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Recombinant Human Growth Hormone (rhGH)

1. Cloning: The human growth hormone gene is either manufactured or extracted from the genomic DNA of humans. A plasmid with a potent mammalian promoter, for example, is used to clone the gene into an expression vector appropriate for mammalian cells.

2. Expression: Mammalian cell lines that supply the required post-translational changes, such as Chinese Hamster Ovary (CHO) cells, are transfected with the recombinant plasmid. To encourage proliferation and protein expression, the cells are cultivated in a controlled environment.

3. Purification: Following cell harvesting, rhGH is purified using techniques such as affinity chromatography, which makes purification simpler by using a tag (such as His-tag). To guarantee high purity, extra purification procedures such ion exchange chromatography could be used.

4. Downstream application: In the medical field, recombinant human growth hormone is used to address growth hormone deficits in both adults and children. It is relevant in sports medicine and healthcare since it is also utilized in some cases to encourage fat reduction and muscular gain.

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Insulin

1. Cloning: The human insulin gene is isolated from the pancreatic cells (Source Gene). The gene is cloned into a bacterial expression vector (like pBR322) that has a selective marker (like antibiotic resistance) and a potent promoter (like the T7 promoter). (Vector)

Expression: The recombinant vector is transformed into an expression host, such as E.coli. (Host System)

Induction: Bacterial cultures are induced with IPTG (Isopropyl β-D-1-thiogalactopyranoside) to promote the expression of the insulin gene.

Purification: The insulin protein is released by harvesting and lysing the bacterial cells.
A number of chromatographic methods, including affinity chromatography, which takes use of the unique binding characteristics of insulin to a ligand, are used to purify insulin.

Downstream application: In the medical field, purified insulin is used to treat diabetes. Patients are given it to control their blood sugar levels, which greatly enhances their quality of life if they have diabetes.

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Challenges:

1. Risk contamination

Mammalian cell cultures are highly susceptible to contamination by bacteria, fungi, and mycoplasma due to their slower growth and complex media requirements. Contaminations can destroy entire batches, causing delays and financial loss, particularly in large-scale bioreactor systems.

2.High production costs

Especially in large scale productions, mammalian cells are expensive to grow as they need costly media, equipment and long culture times. Thus, this increases the overall cost of protein production compared to microbial systems.

3.Cell line stability

Over time, cell lines can lose the ability to express the target protein due to genetic drift, silencing of the gene of interest, or instability in the integrated plasmid.This leads to reduced yields and necessitates the redevelopment of stable cell lines.

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Strategies for the challenges stated:

1.Contamination Prevention

Maintain strict sterile conditions and use antibiotics sparingly to reduce contamination risks. Test routinely for mycoplasma and other contaminants.For example, use closed system bioreactors and conduct regular quality checks to ensure sterile conditions.

2.Use serum-free, chemically defined media

Switch to serum-free or chemically defined media to reduce costs contamination risks and batch-to-batch variability. These media formulations can be customized to enhance cell growth and protein expression. Supplement media with specific nutrients, growth factors, or metabolic inhibitors that improve cell productivity.

3.Development of Stable Cell Lines

Instead of transient expression, create stable cell lines where the gene of interest is integrated into the genome. This ensures consistent protein production over time.For example, use selection markers (e.g., antibiotic resistance) to isolate and maintain clones with high expression levels. Advanced techniques like CRISPR can be used to integrate genes at specific, high-expression loci.

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Key features of the mammalian expression system:

1. Post-Translational Modifications (PTMs):

Mammalian cells perform PTMs such as glycosylation, phosphorylation, and disulfide bond formation, which are critical for the biological activity, stability, and functionality of many proteins.

2. High Protein Quality:

Proteins produced in mammalian systems have folding and functional characteristics similar to native human proteins, making them suitable for therapeutic and clinical applications.

3. Wide Range of Host Cell Lines:

Commonly used cell lines include CHO, HEK293, and NS0 myeloma cells, each optimized for specific protein production needs.

4. Stable and Transient Expression:

Offers both stable transfection for long-term protein production and transient transfection for rapid, short-term protein production.

5. Human-like Protein Secretion:

Mammalian systems are effective for producing secreted proteins, which simplifies downstream purification processes.

