Background

Overview of Microbial Fermentation for Pharmaceutical-grade Proteins

In the early 2000s, the world of microbial fermentation in pharma really took off, thanks to big strides in synthetic and systems biology. These cutting-edge fields let scientists tweak microorganisms' genetics with precision, boosting how much and how efficiently they can churn out stuff through fermentation. The game-changing genome editing tools like CRISPR-Cas9 have made it quicker and way more accurate to develop and fine-tune these microbial strains. At the same time, tools for keeping an eye on and controlling these processes have gotten a lot better, with things like online analytical technology (PAT) and automated systems. This tech leap means the fermentation process is now more stable and predictable. By keeping tabs on fermentation as it happens, researchers can make swift tweaks, ensuring that the product quality and how quickly things get made stay consistent and efficient.

Fueled by regulations and industry policies, microbial fermentation tech has swiftly gained traction and expanded globally. Pharmacies are teaming up with biotech firms to co-create new, fermentation-based pharmaceutical-grade proteins. These partnerships have sped up moving tech from labs to factories and pushed the launch of new drugs. As folks get more eco-conscious and sustainability becomes a bigger deal, microbial fermentation stands out as a greener way to churn out pharmaceuticals. It often uses renewable resources and generates less waste, aligning perfectly with green manufacturing ideals.

These days, microbial fermentation is at the heart of making all sorts of biopharmaceutical goodies like therapeutic proteins, biosimilars, and protein-driven treatments. The sector keeps pushing boundaries, fostering the rise and mass production of cutting-edge biopharmaceuticals, which makes a big impact on health worldwide.

Fig. 1.The process of biopharmaceuticals manufacturing. (Almeida, et al., 2023)

Microbial Fermentation in Pharmaceutical Production Applications

The proper use of microbial fermentation can produce many therapeutic proteins, such as insulin, hormones, cytokines, antibodies. Specifically, there are the following practical cases:

• Antibiotic drugs: Using the nature of microorganisms to produce antibiotics such as penicillin and streptomycin by fermentation, production can be easily increased.
• Amino acid drugs: Using microbial fermentation, we can brew all sorts of amino acids big for pharma, food and animal feed. So it's cool how we could design amino acids for particular diseases, and amino acids that are used for food and fodder.
• Hormonal drugs: Hormone drugs, like insulin, also get the microbial fermentation treatment. This approach not only cuts down the cost but also speeds up how fast we can roll out new meds.
• Enzyme preparations: The tech helps produce enzyme preparations essential for making drugs—think amylase and saccharifying enzymes. They're quicker and more selective than traditional chemical catalysts, making them super useful in drug prep.
• Vaccine production: With microbial fermentation, whipping up the proteins needed for vaccines, like human growth hormone or peptide bits, becomes a breeze.

Creative BioMart Microbe is all about pushing the envelope with microbial fermentation services for crafting top-notch pharmaceutical proteins. By leveraging cutting-edge biotech and fermentation know-how, we churn out essential pharmaceutical proteins, covering the whole gamut from hormones to the enzymes and engineered drugs needed for vaccines. Our services take you from the brainstorming stage right through to mass production, making sure everything stays top-quality and affordable. We're all about helping the pharmaceutical world keep innovating and kicking up efficiency. Adjusting fermentation parameters and sifting the best microbes allows us to customize products for our customers to drive novel drug development and expand the manufacture of already available medications. If you're curious about what else we can do, don't hesitate to contact us for more information.

Service Procedure

Service Details

Screening of Dominant Strains

We use advanced genetic engineering technology to construct target protein genes and select host strains suitable for optimal production through high-throughput screening technology. The screening process uses a variety of cutting-edge technologies, including screening based on fluorescence, cell growth, and biosensors, combined with a droplet microfluidics platform to achieve ultra-high-throughput screening. At the same time, ELISA is used to accurately detect specific proteins, and CRISPR/Cas9 technology is used to perform efficient gene editing of strains to improve production efficiency and adaptability, ensuring that the selected strains have the best production potential.

Fermentation Process Optimization

When we're into fermentation production, we really dive into what makes the strain tick—its growth habits and what it needs to thrive. We get the culture medium just right by tweaking it based on a bunch of experiments. Keeping a close watch on the fermentation process is key. We keep an eye on factors like temperature, pH, and dissolved oxygen, adjusting them on-the-fly while we're scaling up, so everything runs smoothly. Plus, we top up nutrients as needed based on how the fermentation's going. For those top-tier, pharmaceutical-grade proteins, we bring out the big guns like HPLC and MS to track the process, and we use ELISA to get a handle on protein amounts. Once everything's fermented, we clean up the mix by filtering and spinning it to get rid of leftover cells, setting it up for the next steps in extraction and refinement.

