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Protein Production
Supported by the industry-leading high throughput, high expression, and state-of-the-art purification and analysis technologies of WuXi Biologics, we provide production services for a variety of proteins and antibodies, including monoclonal, bispecific and multispecific antibodies, enzymes, fusion proteins, and other recombinant proteins. Combined with our unique codon optimization algorithm and fast DNA synthesis technology, our custom protein generation platforms utilizing mammalian cells, E. coli, insect cells and yeast work to produce high-quality proteins at industry-leading speeds.
WuXi Biologics Solution: One-Stop Shop for Protein and Antibody Generation
Projects can be structured by either a Full-Time Equivalent (FTE) or Fee-for-Service (FFS) model to meet your project needs and timelines.
Ultra 96
WuXi uHTP platform can support AI drug development, this automated protein production platform integrated with expression, purification, and analytical modules. The daily throughput of 1000X transient transfection expression is performed in 96-deep well plates.
96/24 well DWP for 100-1500ug antibodies; Suitable for in vitro assays
Ultra 96 Service Details:
| Service Item | Deliverables | Duration | QC | Request A Quote |
| Gene synthesis, cloning and plasmid prep all included. | ||||
| 96 x 0.8 mL CHO/HEK293 |
100-800 μg |
3 weeks | Titer, A280, Caliper (R/NR), and SEC-HPLC | Request A Quote |
| 24 x 3 mL CHO/HEK293 |
0.5-2 mg | 3 weeks | ||
Case Study: WuXian™ uHTP Platform
Ultra 96 is a well defined high throughput (HTP) protein production platform that can reach up to 1000 molecules in 1 run and can deliver up to 2 mg protein within 2 weeks production time. With a typical final concentration of >1 mg/mL, the Ultra 96 is ideal for any intro assay application. For high throughput antibody production and even some AI designed molecules (853 Abs), Ultra 96 maintains high titer. After only a 1 step purification, enough protein is generated for any intro assay requirement.
7-7-7
WuXi uHTP An efficient high throughput (HTP) platform is crucial for generating small quantity of candidate in large numbers for screening of high-affinity and high-specificity antibody leads. WuXi Biologics “7-7-7” platform takes only 3 weeks for production, 7 days for gene synthesis, 7 days for protein expression, and 7 days for purification and QC. Fc-fusion, monoclonal antibody (mAb), bispecific antibody (bsAb), his-tagged protein, and flag-tagged protein can all be produced by WuXi Biologics “7-7-7” platform.
High quality antibody is good for most in vitro and in vivo studies
- Low aggregation (>99% monomer)
- Low endotoxin (<0.5 EU/mg)
Many scales are available (yields are dependent on molecules)
7-7-7 Service Details:
| Service Item | Deliverables | Duration | QC | Request A Quote |
| Gene synthesis, cloning and plasmid prep all included. | ||||
| 20 mL CHO/HEK293 |
3-10 mg, average 4.5 mg after 2-step purification | 3 weeks | A280, Caliper (R/NR) or SDS-PAGE (R/NR), SEC-HPLC, and endotoxin (<0.5 EU/mg) |
Request A Quote |
| 1 L CHO/HEK293 |
400-1000 mg | 3 weeks | ||
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | |||
Case Study: 170 Regular IgG at 20 mL
Using the 7-7-7 platform, 70 vanilla antibodies were produced. Each antibody was expressed at 20 mL scale with an average titer of 0.7 g/L. After a 2 step automated purification, substantial proteins (average = 4.5 mg) were obtained with < 0.2 EU/mg and > 99% purity.
Quick ’n Clean
Bispecific antibodies (bsAb) are a large family of molecules designed to recognize two different epitopes or antigens and represents a highly promising class of biotherapeutic modality. Structural diversity has been fast-growing in the bispecific antibody field, ranging from single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike vanilla antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. To support our clients and to overcome the challenges associates with bsAb production, WuXi Biologics has established one HTP bsAb production platform “Quick ’n Clean”.
Our platform can produce 1 mg of customized bsAb with 99% heterodimer in 3~4 weeks starting from 20 mL culture medium using a 3 step automated purification process. With the capability to provide high throughput screening of large number of customized bsAb, our “Quick ’n Clean” platform is highly suitable for early stage drug discovery.
