High-Titer CHO Production of T-Cell-Related Proteins

Leveraging a high-titer CHO expression system, T Cell Mate platform enables high-throughput production of a broad range of T-cell-related proteins, including soluble TCRs, TCR-Ab fusions, and single-chain trimers (SCTs), all with high yield and purity.

T Cell Mate Platform: High-Titer CHO Production of T-Cell-Related Proteins

Dawei Zhang, Mengjie Lu, Zhenyang Zhao, Feng Li, Jiansheng Wu Protein Sciences Department, WuXi Biologics CRO Services NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)

Abstract

Case Study 3: High-Throughput (HTP) Production of 10 SCTs in CHO Cells Figure 4: SDS-PAGE (NR/R) analysis and average titers for two-step purification of 10 SCTs

T-cell receptors (TCRs) are important targets in the development of immunotherapies and cancer treatment. However, traditional E. coli production methods for TCRs faces challenges, often leading to limited throughput, low yields, and high endotoxin levels. To address these challenges, WuXi Biologics’ Protein Sciences Department developed the T Cell Mate platform. Leveraging a high-titer CHO expression system, this platform enables high-throughput production of a broad range of T-cell-related proteins, including soluble TCRs (sTCRs), TCR-Ab fusions, and single-chain trimers (SCTs), all with high yield, superior quality, and exceptionally low endotoxin levels, meeting the stringent requirements of therapeutic development.

AC Final

1000

800

250 150 75 100 kDa

600

554

50 37 25 15 10 20

HCCF

400

306

200

Introduction

250 150 75 100 kDa

T-cell receptors (TCRs) recognize antigenic peptides presented by MHCs on target cells, triggering signaling pathways that activate T-cells and initiate a targeted immune response. This mechanism has made TCRs important therapeutic targets in immunotherapies and cancer treatment. However, expressing TCRs in traditional E. coli systems has posed challenges in throughput, yield and endotoxin levels. To tackle these challenges, WuXi Biologics’ PS Department developed the T Cell Mate platform, which utilizes a high-titer CHO expression system to efficiently produce a variety of T-cell-related molecules, including sTCRs, TCR fusion proteins, and SCTs, all achieving high yields, quality and throughput. Notably, the SCT format integrates HLA and β 2-macroglobulin into a single polypeptide, facilitating the secretion of well-folded pMHCs from CHO cells. The platform also supports the production of RF-pMHCs in E. coli ., providing a versatile and scalable solution for therapeutic T-cell-related proteins.

AC-SEC

50 37 25 15 10 20

• SCT in CHO has much higher expression than RF-pMHC from E. coli • SEC is required to remove aggregated SCT

In this case study, the expression of SCTs in transient CHO cells significantly surpassed that of RF-pMHC in E. coli. Among 10 SCTs, the average yield was 554 mg/L using AC purification. Following SEC for aggregate removal, the average yield of final products reached 306 mg/L. Case study 4: High-Purity, High-Yield Production of a Mouse SCT in CHO Cells Figure 5: SDS-PAGE and SEC-HPLC analysis for two-step purification of a mouse SCT

M L FT

250 KDa 100 150

Figure 1: T-Cell-Related Proteins The innovative T Cell Mate platform delivers all the proteins necessary for T-cell related projects, including sTCRs, TCR fusion proteins, RF-pMHCs (in E.coli ), and SCTs.

RT: 8.373 minutes Purity: 99.57%

RT: 8.340 minutes Purity: 82.42%

SCT

Ni: 18.3 mg

15 10 25 20

250 KDa 100 150

SEC

From 20 mL CHO HCCF, a mouse SCT was yielded at 18.3 mg after Ni purification. Subsequent SEC purification increased the target purity from 82.42% to 99.57%, with a yield of 10 mg and an endotoxin level of less than 0.1 EU/mg.