Protein Extraction and Purification

After fermentation finishes, we dive into isolating the target protein using nifty methods like solvent extraction, ion exchange, and membrane separation. We then refine the rough product with techniques such as crystallization, recrystallization, high-performance liquid chromatography (HPLC), ultrafiltration, and affinity chromatography. To ensure the final product's high purity and top quality, we assess its safety and effectiveness for medical purposes using advanced tools like mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Product Analysis

Product performance analysis is a key step to ensure its quality and efficacy. We carry out detailed quality control, including testing indicators such as protein concentration, purity, biological activity and stability, and use precision instruments such as HPLC and mass spectrometry to analyze purity and molecular weight. In order to ensure safety and effectiveness, microbial limit testing is also performed. In addition, according to different types of medical proteins, we apply specialized efficacy testing methods, such as cell function experiments, biomarker analysis and in vitro model experiments, to verify the pharmacological efficacy of the protein. These services include:

• Cell bank testing
• Viral and bulk harvest testing
• Raw material testing
• In-process control and intermediates testing
• Release drug substance, drug product, and assembled device testing
• Stability testing

Our Advantages

• Industry Know-How and Skills. We've got a ton of experience in microbial fermentation and making proteins, so we're all set to offer expert advice and support to our clients.
• Efficient Production Capacity. With advanced fermentation technology and equipment, we can produce high-quality medical proteins in a short time.
• Quality Assurance System. Our strict quality control keeps our products super pure and consistent, meeting all those pharmaceutical industry standards.
• Regulatory Know-How. We know the ins and outs of pharmaceutical regulations in different countries, making sure our products meet legal standards worldwide.
• Sustainable Development and Green Production. Adopt environmentally friendly processes and support sustainable development production models.

Case Study

Case Study 1: The synthesis of antimicrobial peptaibols by Emericellopsis alkalina strains.

The synthesis of antimicrobial peptaiboles A118-35, A118-36, and A118-37 with antifungal properties was examined across 22 strains of the species E. alkalina. It was found that 72% of these strains are capable of producing peptaiboles. Additionally, the production of peptaiboles was identified as a strain-specific characteristic, influenced by cultivation conditions and the concentration of sugars and carbon sources. Specifically, the peptaibol A118-37 exhibited activity against the opportunistic yeast Candida albicans, the mold Aspergillus niger, and clinical fungal isolates of mycosis pathogens that demonstrate multiple resistance.

Fig. 2. Comparison of the isolation profiles of ethyl acetate extracts of the E. alkalina A118 strain. (Baranova, et al., 2019)

Case Study 2: Enhancing cephalosporin C yield by modulating Acatg8 autophagy in Acremonium chrysogenum.

Autophagy is like the cell's way of taking out the trash and reusing what's left, but we don't really get how it works in filamentous fungi as well as we do in yeast. In A. chrysogenum, if you mess with the autophagy-related gene Acatg1, it actually boosts the production of cephalosporin C because it stops breaking down the proteins needed to make it. This study identified and characterized the Acatg8 gene in A. chrysogenum, a homologue of S. cerevisiae's ATG8, critical for autophagosome formation. Disrupting Acatg8 reduced conidiation and delayed germination but increased cephalosporin C yield by accumulating biosynthetic enzymes and peroxisomes. However, the biomass declined during late fermentation stages due to disrupted autophagy leading to increased reactive oxygen species (ROS) and premature fungal death. To mitigate this, a xylose-inducible Acatg8 expression system was introduced, restoring normal growth while maintaining high cephalosporin production during extended fermentation.

Fig. 3. Cephalosporin C production was detected during fermentation. (Li, et al., 2018)

Case Study 3: Scalable production of ncAA-modified proteins in E. coli.

Engineering proteins with non-canonical amino acids (ncAAs) offers various applications but faces scaling challenges in manufacturing. Efficient large-scale production of ncAA-incorporated proteins is not well-established. In this study, researchers incorporated the ncAA N6-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into a Fab fragment in E. coli, using an orthogonal system from Methanosarcina mazei. They tested different fermentation conditions to optimize Azk uptake and Fab production. Results show efficient Azk uptake in batch phases and that minimizing time between Azk uptake and incorporation is crucial to prevent its degradation. This study advances scalable methods for producing Azk-incorporated proteins, achieving 2.95 mg/g CDM yields, 80% of the wild-type Fab yield. Researchers identified Azk uptake as dependent on the cell's physiological state as a production bottleneck.

Fig. 4. Calculated CDM (grey line) and calculated Azk concentration (blue line). (Hanaee-Ahvaz, et al., 2024)

FAQs

References:

1. Jozala AF.; et al. Biopharmaceuticals from microorganisms: from production to purification. Braz J Microbiol. 2016;47 Suppl 1(Suppl 1):51-63.
2. Baranova AA.; et al. Antimicrobial peptides produced by alkaliphilic fungi Emericellopsis alkalina: Biosynthesis and biological activity against pathogenic multidrug-resistant fungi. Appl Biochem Microbiol. 2019;55:145–151.
3. Li H.; et al. Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum. Microb Cell Fact. 2018;17(1):175.
4. Hanaee-Ahvaz H.; et al. Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein. Sci Rep. 2024;14(1):25375.