Ultra-high-titer CHO expression and tailored engineering strategies for antibody fragments, including Fab, Fab’, F(ab’)2, scFv, VHH, and their corresponding bsAbs
Innovative Production & Engineering Strategies for Fab, ScFv and VHH Mengjie Lu, Li Zhang, Zhumei Feng, Jiansheng Wu WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)
Antibody fragments, such as Fab, scFv and VHH, are gaining significant attention in antibody drug development. These fragments can function as standalone molecules, or as building blocks for bispecific antibodies (bsAbs). CHO cells have become an emerging host for producing antibody fragments due to low endotoxin levels and proper protein folding capabilities. However, current CHO expression methods encounter several challenges, such as time-consuming processes, low yields and insufficient throughput. Specifically, some fragments lack optimized engineering strategies, leading to high impurity levels caused by aggregation or adduct formation. To address these challenges, WuXi Biologics’ Protein Sciences (PS) Department utilizes ultra-high titer CHO expression for the production of antibody fragments, including Fab, Fab’, F(ab’) 2 , scFv, VHH, and their corresponding bsAbs. Our innovative platform ensures high-titer and throughput production, consistent high purity, and scalability for in vivo testing. Furthermore, we have implemented tailored strategies for each fragment type, such as deblocking methods for Fab’, stabilization techniques for scFv, and prediction of Protein A binding for VHH. Abstract Antibody fragments are smaller parts of antibody molecules and retain the ability to bind to specific antigens. Widely used in research, diagnostics and therapeutics, they offer advantages such as smaller size and improved tissue penetration. However, their production and engineering encounter numerous challenges, including issues related to yield, purity, throughput and stability. To address these challenges, WuXi Biologics’ PS Department leverages an ultra-high titer CHO expression system for antibody fragment production with tailored engineering strategies. Introduction
Results (cont.)
Figure 7. SEC-HPLC and Titer Analysis of Four scFv Structures with Different Linkers and VH & VL Orientations Case Study 4: High-Purity Production of ScFv Reformatted from Regular IgG
57% RT: 10.1 min
32% RT: 10.2 min
80% RT: 10.0 min
77% RT: 10.2 min
Linker 1:
Linker 2:
390 mg/L
290 mg/L
350 mg/L
130 mg/L
This case study highlights our capability to reformat IgGs to scFvs. Among four structures, VL-VH orientation exhibited higher titers, while Linker 2 showed higher purity.
Case Study 5: Extensive Experience in Affinity Resin Selection for Untagged VHH
Results
Figure 8. Scheme of VHH (Monomer, Dimer and Bispecific), VHH-Fc, and VHH-Related BsAb
Case Study 1: High-Throughput (HTP) Production of Fab in 1 mL Transient CHO
Figure 1. Scheme of Fab, Fab’ and F(ab’) 2
Human • KanCapG • Capture CH1 • CaptoL, etc.
Monomer Dimer Bispecific VHH-Fc
Building Blocks for BsAb
Figure 9. ProA Binding Prediction and Resin Capacity Comparison for Three Bispecific VHHs
L FT E
kDa M
Molecules
Predicted ProA Binding Measured Capacity of Resin 1 (mg/mL)
Measured Capacity of Resin 2 (mg/mL)
Cys Fab’ through deblocking
Human, mouse, rat, rabbit • Ni • Anti-Flag
190 115
F(ab’) 2 from enzyme digestion of full IgG
A (Process 1) B (Process 1) C (Process 1) C (Process 2)
+++ / +++
6.1
21.9 20.6
80 70 50 30 25
Fab
Fab/Flag
+ / +++ ++ / ++ ++ / ++
16.1
3.1
0.9
Figure 2. Average Yields of 557 Flag-Tagged Fabs with Single-Step Purification
10.3
13.8
ProA Binding: ‘+’ extremely weak binding; ‘++’ weak binding; ‘+++’ normal binding
15 10
In this case study, ProA binding affinity for each VHH domain was predicted, and resin capacity using different processes was measured. Based on the results, Resin 2 & Process 1 were selected for molecule A and B purification, while Resin 2 & Process 2 were chosen for molecule C purification with greatly enhanced binding capacity.
