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