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Poster

Powerdoma™: An Upgraded Hybridoma Platform

Powerdoma™: an upgraded hybridoma platform  
boasting fast timeline and improved  
cost-effectiveness for antibody discovery  
Derek Chen, Yu Liang, Lindi Wang, Wenwan Fang, Jie Ma  
ProBio Inc. USA  
ABSTRACT  
ProBio’s  
Conventional  
TM  
Powerdoma  
Hybridoma  
As one of the most traditional and well-established technologies,  
hybridoma approach contributed to more than three quarters of antibody  
drugs approved by FDA over the last decade. However, this technology is  
limited by several bottlenecks ever since its first appearance, including  
time-consuming process and loss of diversity in in cell fusion and  
subcloning. To address these limitations, here we developed the  
Monoclonal  
14-20 days  
In-vitro  
function  
assays  
Recover  
Recover  
Expanding  
cell culture  
Subcloning  
splenocyte  
splenocyte  
TM  
Powerdoma platform, an upgraded version of the conventional  
hybridoma technology, which allows us to discover functional Ab leads with  
good diversity in a much faster and more cost-effective way.  
3-4 4-6  
7 days  
7-14 days  
Sequencing  
of desired  
binders  
Sequencing  
of desired  
binders  
months  
Plasma B cell  
enrichment  
Binder  
Cell fusion  
screening  
7 days  
7 days  
From  
immunization  
to  
BOTTLENECKS & SOLUTIONS  
96-well plate,  
seeding 5-10  
clones per well  
In-vitro  
function  
assays  
Plasmid  
Plasmid  
Cell fusion  
preparation  
preparation  
recombinant  
Ab  
Diversity loss in conventional hybridoma workflow  
7 days  
Due to the nature of cell fusion and genomic instability of hybridoma cells, the  
diversity of Ab sequences carried by hybridoma cells suffers significant loss  
during each step of the screening process, leading to much fewer surviving  
candidates for functional screening and reduced chance of identifying  
functional Ab hits.  
384-well plate,  
seeding 1-3  
Antibody  
HTP Ab  
Binder  
production  
production  
screening  
clones/ well  
7 days  
7-8 days  
Recombinant  
Ab protein  
Binder  
Recombinant  
Ab protein  
screening  
Monoclonal  
Figure 2A  
Figure 2B  
TM  
Powerdoma project statistics  
TM  
Comparing to conventional hybridoma workflow, Powerdoma allows ~40%  
more hybridoma clones to be screened (figure 3A), with a shortened timeline  
decreasing from 58 to 30 days in average (figure 3B), and a four-fold higher  
positive hit rate (2000+ vs. 500+) in average from 11 projects with  
head-to-head comparison (figure 3C). Based on the 40+ previously delivered  
projects, for Powerdoma workflow we observed a monoclonal rate of 80%, a  
sequencing success rate of 97%, and a 100% confirmation rate by recombinant  
expression (figure 3D).  
Shorter Turnaround Time to Get  
More Clones Available for Screening  
Confirmed Binder Sequence  
250 00  
PowerdomaTM  
Hybridoma  
PowerdomaTM  
Hybridoma  
200 00  
150 00  
100 00  
500 0  
80  
70  
60  
50  
40  
30  
20  
10  
Avg.  
Avg.  
58 d  
14810  
Avg.  
10437  
Avg.  
Figure 1  
30.5 d  
0
0
Project1 Project2 Proj-  
Project4 Project5 Project6  
Project  
1 2 3 4 5 6 7 8 9 10 11 12  
Figure 3A  
Figure 3B  
TM  
Powerdoma vs conventional hybridoma workflow  
Conventional hybridoma (figure 2B) workflow heavily relies on the subcloning  
steps, which may take about 6 weeks, to reach the monoclonal stage of  
hybridoma clones for downstream screening. In contrast, this time-consuming  
subcloning process is eliminated in the Powerdoma workflow (figure 2A);  
instead, an optimized process is developed to allow the direct achievement of  
monoclonal stage of hybridoma clones. This optimization leads to a shortened  
timeline by 1-1.5 months and enables functional screening using hybridoma  
supernatant at much earlier stage of Ab lead discovery, allowing the  
identification of functional Ab hits with a much faster turnaround time,  
improved cost-effectiveness and better chance of success.  