6. Regulatory Compatibility:

Proteins produced using mammalian systems are often suitable for FDA and EMA regulatory standards for biologic drug production.

7. Scalable Production:

Suitable for small-scale laboratory research and large-scale commercial biomanufacturing using bioreactors.

8. Complex Protein Production:

Capable of producing antibodies, hormones, growth factors, and other complex therapeutic proteins.

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-video from 4:16 to 5:23-

In biotechnology, the mammalian expression system poses as an effective tool for creating high-quality, biologically relevant proteins. However, like any other system, it has its advantages and limitations.

Advantages:

1. Presence of Post-Translational Modification (PTMs)

In mammalian expression systems, PTMs are crucial for generating high-quality, stable, and biologically relevant proteins. Protein folding, function and stability are ensured by PTMs such as disulfide bond formation, phosphorylation and glycosylation, which also increases protein solubility and reduces immunogenicity. Additionally, they are essential for signal transduction, protein localization and regulatory processes. Unlike bacterial or yeast expression systems, mammalian cells are able to replicate human-like PTMs, making them ideal for manufacturing therapeutic proteins including hormones, enzymes and monoclonal antibodies.

2. Produce high-quality complex proteins

As mammalian expression methods mimic human-like folding and PTMs such as phosphorylation and glycosylation, they are excellent at creating high-quality proteins. These processes ensures that proteins closely resemble their natural counterparts in terms of function, stability and biological activity. This accuracy promotes the synthesis of complex proteins such as multidomain and membrane-bound proteins, reduces immunogenicity and improves therapeutic efficacy. In applications where functionality and safety are crucial, high-quality proteins from mammalian systems provide enhanced stability, activity and dependability, making them indispensable for research, therapeutics and diagnostics.

3. Produce soluble secreted proteins

The production of secreted proteins, which are made inside the cell and transferred into the culture medium, is a highly effective function of mammalian expression systems. This minimizes contamination from internal components and removes the requirement for cell lysis, simplifying downstream processing. During their passage through the secretory pathway, secreted proteins undergo proper folding and PTMs, ensuring their solubility, stability and functionality.

Additionally, by preventing protein accumulation-induced cellular toxicity, this process promotes the synthesis of complex therapeutic proteins such as hormones and antibodies. The efficient secretion of high-quality proteins makes mammalian expression systems ideal for pharmaceutical and research purposes.

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Educational Seminar: Expression Hosts for Recombinant Proteins

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Limitations:

1. High cost

The need for specific growth media, like serum supplements or chemically specified media, which are costly and required in large quantities for scaling up, is the reason why mammalian expression systems are so expensive. Furthermore, the systems require sophisticated infrastructure, such as bioreactors with exact environmental controls, which raises the initial setup and maintenance costs.

Compared to microbial systems, mammalian cells grow more slowly and yield less, requiring longer production cycles and more resources to reach the target output. These procedures are also labor intensive, needing skilled professionals to monitor and optimize conditions, further increasing operational expenses.

2. Slow growth and lesser protein yields

Slow growth and a lower protein production than microbial expression systems are the major drawbacks of mammalian expression systems. With doubling times ranging from 12 to 36 hours, mammalian cells grow considerably more slowly, resulting in lengthier production schedules and culture times. Furthermore, the numerous PTMs that mammalian cells require, which employ more energy and biological resources, result in generally lower protein yields. Larger scale production is required due to the slower growth and lower yield, which raises costs and lengthens production times.

Although mammalian expression systems are perfect for creating sophisticated, biologically relevant proteins with human-like modifications, these drawbacks make them less appropriate for applications needing high-volume, cost-effective protein production.

3. Risk of contamination

A major limitation of mammalian expression system is the possibility of contamination, which can compromise cell viability, protein yield and the overall safety of the processes. Compared to simpler prokaryotic systems, mammalian cells are more vulnerable to bacterial, fungal and viral contamination. While viral infections can spread quickly and cause cell lysis, which compromises the integrity of the culture, bacterial and fungal contamination can outcompete mammalian cells by causing cell death, leading to substantial economic losses.

References:

Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2015). Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical Reviews in Biotechnology, 36(6), 1110–1122. https://doi.org/10.3109/07388551.2015.1084266

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Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives

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Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind…