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1. Candidates Screening (HTP Mini Scale)
Expression Volume: 4-50mL
Capacity: Up to 500 samples per day
Evaluate protein expression level and solubility
Enzyme activity can be tested in crude material or with low purity
2. Candidates Screening (Small Scale)
Expression Volume: 1L (Flask) or 5L (Fermenter)
Capacity:
200 samples/week
Yield and quality data after purification
Some impurities affect the performance of enzyme
candidates
Performance of some candidates need to be confirmed again at small scale
3. Cell Banking
Vector and strain screening，and codon optimization
PCB, MCB, and WCB Construction
Stability After 10-50 Generations of WCB，plasmid loss rate，expression level will be tested
Use Double-Layer Agar (DLA) technique to test phage of master cell bank and working cell bank.
The viability and expression level of the working cell banks will be tested termly to determine the storage time.
4. Upstream Process Development
Expression Volume: 20-500L (Fermenter);
Fermentation Process: High density; Cell Density (OD 600) > 200
5. Downstream Process Development
His tag or tag-free protein; HPLC purity: up to 99%;
Endotoxin level: <1Eu /mg; Yield: Up to 15g/L)
6. Analytical Methods Development
7. Formulation Development
The performance of buffer candidates will be tested under stress condition, and check purity or activity at some points
Freeze-thaw test by 10 times
8. Stability Assessment
Two stress conditions, check purity or activity 4 times in one month
Two accelerated conditions, check purity or activity 3 times in 6 month
Under storage condition, check purity or activity 9 times in 24 month
Shipping condition confirmation
9. Scale-Up Production
Zinc Finger Protein Screening
Customer Requirement: Deliver one-step purified proteins
| Zinc finger protein | Number of items | Timeline | Expression Scale | QC | ||
|---|---|---|---|---|---|---|
| Deliver one-step purified mutants | 156 | 8 calendar days | 50 mL | A280 | SDS-PAGE | Intact Mass (Random 5% samples) |
Summary of 156 Items of Zinc finger proteins
T7 RNA Polymerase Screening (1L scale)
Customers Expectations：90% purity, 5mg, Nucleic acid & Nuclease removal
X Enzyme for GCT
Project Goal: Deliver work cell bank for this GCT enzyme
Comment: The clone with the highest expression level was selected to construct MCB and WCB
1. Expression Evaluation & Restriction Map
Result: MCB and WCB were confirmed by expression level evaluation and restriction map of plasmid
2. Phage Test
Use Double-Layer Agar (DLA) technique to test phage of master cell bank and working cell bank.
Result: No phage contamination in the master cell bank and working cell bank.
3. Plasmid Stability Test
Passage the WCB cells until P20 with antibiotics and test the expression level.
Result: The expression level of the working cell bank remained stable and did not show a significant reduction from the SDS-PAGE result of P1, P5, P10, P15 and P20.
Passage of the WCB cells until P20 without antibiotics to test plasmid stability
Result: There was no obvious plasmid loss in the passages of the work cell bank (P1, P5 and P10).
4. Long-Term Viability Test
The viability and expression level of the working cell banks are tested to determine the storage time.
| Parameters | Check point of storage time (Months) | |||||
|---|---|---|---|---|---|---|
| 0 | 3 | 6 | 12 | 18 | 24 | |
| Viability | PASS | PASS | On-going | On-going | On-going | On-going |
| Expression level | PASS | PASS | On-going | On-going | On-going | On-going |
X Enzyme for GCT
Project Goal: Optimize the cell culture and purification process for this GCT enzyme
| Phase | Item | Lot 1 |
|---|---|---|
| Upstream Process Development (Cell Culture) | Cell Density (OD 600) | 102 |
| Cell Pellet | 234 g/L | |
| Fermentation Period | 22 h | |
| Downstream Process Development (Purification) | Appearance | Clear, Color less, Liquid |
| Concentration | 12.45 mg/ml | |
| Purity (SDS-PAGE) | >90% | |
| Purity (RP-HPLC) | >95% | |
| Purity (SEC-HPLC) | >95% | |
| Endotoxin | 1.7 EU/mg | |
| HCP | 35.49 ng/mL | |
| HCD | 0.06 ng/mL | |
| Residual DNAse | < LOD | |
| Mycoplasma Detection | Negative | |
| Yield | 29.2 mg/L |
X Enzyme for IVD
Project Goal: Optimize the cell culture and purification process for this IVD enzyme
| Phase | Item | Lot 1 |
|---|---|---|
| Upstream Process Development (Cell Culture) | Cell Density (OD 600) | 162 |
| Cell Pellet | 250 g/L | |
| Fermentation Period | 22 h | |
| Downstream Process Development (Purification) | Appearance | Yellow Powder |
| Concentration | 50 mg/ml | |
| Purity (SDS-PAGE) | >95% | |
| Purity (SEC-HPLC) | >99% | |
