extending half-life, introducing targeting functions, and providing specific biological activities. At least two of these three elements coexist within the fusion protein. Antibody derivatives, fragments, or domains are commonly utilized as building blocks for fusion proteins. Additionally, many non-antibody building blocks exhibit high affinity and selectivity for specific epitopes, and these building blocks can be employed as single modules or combined in pairs with different specificities.
Fusion proteins are highly appealing due to their numerous advantages. The integration of two functionalities into a single molecule streamlines drug production and delivery. When two molecules are linked together, they inherently share the same biodistribution characteristics, unlike two separate molecules that may exhibit completely different properties. Furthermore, this fusion can generate new functions not present in the native or free proteins, such as alterations in half-life or target specificity. The therapeutic benefits, including reduced side effects, extended dosing intervals, and enhanced efficacy, make the development of fusion proteins particularly attractive.
However, alongside these significant benefits, several challenges arise. In some instances, combining unrelated proteins can be problematic due to the incompatible properties of the fusion partners. For example, one domain may misfold or aggregate. Although some fusion protein modules are components of other mature molecules, such as antibodies, standard platform processes may not be applicable because other elements may substitute for essential domains. This can impede successful formulation due to conflicting characteristics. Additionally, controlling and fine-tuning the relative amounts of each component can be complex, making it challenging to administer the drug within the optimal efficacy and safety window. The most critical issue is the high potential for immunogenicity, even when only human protein domains are utilized, due to the formation of new epitopes at the junction between the fusion partners.
Production: At first glance, the production of fusion proteins may appear similar to that of conventional therapeutic proteins; however, it becomes challenging due to the differing physicochemical properties of the artificially combined protein subunits. The orientation of the fusion partners can significantly impact both activity and yield. The production of fusion proteins typically involves several stages: upstream processing (which encompasses all aspects of cell culture), downstream processing (which includes steps to capture and purify the target protein from impurities), and formulation steps to convert the protein into a storable and administrable form. One of the many advantages of fusion proteins is the ability to manufacture monomeric yet multifunctional molecules without interruption. However, fusion proteins also present numerous challenges that can complicate production. The combination of two distinct proteins with varying properties may not always be compatible regarding pH preference, hydrophobicity, native cellular localization, and other factors. Additionally, some fusion proteins are sensitive to low pH, which can hinder conventional viral inactivation methods. Furthermore, certain fusion proteins may be larger than well-characterized molecules such as antibodies, resulting in potentially lower expression levels.
Upstream: Due to their size and complexity, most modern fusion proteins are expressed in eukaryotic cells. This environment facilitates the proper formation of post-translational modifications, such as glycosylation and disulfide bonds. The primary host cell used for these proteins is the Chinese Hamster Ovary (CHO) cell line, which is frequently employed for Fc fusions. Yeast cells are predominantly utilized for albumin fusions. Prokaryotic expression systems are advantageous for relatively small proteins with a molecular weight below 30 kDa or for proteins that form inclusion bodies, allowing for the production of toxic proteins, such as immunotoxins.
In principle, the upstream process consists of three key elements: a DNA construct that encodes the gene of interest along with its regulatory sequences, a host cell, and specific culture conditions. Each of these elements significantly influences the expression level or titer that can be achieved during the upstream process. Prokaryotic expression can be accomplished by directly introducing a plasmid that encodes the fusion protein into microbial cells. This plasmid typically includes at least a promoter sequence, an origin of replication, and often a selection marker, such as an antibiotic resistance gene. In contrast, in mammalian cells, the expression construct is integrated into the cell's genome. This integration usually occurs at multiple sites simultaneously, which enhances the overall expression level. To generate a production cell line, a selection process is necessary to identify the most suitable cell clones based on expression yield, growth characteristics, and genomic stability.
Temperature significantly influences both cell growth and product titer. It has been observed that the titer of certain products is several-fold higher at 30°C compared to 37°C; however, increasing viable cell density at 30°C can lead to reduced productivity. Therefore, both effects must be systematically evaluated to identify optimal expression conditions. One effective strategy is to implement a two-phase process: initially establishing culture conditions that promote rapid cell expansion, followed by a transition to parameters that limit growth but enhance productivity in the second phase. The drawback of incomplete sialylation at lower temperatures can be mitigated by simultaneously lowering the pH. Additionally, the two-phase culture strategy may help reduce the formation of aggregates. This synergistic effect can also decrease the consumption of glucose and glutamine while minimizing the production of lactate and ammonium.
Another important eukaryotic expression host is yeast. Both Saccharomyces cerevisiae and Pichia pastoris can be utilized to produce fusion proteins that contain albumin or transferrin. In addition to optimizing the feeding strategy, lower fermentation temperatures have been shown to decrease the presence of proteases, while a neutral pH of 7.0 is optimal for maximizing protein expression. Furthermore, the structure of the fusion protein to be expressed can also negatively impact expression yields in Pichia pastoris.