More Positive Clones After Primary Screening  
40+  
~80%  
PowerdomaTM  
Hybridoma  
PowerdomaTM projects  
successfully completed  
Monoclonal rate  
Avg.  
100 00  
100 0  
100  
2143  
Avg.  
535  
>97%  
100%  
10  
Success rate of  
sequencing  
Recombinantly  
1
confirmed  
Project  
1
2
3
4
5
6
7
8
9
10 11  
Figure 3C  
Figure 3D  
ProBioCDMO.com  
Powerdoma™: an upgraded hybridoma platform  
boasting fast timeline and improved  
cost-effectiveness for antibody discovery  
Derek Chen, Yu Liang, Lindi Wang, Wenwan Fang, Jie Ma  
ProBio Inc. USA  
FUNCTIONAL CHARACTERIZATION CASE STUDIES  
TIGIT/CD155 blockade  
assay  
FACS Binding-CHO-  
TIGIT Blockade bioassay on  
K1/Human TIGIT stable cells(+) Opti-TIGIT effector cell+ GS-  
C2/CD155  
TM  
1. Affinity ranking by KD-ELISA using Powerdoma supernatant  
BMK  
Affinity ranking by SPR/BLI is expensive, especially when it comes to deal with  
hundreds of samples. As a cost-effective substitute, kD-ELISA utilizes a  
capturing Ab coated to the plate to capture the Abs being tested from the  
supernatant, followed by detection with a biotinylated antigen (figure 4A). In a  
primary screening assay using Powerdoma supernant based on kD-ELISA, a  
number of potentially strong binders (green box) and weak binders (red box)  
were identified (figure 4B). Then these selected candidates were  
recombinantly expressed and characterized by full curve ELISA. Consistently,  
the previously identified strong binders (clone 4, 7, 13, 15 in green box)  
showed a high binding affinity similar to benchmarks (figure 4C), while those  
weak binders (clone 1, 3, 14) showed much lower affinity than benchmarks  
(figure 4D).  
Figure 5B  
Figure 5C  
Figure 5D  
TM  
GPCR assay using Powerdoma supernatant  
cAMP assay is a well-recognized approach to evaluate the agonist /antagonist  
activity of antibody against GPCR targets. By such an assay using  
TM  
powerdoma supernatant (figure 6A), a number of potential antagonist Abs  
against target A were identified. Consistently, after recombinant expression  
and purification, these Abs were confirmed to block target A mediated cAMP  
production in a dose-dependent manner (figure 6B).  
Neutralization Assay on CHOK1/Gα15/GPCR target A  
Figure 4B  
Figure 4A  
FACS Binding-CHO-K1/CD117 cell pool  
FACS Binding-CHO-K1/CD117 cell pool  
Figure 6A  
Neutralization Assay on CHOK1/Gα15/GPCR target A  
Figure 4C  
Figure 4D  
TM  
2. Reporter Gene Assay (RGA) using Powerdoma supernatant  
TM  
TIGIT reporter gene assay was conducted with powerdoma supernatant  
(figure 5A), identifying four potential functional blockers of TIGIT/CD155  
signaling (clone 3, 7, 15, 19, in green box), and one non-functional binder  
(clone 10, in red box). These candidates were recombinantly expressed and  
the purified Abs subjected to full curve blocking assays. As shown in figure 5B,  
these previously identified functional blockers are confirmed to potently block  
TIGIT/CD155 signaling, while the non-functional binder (clone 10) binds to  
TIGIT (figure 5C) without signaling blockade (figure 5D).  
Figure 6B  
TIGIT/CD155 Blockade assay  
0.3μg /mL  
CONCLUSION  
Blank  
TM  
Taken together, the Powerdoma workflow is an upgraded hybridoma  
platform for Ab lead discovery, with substantially shortened timeline and  
improved cost-effectiveness. More importantly, it makes it feasible to  
conduct functional screening at very early stage of Ab lead discovery, which  
may greatly improve the chance of success in discovering functional leads  
with desired target product profile.  
Figure 5A  
ProBioCDMO.com