Ultra 96 HTP Antibody Production Platform

A cost-effective, ultra-HTP antibody production platform that uses 96- and 24-well DWP format, resulting in a daily throughput of 1000x antibody production at 100ug -1,500ug per antibody, suitable for in vitro assays.

Establishment of an Ultra High-Throughput Protein Production Platform (Ultra 96) for Early Stage in vitro Studies Kai Jiang, Huishuai Li, Mengjie Lu, Chuanlong Tang, Xuejian Zhang, Ying Zhang, Zhengyang Zhao, Jun Zhou, Jiansheng Wu

WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)


Fig 6. One step purification result of the 24 constructs expressed in 24 DWP. For each molecule (M1 to M12), the left lane corresponds to SDS-PAGE reducing condition and the right lane corresponds to SDS-PAGE non-reducing condition. Case study: 24 DWP production of His-tagged and Fc-tagged recombinant protein To meet the requirement of our clients, 1 to 3 mL expression scale was chosen to achieve high quantities of protein for construct screening. In this case, 12 different recombinant proteins with His-tag and Fc-tag were produced in 24 well DWP.

Fig 3. The average titer and recovery rate of “Ultra 96” platform The left histogram shows the average titer of each molecule expressed in 96 well DWP at 1 mL scale. The average protein recovery rate after one-step purification is above 83%.

Proteins are known as the building blocks of life which play many critical roles in the body. In early stage drug discovery, numerous μg to mg scale high quality proteins need to be produced for in vitro functional studies and therapeutic evaluation, including antigens, monoclonal antibodies (mAb), bispecific antibodies (bsAbs) as well as other Fc-fusion proteins. High-throughput production (HTP) of various proteins with high quality in very short time is a big challenge in industry. Here we present how WuXi Biologics has created a powerful ultra HTP protein production platform (termed as “Ultra 96”) that can automatically produce proteins within 3 weeks with one step purification at 1~ 3mL expression scale by using 96 and 24 well DWP. High titer of expression, high quality of products, short timeline as well as low cost make “Ultra96” a robust platform which can empower our clients and dramatically accelerate their early stage drug discovery projects.

Titer Variation in 96 DWP

50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 1 2 3 4 5 6 7 8 9 101112131415161718192021222324 Recovery Rate (24DWP) Average 84% 50.0% 55.0% 60.0% 65.0% 70.0% 75.0% 80.0% 85.0% 90.0% 95.0% 1 3 5 7 9 11131517192123252729313335373941434547495153555759 Recovery Rate (96DWP) Average 83%


• 12 different proteins • 24 constructs • 293 system • 24DWP • 3mL cell culture





All proteins vary at some level in its cell culture requirements to achieve high protein expression and also unique purification conditions to obtain a high enough purity to function properly within in vitro and analytical screening methods. Because of this variability, establishing one robust high- throughput system has been a major challenge. WuXi Biologics’ expert cell line engineering, protein purification and analytical scientists have established a unique HTP platform using our proprietary WuXian™ Transient protein expression system, unique HTP single-column purification strategy and streamlined analytical quality control (Fig. 2) to overcome these obstacles and to rapidly provide high quality products for our clients.


mAb Bi-specific Fc fusion

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 see us at wuxibiologics.com About WuXi Biologics We thank all scientists from Department of Protein Sciences of WuXi Biologics for their great support during the development of the “Ultra 96” platform. We also thank our legal department, public relations department and marketing department for their great help in patent filing and poster preparation and revision. Acknowledgment • WuXi Biologics has established a powerful ultra-high-throughput protein production platform (“Ultra 96”) which can deliver various types and formats of proteins and greatly supports our client’s early-stage drug discovery projects. • The “Ultra 96” platform can provide mg proteins from 1 to 3 mL cell culture scale for construct screening and in vitro studies. • Tens of thousands of antibodies have been produced in the “Ultra 96” platform and our data shows the platform is robust, rapid and can provide proteins at low cost. Conclusions Method All experiments were conducted by the department of Protein Sciences at WuXi Biologics. In summary, for each protein, codon optimization of the DNA sequence was conducted utilizing the WuXi Biologics lab data management system (LDMS) and plasmids were constructed using WuXi Biologics’ proprietary vector. WuXian™ Transient cell culture platform was used for protein expression and performed based on SOPs and the culture medium was collected after 7 days. After clarification of the culture medium, all supernatant was loaded onto an appropriate affinity column. Final product was analyzed by Caliper-SDS and SEC-HPLC.


Fig 4. High-throughput production and screening of 1016 antibodies in the Ultra 96 platform Case study: HTP antibody screening In this case, 1016 antibodies were produced in 96 well DWP at 1 mL scale. The average titer was 0.6 g/L. Subsequently, 60 antibodies were selected and further evaluated after production in 24 well DWP at 3 mL scale.

Fig 1. Therapeutic molecules

60 Antibodies 24DWP

1016 Antibodies 96DWP

Monoclonal Antibody

Bispecific Antibody

Antibody Drug Conjugate

Fc-fusion Protein

Antibody Fragment

Recombinant Protein

Fig 2. The scheme of “Ultra 96” platform In the Ultra 96 platform, proteins, with only one step purification, can be delivered within 3 weeks. The expression scale is 1to 3 mL using 96 and24 well plates. We typically obtain 100 to 1,500 μg protein which is of sufficient quantity and quality for in vitro studies.

Fig 5. Average titers of different molecules in Ultra 96 platform

50 Antibodies, 1 mL

56 Fc fusion, 3 mL

88 VHH-His, 3 mL




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