SCT

sTCR TCR-Ab Fusion RF-pMHCs, E.coli

SCT

Final: 10 mg LAL: < 0.1 EU/mg

15 10 25 20

Results

Case Study 5: High-Yield Production of MHCII with Different Constructs in CHO Cells Figure 6: SDS-PAGE (NR/R) analysis and titers for one-step purification of 4 MHCII molecules

Case Study 1: High-Titer Expression of sTCR in CHO Cells Figure 2: SDS-PAGE (NR/R) analysis for one-step affinity purification of 3 sTCRs

8EUQ: HLA-DRA*0101 Peptide-HLA-DRB1*0401 5V4M: HLA-DRA*0101 Peptide-HLA-DRB1*1501

V 

His

ScFv

C 

V 

V 

KDa

C 

KDa

C 

250 150 100

V 

250 150 100

C 

HCCF

Degly

L FT E M L FT E

L FT E

M L FT E

L FTE M L FTE

KDa

100 150 250

15 10 25 20

100 150 250

15 10 25 20

100 150 250

 1

 1

Fc −

ScFv −



8EUQ

5V4M





PNGase F

600

37 25



 Fc



400



200

Zipper1

Zipper2

Zipper1

Zipper2

Zipper1

Zipper2

AC yields: 1,100 mg/L

130 mg/L

100 mg/L

In this case study, three sTCRs were produced using the T Cell Mate platform. Following one-step affinity purification, the yields for one-arm Fc fusion, His-tagged TCR, and scFv-infused TCRs achieved 1092.0 mg/L, 112.2 mg/L, and 90.7 mg/L, respectively.

All experiments were conducted within the Protein Sciences (PS) Department at WuXi Biologics. The DNA sequence codons were optimized using WuXi Biologics’ laboratory data management system (LDMS), followed by plasmid construction into WuXi Biologics’ proprietary vector. Using the WuXian Transient expression system as per SOPs, the culture medium was collected after 7 days. Following clarification of the culture medium, all supernatant was loaded onto appropriate purification columns based on titer. Purification involved either Nickel column or protein A purification, followed by size exclusion chromatography (SEC). The purified products were then characterized through SDS-PAGE, SEC-HPLC, intact mass analysis, and endotoxin detection. Method WuXi Biologics has revolutionized the high-titer production of T-cell-related proteins with the T Cell Mate Platform, which encompasses sTCRs, TCR fusions, SCTs, and RF-pMHCs (in E. coli ). • The ultra-high titer CHO expression system ensures outstanding yields and enhanced throughput. • Flexible purification and advanced QC techniques guarantee exceptional protein quality, achieving high purity, low endotoxin levels, and robust complex integrity. • Efficient workflow allows for rapid project completion, typically within just 3-4 weeks. Conclusions We extend our gratitude to all scientists from the Department of Protein Sciences at WuXi Biologics for their invaluable support during the development of the T Cell Mate platform. Additionally, we express our appreciation to the legal department, public relations department, and marketing department for their assistance with patent filing, poster preparation, and revision. Acknowledgment WuXi Biologics is a global leading Contract Research, Development and Manufacturing Organization (CRDMO) offering end-to-end solutions to enable partners to discover, develop and manufacture biologics from concept to commercialization. For more information, visit us at wuxibiologics.com About WuXi Biologics

Case Study 2: Extensive Production of MHCI in E. coli Figure 3: MHCI production workflow, detailing purification and QC methods for purity evaluation

Over 260 of MHC production from 0.1 to 50 L E. coli

MHC Biotinylation

Purification

HLA

B2M Peptide

MHC Complex

Purification Profile

Mass Spectrum

Final Product

…

3 x10 0 1 2 3 4 5

+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr

M 1 μ g 2 μ g 5 μ g 10 μ g

37421.48

mAU

HLDb Biotinylated

3000

Q HP Gradient

B2M

11861.29

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10000 20000 30000 40000 50000 60000 70000 80000 90000

Counts vs. Deconvoluted Mass (amu)

1500

2 x10

…

+ESI Scan (4.497-5.723 min, 74 Scans) Frag=200.0V H2-Db-1.d Subtr

1000

1027.4328

Peptide

500

1028.4333

1029.4316

100

200

300

400

500

600

700

Counts vs. Mass-to-Charge (m/z) 1026 1027 1028 1029 1030 1031 1032

Poster modified on 10/24/2024

To discuss this poster: PS_BD@wuxibiologics.com

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