Quick ‘n Clean Service Details:
| Service Item | Deliverables | Duration | QC | Request A Quote |
| Gene synthesis, cloning and plasmid prep all included. | ||||
| 20 mL | • Average 1 mg • 3-step automatic purification • 99% heterodimer |
3-4 weeks | <0.5 EU/mg endotoxin control, QC incl. Titer, A280, Caliper (R/NR) or SDS-PAGE, SEC-HPLC | Request A Quote |
| 1 L CHO/HEK293 |
50 mg | 3-4 weeks | Request A Quote | |
| Additional QC |
RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing |
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Case Study: Customized Formats to Screen Large Number of BsAb at Early Stage
Generation of bispecific antibodies (BsAbs) often require a complex and time-consuming purification process in order to separate the impurities formed as a result of HC mispairing, LC mispairing, fragmentation and aggregation. Through our “Quick ’n Clean” platform fortunately, customized formats of bispecific antibodies which comprise an scFv or VHH and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. Using innovative purification strategies, high purity customized BsAbs can be purified in high throughput, automatic mode.
Transient (CHO)
Chinese hamster ovary (CHO) is the most widely used host cell lines in biopharmaceutics production, as they are capable of producing proteins with bioactive PTMs similar to those produced in human cells. Over 70% biologics and nearly all monoclonal antibodies (mAbs) are produced in CHO cells.
Features of the WuXianTM Transient CHO system that facilitate the early stage drug development include:
- High expression level — over 1 g/L on Day 7
- Short timeline — gram level material can be delivered within 5 weeks
- Quality consistency — WuXianTM CHO was derived from WuXiaTM CHO stable cell line
Transient (CHO) Service Details:
| Service Item | Deliverables | Expression Scale | Duration | QC | Request A Quote |
| Highly customizable package with multi-step purification | Any antibody, tagged or untagged protein with need for scale up manufacturing | 1-50 L | 3-5 weeks |
<0.5 EU/mg endotoxin control, QC incl. Titer, A280, Caliper (R/NR) or SDS-PAGE, SEC-HPLC |
Request A Quote |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||||
Case Study:
Titer result of multiple molecules were presented in the figures above. All titer result were measured on Day 7 post the transient transfection. The volume was from 20 mL-10 L. The average titer would be around 1 g/L. WuXian™ Transient (CHO) platform will be capable of deliver >5 g protein with in 5 weeks.
Transient (HEK293)
Human Embryonic Kidney 293 (HEK293) cell line is one of the most common cell lines used in biotechnology and research studies. It can provide a more compatible type of post-translational modification close to human body, with short expression cycle and high quality. HEK293 cell line is popular for the expression of humanized antibodies, recombinant proteins, membrane proteins, etc.
Transient (HEK293) Service Details:
| Service Item | Deliverables | Expression Scale | Duration | QC | Request A Quote |
| Highly customizable package with multi-step purification | Reagent protein, extracellular domain, membrane protein, antibody with clipping issue | 1-50 L | 3-5 weeks |
<0.5 EU/mg endotoxin control, QC incl. Titer, A280, Caliper (R/NR) or SDS-PAGE, SEC-HPLC |
Request A Quote |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||||
Case Study: Small Scale Construct Screening for Non-Antibodies
- 12 Different Proteins
- 24 Constructs
- 293 System
- 24 DWP
- 3 mL Cell Culture
CHO
WuXian Express™ is a fast stable pool production for CHO K1 cell line that can be used to provide large amount of antibodies and proteins for developability, in vitro, and in vivo studies. The average titer for regular antibody is 3.5 g/L, and the average timeline is around 6-7 weeks.
Express (CHO) Service Details:
| Service Item | Deliverables | Expression Scale | Duration | QC | Request A Quote |
| Gram-level protein generation for animal study and developability study in CHO cells | Any antibody, tagged or untagged protein with need for scale-up manufacturing | 20 mL-50 L | 6-7 weeks | A280, Caliper (R/NR) or SDS-PAGE (R/NR), SEC-HPLC, and endotoxin (<0.5 EU/mg) |
Request A Quote |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||||
Comparable Product Quality of Different Scales
Excellent stability and reproducibility among different batches is seen. As the production of the target protein was scaled up from 200 L to 2,000 L, SEC-HCPLC, cIEF, SDS_CALIPER and Glycosylation analysis of three final product batches show very high consistency.