Downstream: Downstream processing (DSP) begins with a feed containing the target product. Depending on the expression system, this feed can consist of either extracellular or intracellular material. Microbial expressions using E. coli often leads to the formation of inclusion bodies (IBs), which contain denatured aggregated proteins. Inclusion bodies can account for 10% to 50% of the total cellular protein and may contain up to 95% of a single protein species. Consequently, the cells are harvested and then mechanically or chemically disrupted to release the inclusion bodies. A common impurity at this stage is endotoxins, which are present in significant quantities due to the disruption of the cell wall. The inclusion bodies are subsequently washed and solubilized under reducing conditions using agents such as guanidine hydrochloride or urea to break all disulfide bonds. Following unfolding, the refolding process is initiated, which involves the gradual removal of the denaturing agent while slowly establishing oxidative conditions to reform the disulfide bonds, thereby restoring the correct native secondary and tertiary structures. Alternatively, the recombinant protein can be secreted into the periplasmic space of E. coli, which eliminates the need for refolding but typically results in lower expression yields.
In the case of extracellular expression, the protein of interest is secreted into the cell culture medium. Harvesting involves removing the cells through centrifugation, depth filtration, or a combination of both methods to obtain a cell-free fluid (CFF) for further processing. Hosts for secretion are typically eukaryotic cells, such as yeast or mammalian cells. Secretion is preferred because, in modern serum-free, chemically defined culture media, only a limited number of host cell proteins are present alongside the secreted product.
Fusion partners can sometimes benefit from platform purification processes. The most commonly used fusion partner, IgG Fc, is typically processed similarly to conventional antibodies. However, Protein A resin has been reported to exhibit low dynamic binding capacity for Fc fusion proteins, primarily due to steric hindrance resulting from the larger size of the fusion partner. The remainder of the capture process is comparable to that of antibodies, involving binding at neutral pH and elution at an acidic pH below 3.7, which also inactivates potential viruses. However, aggregation can occasionally occur during elution at low pH, caused by high local protein concentrations or unnatural charges induced by the acidic environment. In such cases, the addition of a chaotrope during elution can help maintain monomeric material. Other antibody derivatives, such as Fab fragments or single-chain variable fragments (scFv), do not bind to the Protein A ligand and require specific affinity resins, such as Protein L or synthetic ligands that are specific for kappa light chains. A procedure for separating Fab fragments using a series of Protein G and L chromatography has recently been described, although some product-related impurities, such as fragmented light chains, may co-purify on the Protein L resin.
Other non-traditional fusion proteins, which lack a well-known fusion partner, cannot be enriched from the crude supernatant using affinity chromatography. Instead, they must be collected through a non-specific method characterized by high loading capacity but limited selectivity: ion exchange chromatography. In this process, the protein of interest binds to the fixed charges of the stationary phase at pH values below or above its isoelectric point, thereby exposing sufficient surface charge. Elution is achieved by altering the pH to modify the protein's charge or by adding salt ions that disrupt ionic binding. In addition to the bind-and-elute mode, proteins can also be purified using a flow-through method, which allows the protein of interest to pass through while impurities bind to the resin.
By combining various purification principles, all types of impurities can be selectively removed while enriching the target protein, thereby achieving a high purity of the fusion protein. Although a two-step approach may seem more economical, a three-step approach can yield higher purity. Therefore, costs must always be balanced at a reasonable level. Finally, the protein needs to be concentrated and mixed with appropriate excipients for stable administration. Stabilizing fusion proteins can be particularly challenging, as the two different fusion partners may have varying preferences for additives.
Glycosylation: In addition to the proper formation of disulfide bonds that determine a protein's three-dimensional structure, glycosylation is another crucial post-translational modification. This process enhances the solubility of proteins by adding highly hydrophilic glycans and can improve stability by shielding protease-sensitive regions or preventing aggregation through hydrophobic patches. Furthermore, glycosylation plays a significant role in the recognition of proteins by specific receptors, facilitating tissue targeting and the recruitment of immune cells. Glycosylation can be regulated at multiple levels and should be incorporated into the protein design from the outset. An important consideration is the choice of host cell or the potential use of strain engineering to enhance the uniformity and intensity of glycosylation. In upstream processing, the glycosylation pattern can be influenced by growth conditions or the presence of specific substances in the culture medium. During downstream processing, it is essential to separate the ideal glycosyl isomers from incorrect or incomplete forms to achieve a product with the highest possible homogeneity in glycosylation.
Aggregation: Another factor to consider is aggregation. When investigating the causes of aggregation, one can differentiate between extrinsic and intrinsic factors. For instance, temperature fluctuations during freeze-thaw cycles can induce the formation of high molecular weight species. The same applies to physical stresses such as stirring, filtration, or high flow rates. Additionally, solution conditions can also contribute to aggregation, including pH, ionic strength, organic solvents, and metal ions. Intrinsic factors are molecular in nature and may depend on the presence of residues that are sensitive to shearing, oxidation, deamidation, or isomerization. Furthermore, the presence of hydrophobic patches, charged residues, unpaired cysteines, or promiscuous disulfide bonds can trigger aggregation. Sensitivity to low pH is often observed in Fc-fusion proteins. Incorporating chaotropic agents, such as urea, during elution can help prevent the formation of high molecular weight species.