An Example of Affinity Capture
His or Flag tag is required for 96/24
0.55 mg
DWP-based production
Figure 10. SDS-PAGE (NR/R) and Intact Mass Analysis of Multi-Step Purification for a VHH BsAb Case Study 6: 10 L Stable Poll Production of VHH BsAb with High-Purity
Flag
M R NR
96-well DWP (1 mL/well)
kDa
MSS
MMC
CEX
UF/DF
190 115
70 80 50 30 25 15
Figure 3. SDS-PAGE (NR/R), SEC-HPLC and Intact Mass Analysis of a Fab’ Following Two-Step Purification Case Study 2: Scale-up, High-Purity Production of 1 L Deblocked Fab’
MMC
CEX
Intact MS
AC
Deblocking
CEX
Intact MS
AC Chromatogram
SEC-HPLC
VHH2
VHH1
8 g bispecific antibody was delivered within 9 weeks.
Cys
In this case study, 8 g high-purity bsAb was produced from 10 L stable pool within 9 weeks following MSS-MMC-CEX-UF/DF purification steps. The high purity was confirmed by SDS-PAGE and Intact Mass analysis.
WuXi Biologics has established a high titer, high quality production platform and efficient scale-up processes for Fab, scFv and VHH, as well as their related bsAbs. This platform facilitates the development of antibody fragments in therapeutic contexts. Conclusions Method All experiments were conducted in the PS Department at WuXi Biologics. DNA sequences were optimized using WuXi Biologics’ laboratory data management system (LDMS), and plasmids were constructed into WuXi Biologics' proprietary vectors. A WuXian Transient procedure was conducted according to SOPs, with CHO cell culture medium collected after 7 days. After clarifying the medium, the supernatant was loaded onto purification columns based on titer. Purification steps included nickel column, Protein A column, cation exchange chromatography (CEX), and size exclusion chromatography (SEC). Final products were analyzed by SDS-PAGE, SEC-HPLC, Intact Mass analysis, and endotoxin detection. • Our platform achieves exceptional titers through innovative CHO expression system, ensuring excellent yield and unmatched efficiency. • We possess extensive experience in each fragment with tailored engineering strategies, including deblocking Fab’, stabilizing scFv, reformatting IgG to scFv, and predicting ProA binding for VHH.
This case study demonstrates the large-scale production of a Fab’ molecule. Following AC, deblocking, and CEX, 400 mg Fab’ was obtained from 1 L of transient CHO cells, eliminating F(ab’) 2 and adduct formation.
Case Study 3: 99% Purity of Engineered ScFv at High Concentration & Low Temperature
Figure 4. Scheme of Untagged ScFv, His-Tagged ScFv, and ScFv-Related BsAb
Untagged: CaptoL, Protein A, or non-affinity methods
His tagged: Early discovery Building blocks for BsAb
Figure 5. Scheme of a Disulfide Bond Added to the VH-VL Interface to Prevent Aggregation
Figure 6. Comparison of Monomer % among Four scFv Molecules, and SEC-HPLC Analysis of mt4 under Different Conditions
Acknowledgments
We extend our gratitude to all scientists from the Protein Sciences Department at WuXi Biologics for their invaluable support during the development of antibody fragments (Fab, scFv & VHH). Additionally, we would like to thank the legal department, public relations department, and marketing department for their assistance with patent filing, poster preparation, and revisions.
Mt4
Poster modified on 9/17/2024
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This case study shows the successful stabilization of scFv by introducing a disulfide bond to the VH-VL interface to prevent further aggregation. ScFv mt4 was selected for its highest purity at high concentration and low temperature. SEC-HPLC analysis demonstrates that mt4 maintained 99% purity at 5 mg/mL, even after storage at 4°C.
To discuss this poster: PS_BD@wuxibiologics.com
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