NS0 Expression
NS0 (Non-secreting Null) cell line is a model cell line derived from murine myeloma cells used in biomedical research and commercially in the production of therapeutic proteins. NS0 cells are well equipped for producing and secreting exogenous proteins.
NSO Expression Service Details:
| Service Item | Deliverables | Duration | QC | Request A Quote | |
| 1-50 L | Challenge protein or cystine rich protein production | 6-7 weeks |
<0.5 EU/mg endotoxin control, QC incl. Titer, A280, Caliper (R/NR) or SDS-PAGE, SEC-HPLC |
Request A Quote | |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||||
Case Study: A Cysteine Rich Protein Expressed in NS0 Cells
| Express Host | CHO | NSO |
| AC Yield(mg/L) | 312 | 327(correct disulfide bonds confirmed) |
| SEC-HPLC & SDS-PAGE gel of AC eluate |  |
 |
- Target protein: 39 kDa protein with 10 cysteines, fused to Fc
- The Fc fusion is aggregated in CHO cells, and with scrambled disulfide bonds
- The yield and quality of the target protein was significantly improved in NS0 cells
Insect Platform
The baculovirus–insect cell expression system is a powerful and versatile platform for the production of a wide range of proteins. This platform offers several unique advantages for recombinant protein expression , such as post-translational modifications, higher yield for secreted protein, and proved solubility and relatively high cell culture density, which render it an excellent choice for the expression of intracellular proteins, protein complexes, kinases, membrane protein and viral proteins. A wide variety of commonly used insect cell lines, such as Sf9, and High Five, are available at WuXi Biologics. By utilizing optimized expression vectors , host and high-titer virus packaging technology, WuXi Biologic can effectively increase protein expression levels and deliver highly quality proteins.
Expression
- Cell line Screening: Sf9, HighFive
- Flashbac system to reduce timeline
- Optimization of expression conditions at small scale(construct screening, MOI, harvest time… )
- High-throughput purification system for purification of many variants
- Scale up volumes up to a 20 L
Protein type
- G protein-coupled receptors (GPCRs)
- Ion Channel
- Cell Surface Receptor
- Viral Envelope Protein
- Kinases
- Proteases and enzymes
- Protein complex
- Other intracellular proteins
Routine Soluble Protein Production: 4-5 weeks
Insect Service Details:
| Service Item | Description | Duration | QC and Deliverable |
| Baculovirus generation | P0, P1, P2 | 1 weeks | Titer: 1 x 109 PFU/mL |
| 24 x 3 mL | 1 step purification, screening | 2 weeks | Purified protein |
| 1 L – 20 L | 2-3 step purification | 2-4 weeks | SDS-PAGE, LC/MS |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, cIEF, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||
Case Study: Small Scale Protein Kinase Production
- 17 different protein kinases
- 5 mL scale insect cell expression
- One step purification with high purity
- Evaluate expression levels
- LC-MS confirmed
- Good for different biochemical assays
E.Coli Platform
Escherichia coli (E. coli) is a popular host option for recombinant protein expression because of its high yield, low cost, and ease of scaling . Thus, bacterial expression systems play an important role in both laboratory investigations and industrial-scale production.
WuXi Biologics have extensive experience with difficult proteins in E. coli expression, both for soluble proteins and inclusion bodies refolding, and provide customers with high-quality recombinant proteins from research to GMP.
E.coli Platform Features
Service Scope
- Plasmid Construction
- Shake Flask Optimization
- 1-5 L Scale up
- Up to 50 L
Advantage
- Various Promoter Systems
- Different Feed Control: DO/pH-stat, Exponential
- Higher Titer (1-20 g/L)
- Short Culture Period (1-5 days)
Protein Type
- Intracellular & Extracellular Protein
- Soluble & Inclusion Body
- Cytokines refolding
- FAB
- Large scale enzyme production
E.Coli Service Details:
| Service Item | Deliverables | Expression Scale | Duration | QC | |
| Expression condition screening (4~8 conditions per set) |
Report in PPT format | 5-10 mL |
1 week |
SDS-PAGE | |
| 96DWP refolding condition screening | Report in PPT format | NA |
2-3 weeks |
NA | |
| Scale-up production in shake flask | Tagged or untagged protein, biotinylation | 1-20 L | 3-4 weeks | Concentration, SDS-PAGE (R/NR), and LC/MS Endotoxin removal upon request |
|
| Scale-up production in bioreactor | Tagged or untagged protein, biotinylation | 1-10 L | 4-5 weeks | ||
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||||
Case Study: E.coli Expression Optimization
In this case, the inclusion body has a very low expression and the protein was partially truncated after purification in the pilot test. The expression condition was optimized with different host, culture medium and temperature. With the optimized expression condition, the expression was significantly increased and proteins with a purity of >95% were obtained after the final purification.
