The Significance of Upstream Process Development
Upstream process (USP), also known as fermentation process, is one of the initial phases of upstream bioprocessing. The microbial fermentation process is crucial for high-density cell culture and exogenous gene expression, which directly affects the biologics yield and cost.
In the early stages of biologics development (pre-clinical or phase 1/2), the focus is to develop scalable animal-free processes with intermediate yield and meet all product quality standards.
While in the late stages (phase 3 or commercial), we will focus on yield, robustness, scalability, and reproducibility, generating a cost-efficient process. The activities that address these issues are process characterization and process validation studies. Meanwhile, quality by design (QbD) and design of experiments (DoE) tools are typically applied.
Keywords: process development, optimization, and validation, process of fermentation, microbial cell fermentation, bacteria fermentation, yeast fermentation, high-density fermentation, engineered strain fermentation
Application: Biopharmaceutical industry, human medicine, animal medicine, vaccine, recombinant large molecule biologics, recombinant biologics, biological reagent
USP Solutions of Yaohai Bio-Pharma
Benefiting from over 10 years of microbial fermentation experiences, Yaohai Bio-Pharma has the expertise to help solve fermentation process challenges. We can rapidly establish high-density fermentation strategies for Escherichia coli (E. coli) periplasmic secretion, intracellular soluble or inclusion body expression, and Yeast extracellular or intracellular expression.
Our available general fermentation development services include:
- Fermentation process design performed at a small scale of 7L in fed-batch mode
- Three-batch process validation in 7L fermenters
- An intermediate process scale-up to 30L/70L
We can flexibly perform multi-parameter optimization to meet the desired yield or proportion, using One-Time-A-Factor (OTAF) or Design-of-experiments (DoE).
Key fermentation parameters are listed as follows:
Animal-free fermentation media compositions
Antibiotic-free or not, Inoculum volume
Growth temperature, pH, Dissolved oxygen (DO) level
Feeding mode (e.g., fed-batch)
Concentration of inducer (e.g., IPTG, methanol), Induction point (cell wet weight/OD600),
Induction temperature, Induction time
etc.
Service Details
| Services |
Process Flow |
| Cultivation in Shake Flask(500 mL) |
Animal-free media preparation → Cultivation of microbial seed → Cultivation in shaker → Induction → Harvest |
| Cultivation in Fermenter(7 L) |
Animal-free media preparation → Cultivation of microbial seed → High-density cell fermentation in 7L bioreactor → Induction → Harvest |
| Quality Control of Proteins/Peptides |
Pretreatment Intracellular soluble expression: Microbial cells harvest and resuspension → Ultrasonic crushing → Cell debris removal → Crude extract Inclusion body expression: Microbial cells harvest and resuspension → Ultrasonic crushing → inclusion body preparation→ Crude extract Extracellular soluble expression: Culture supernatant collection → Concentration and buffer exchange → Crude extract Quality analysis Crude extract as sample → Target protein analysis by SDS PAGE or Western blotting (WB) |
| Quality Control of Plasmids |
Pretreatment and Quality AnalysisPlasmid extraction → DNA analysis by agarose gel electrophoresis |
Case study
We are commissioned by our customer to increase the target polypeptide hormone yield from 8 g/L to 10 g/L.
Based on the Full Factorial Design and Response Surface Design in the DoE tools, we rapidly recognized and optimized the key fermentation parameters. Under the optimized high-cell density fermentation process, the expression level increased to 10 g/L.
Our experiences
- We have worked on various types of microbial hosts, e.g., E.coli DH5α, TOP10, Trans10, BL21; Pichia pastoris (P. pastoris) SMD1168H, X-33, GS115, PichiaPink strain1/2/3/4; Saccharomyces cerevisiae (S. cerevisiae) and Hansenula polymorpha (H. polymorpha).
- We are experienced with E. coli periplasmic secretion, soluble and inclusion body expression, as well as Yeast intracellular or extracellular expression. The yield of inclusion body is up to 10g/L, while soluble protein yield ranges from 0.5 to 15g/L.
- We have been involved in the development and manufacturing of various large molecules, including recombinant subunit vaccines, virus-like particles (VLPs), hormones (insulin, GLP-1, growth hormone), cytokines (Interleukin-2/IL-2, IL-15, IL-21), growth factors (EGF, FGF, NGF), nanobodies/ single-domain antibodies (sdAbs), enzymes, etc.
- Equipment
7L*16 Fermentation System
Cedex Bio Analyzer, Bioprocess analyzer
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