Fusion proteins represent a highly successful yet heterogeneous class of recombinant therapeutics. While some foundational components are now well-established and serve as straightforward tools, a substantial number of new variants are developed each year. Although these proteins are complex to produce and necessitate significant investment in their development, they also provide innovative and enhanced therapeutic options.
Duoning Biotech offers comprehensive solutions for fusion protein production utilizing both microbial and mammalian cell host systems, with a particular focus on essential process unit operations. Our process systems and consumables are designed to meet the demands of scaling up from laboratory to pilot and commercial levels, ensuring a smooth transition between each stage. With high-quality, stable, and rapidly supplied products, along with years of industry experience, we are well-equipped to meet all of your needs with precision and excellence.
CHO Cell Serum-free Culture Media
Based on the characteristics of the industry production process, the Duoning Culture Medium Product Development Team focuses on the formulation mechanisms of culture media, the metabolism of CHO cells, and the stability of culture media. Through the CHO cell metabolism model and rigorous scientific experimental design, the team has successfully designed and developed the Media C series and DN feed series of culture medium products. These products are tailored to meet the growth, metabolism, and expression requirements of various CHO cell types, including CHOK1, CHOS, and DG44. The Media C series and DN feed series are characterized by their high performance and superior quality. By integrating upstream and downstream production processes, we offer customers comprehensive solutions for diverse bioprocess scenarios.
Animal-free, fully chemically defined (CD) culture medium.
Support high-density suspension cultures and meet high-yield requirements.
Suitable for various CHO cell types, including CHOK1, CHOKISV, CHOZN, CHOS, DG44, DUKB11, CHO-M, and others.
Provide GMP production and convenient preparation methods.
The product possesses independent intellectual property rights.
DuoBioX® Elite Benchtop Bioreactor
DuoBioX® Elite is a benchtop bioreactor system that supports dual culture modes. Users can quickly establish and develop bioreactor cell culture processes ranging from 3 to 15 liters on this system, achieving process establishment, development, and optimization with a highly cost-effective investment. The DuoBioX® Elite system is ideal for the early development of cell culture processes in scientific research institutions or the biopharmaceutical industry. This system fully meets the technical performance requirements of bioreactors in the biopharmaceutical sector, and its process software features a user-friendly design language that simplifies operation. The equipment operates stably and reliably, occupies minimal space, and offers users options for touch operation and a mobile operating platform.
High-Pressure Homogenizer
Antuos Nanotechnology (Suzhou) Co., Ltd., a subsidiary of Duoning Biotech, offers a comprehensive range of high-pressure homogenizer products suitable for various applications, from laboratory to production scale, with a maximum processing speed of up to 4,000 L/H. This series of equipment achieves a high bacterial crushing rate, exceeding 95% in a single pass, and features a specialized feed valve design that eliminates the need for exhaust, allowing for direct feeding. Additionally, the homogenizers are equipped with a frequency conversion control system, enabling flow rate adjustments based on specific requirements. The equipment also includes a built-in cooling system that effectively dissipates heat generated during the crushing process, preserving the activity of intracellular substances. Furthermore, the equipment adheres to GMP design standards and can be enhanced with pneumatic pressurization, PLC control, and other advanced functionalities.
Hollow Fiber Filters
Hollow fiber technology is one of the most widely utilized methods for ultrafiltration and diafiltration. In a hollow fiber filtration module, the feed liquid flows in a mild laminar state, effectively reducing shear stress on the target product. This preservation of structural and functional integrity is crucial for shear-sensitive processes. Duoning offers a comprehensive range of hollow fiber filter module products. The membrane material is hydrophilically modified, featuring low adsorption, high yield, and excellent linear scalability. Additionally, the module is designed with a standard connection ports, ensuring compatibility with various systems on the market.
DuoFill Single-Use Bulk Fill System
DuoFill single-use bulk fill system effectively addresses the challenge of rapidly and aseptically filling bulk solutions in standard cleanroom environments. It ensures data integrity throughout all process steps, providing an enhanced solution for subsequent product data audit tracking. The system features a single-use design principle, fully enclosed operation, and a valve-free design that minimizes residue. It supports rapid filling using either the weight method or flow rate method, achieving filling accuracy of better than 1%. Additionally, the system allows for flexible parameter modification and recipe editing. Once the parameters are set, it can automatically identify whether to operate in weight-increasing or weight-reducing filling mode. It includes a built-in automatic filtration program, supports constant flow and constant pressure filtration, and accommodates sterilization filtration operations. The system is versatile, capable of liquid filling for any total batch size, and supports freeze-thaw bags, liquid storage bags, or bottle filling. It features quick disassembly and occupies a minimal footprint, making it suitable for benchtop use or operation within a conventional laminar flow hood or clean bench.
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