| Parameters | Original | Optimized |
| Medium | Platform Medium | Optimized Medium |
| Strain | BL21 (DE3) | BL21 star (DE3) |
| Temperature | 20 | 37 |
| Titer (mg/L) | 1-5 | >100 |
| Time (h) | 24 | 24 |
Case Study: MHC Production
Over 260 of MHC refolding and production were performed at scales from 0.1 L to 50 L. A robust MHC refolding platform and suitable purification strategy including in vitro biotinylation was used. The refolding buffer and pH conditions were optimized, and a high-quality protein sample was obtained after final purification.
E.Coli Scaleup Feasibility
High-density Escherichia coli fermentation in bioreactor can achieve much more biomass by nutrient feeding, sufficient oxygen supplementation along with stable pH control, resulting in higher yield (up to several grams of proteins per liter) of protein expression than shake flask cultivation. Fermentation conditions are highly scalable and reproducible with reducing risk and delays when the process applies to commercial manufacturing. The short manufacturing times ensure a cost-effective production approach for both soluble protein and inclusion body production.
E.coli Scaleup Service Details:
| Service Item | Deliverables | Duration | QC and Deliverable |
| Fermentation conditions optimization |
• Different Feed Control: DO/pH-stat,
|
1-2 weeks | Expression report including fermentation profile and titer evaluation |
| 1-20 L Fermentation |
• 2-3 steps purification • Tag removal, biotinylation, endotoxin |
2-4 weeks | Concentration, Caliper (R/NR) or SDS-PAGE, LC/MS |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, glycan profiling, cIEF, CHO HCP, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | ||
Case Study: Large Scale Production of an Enzyme for Bioconjugation
Background
- Target is an enzyme with 20 kDa molecular weight for bioconjugation.
- Key challenge: non-tagged protein purification process development.
Yeast Platform
Yeast is a commonly used eukaryotic protein expression system for both endogenous and heterologous proteins with various advantages. Yeast genome is easy to engineer in order to optimize the production performance. The strains with genetically modified metabolic and secretory pathways have great protein expression. Compared to mammalian cell culture, yeast grows faster, and the culture medium is less expensive. Yeast cells also have multiple post-translational mechanisms, such as acetylation, amidation, hydroxylation, glycosylation (e.g., N-glycosylation and O-glycosylation), methylation, phosphorylation, and ubiquitylation. The post-translational modifications machinery makes proteins functional in yeast cells, but not in bacterial expression systems.
Yeast Service Details:
| Host Strain | Phenotype | Application | Fermentation Cycle |
| GS115 | Mut+His4 | Enzyme, vaccine antigen, cytokines, growth factor | 4-5 days |
| KM71 | MutsHis4 | 5-7 days | |
| SMD1168 | Mut+His- Protease-deficient | Degradable protein | 3-4 days |
Routine Soluble Protein Production: 8-11 weeks
Our yeast platform uses Picha and focuses on secreted expression. We start with expression optimization and can scale up to 50L in fermentation.
Yeast Scaleup Service Details:
| Service Item | Deliverables | Expression Scale | Duration | QC |
| Expression screening (3 transformants per set) |
PCR result | 20 mL | 2 weeks | SDS-PAGE |
| Scale-up production in shake flask | Tagged or untagged protein | 1-20 L | 2 weeks | Concentration, SDS-PAGE (R/NR), SEC-HPLC, and endotoxin (<1 EU/mg) |
| Scale-up production in bioreactor | Tagged or untagged protein | 1-10 L | 2-4 weeks | |
| Additional QC | RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, cIEF, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing | |||
Case Study: Protease
Background
- Target protein is a specific protease with 26.9 kDa molecular weight
- Key challenges: CHO and 293 fail to produce an active enzyme
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