| Water Content | 4.0 % | |
| Specific Activity | > 40 KU/g | |
| Yield | > 4.5 g/L |
SDS-PAGE
SEC/RP-HPLC
E.coli Host cell DNA test
qPCR assay kit
Mycoplasma DNA detection
qPCR assay kit
Endotoxin Level
Endotoxin LAL test
E.coli Host cell protein test
ELISA assay kit
Residual DNase detection
DNaseAlert™ QC System
Residual RNase detection
RNaseAlert™ QC System
Enzyme Activity Method Development for MAD7
| cleavage in buffer 1 | cleavage in buffer 2 | |
|---|---|---|
| sgRNA1 | 82.3% | 91.3% |
| sgRNA2 | 62.6% | 87.9% |
| sgRNA3 | 84.3% | 93.3% |
| sgRNA4 | 42.7% | 66.8% |
| sgRNA5 | 68.8% | 93.0% |
| sgRNA | Enzyme | 10 min | 60 min |
|---|---|---|---|
| sgRNA3(30nM) | MAD7(5 nM) | 84.3% | 90.1% |
| sgRNA3(30nM) | MAD7(15 nM) | 93.1% | 98.2% |
| sgRNA3(30nM) | MAD7(30 nM) | 97.9% | 98.4% |
Storage Buffer Selection for MAD7
| Short name | Buffer | Component | Source |
|---|---|---|---|
| P | Buffer 1 | 50 mM sodium phosphate, 300 mM NaCl, 0.1 mM EDTA, pH 7.5, 50 % (v/v) glycerol and 1 mM TCEP | Rojek et al., 2021 |
| TA | Buffer 2 | 10 mM Tris (pH 7.4), 300 mM NaCl, 1 mM TCEP, and 50% (v/v) glycerol. | GenScript |
| TB | Buffer 3 | 10 mM Tris (pH 7.4), 300 mM NaCl, 1 mM TCEP, 0.1 mM EDTA, and 50% Glycerol | ABM |
Freeze-Thaw Test for MAD7
| Start Material | -80°C Mild Freeze-Thaw (10 Times) |
LN2 Fast Freeze-Thaw (10 Times) |
|
|---|---|---|---|
| Appearance | Clear transparent liquid | Clear transparent liquid | Clear transparent liquid |
| HPLC purity | >99% | >99% | >99% |
| Concentration by A280 (mg/ml) | 11.40 | 11.55 | 10.76 |
| SDS-PAGE purity | 90% | 90% | 90% |
Stress Test for MAD7
| 0 day | 5 day | 10 day | 30 day | |
|---|---|---|---|---|
| Appearance (15°C) | Clear | Clear | Clear | Clear |
| Purity (15°C) | 99% | 99% | 99% | 99% |
| Appearance (37°C) | Clear | Whitish | Whitish | Whitish |
| Purity (37°C) | 99% | 91% | 85% | 71% |
Accelerated Test for MAD7
| 0 month | 3 month | 6 month | |
|---|---|---|---|
| Appearance (5°C) | Clear | On-going | On-going |
| Purity (5°C) | 99% | On-going | On-going |
| Appearance (25°C) | Clear | On-going | On-going |
| Purity (25°C) | 99% | On-going | On-going |
Long-Term Stability Test for for MAD7
| 0 month | 1 month | 2 month | 3 month | 6 month | 9 month | 12 month | 18 month | 24 month | |
|---|---|---|---|---|---|---|---|---|---|
| Appearance | Clear | Clear | Clear | On-going | On-going | On-going | On-going | On-going | On-going |
| Concentration by A280 (mg/ml) | 11.40 | 11.36 | 11.11 | On-going | On-going | On-going | On-going | On-going | On-going |
| HPLC purity | >99% | >99% | >99% | On-going | On-going | On-going | On-going | On-going | On-going |
Q: What is the typical timeline for developing and delivering customized enzymes?
A: The timeline varies depending on the complexity of the project and the specific requirements. However, we aim to provide efficient and timely solutions, often delivering customized enzymes within a matter of months.
Q: What role does machine learning play in your enzyme development process?
A: Machine learning is integral to our platform, enabling us to analyze vast datasets, identify patterns, and predict enzyme behavior. This helps us design enzymes with improved properties more efficiently
Q: How does GenScript handle intellectual property and confidentiality concerns when collaborating on enzyme development projects?
A: We take intellectual property and confidentiality seriously. Our collaboration agreements include provisions to protect your interests and ensure that sensitive information remains confidential. We are committed to maintaining the highest standards of ethical conduct in our collaborations
Q: What production scale can you accommodate for customized enzymes, and how do you ensure consistency at different production scales?
A: We can scale up production to meet your specific needs, whether it's for research-scale quantities or large industrial volumes. Our rigorous quality control processes ensure consistency in enzyme production regardless of scale.
Q: How does GenScript stay up-to-date with the latest advancements in enzyme development and biotechnology?
A: We have a dedicated research and development team that continuously monitors the latest advancements in the field. We also collaborate with leading experts and academic institutions to stay at the forefront of enzyme development and biotechnology innovations
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Tackling protein expression and purification challenges in E. coli
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Industry-Leading Enzyme Production Expert from RUO to GMP
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