Rapid Vaccine Development & Biomanufacturing Using a Scalable, Non-viral Delivery Platform ®

for Cell Engineering.

James Brady, Weili Wang, Rama Shivakumar, Pachai Natarajan, Krista Steger, and Madhusudan Peshwa. MaxCyte, Gaithersburg, MD, USA.

Abstract
Researchers have looked to recombinant technologies to develop innovative types of vaccines and new cell culture-based means High Level Cell Viability and Transfection Efficiencies
of production that offer shorter lead times and greater production flexibility while maintaining vaccine safety. Transient
Figure 1. High Efficiency Transfection of Cell Types
transfection offers a means of rapidly producing an array of proteins, including antibodies, vaccines, viral vectors, and virus-like
Commonly Used for Protein and Vaccine Production.
particles (VLPs). Although a variety of transient transfection methods are available, most do not meet the requirements of
Various cells were transfected with 2 µg/1E6 cells of pGFP
scalability, consistency, and cell type flexibility for use in vaccine development and manufacturing. MaxCyte’s electroporation- DNA using the appropriate MaxCyte STx protocol. Cells
based delivery platform reproducibly transfects a broad range of biorelevant adherent and suspension cell types with high cell were examined for GFP expression using fluorescence
viabilities and transfection efficiencies using single-use processing assemblies for cGMP, “plug-and-play” production of microscopy 24 hrs post electroporation (EP).
recombinant proteins and vaccines. In this poster we present data for large-scale production of antibodies, recombinant
antigens, VLPs, and lentiviral vectors using the MaxCyte STx® Scalable Transfection System. Data are presented for high-
efficiency transfection of cells commonly used in protein production including CHO, HEK293, and insect cells—without the use of
baculovirus—with a timeline of just a few days from plasmid to gram quantities of protein.

Expression of Multiple Protein Types Using MaxCyte’s Delivery Platform

High Cell Viability Leads to Strong HIV gp145 Expression Multi-Gram, CHO-based Antibody Production Rapid, More Efficient VLP Production Compared to
Baculovirus Expression Systems
Electroporation Cell Media Control
Cell Media
d
cte
fe ia
In ed
BV ell M
C

h
24

48

72

96

24

48

72

96
M
Antigen 1

Antigen 2

Antigen 3
BV Contaminants

Figure 2: Cell Viability and Protein Titer Data Following Transfection of CHO Cells with an
Figure 3: Post Electroporation Culture Process Optimization Produces CHO-S Antibody Titers >2.7 g/L. Figure 4. Sf9 VLP Production Using MaxCyte Electroporation: Plasmid to Product
HIV Envelope Protein Expression Plasmid. CHO-S cells were transfected with a gp145
CHO-S cells were transfected with an antibody expression plasmid (1μg DNA/1E6 cells) via small scale EP in Two to Four Days. Sf9 cells were transfected via static EP with a single plasmid
expression plasmid via small scale EP (8e7 cells) and large scale EP (2e9 cells). Transfected
on the MaxCyte STx. Cells were plated at approximately 4E6 cells/mL post EP. Transfected cells were encoding three antigens that co-assemble into VLPs. Culture media was collected
cells were inoculated into shake flasks at the same density and cultured for 10 days. Cells
cultured in media with different additives and culture conditions. Titer was verified by both ELISA and at various times from cells post EP or following baculovirus infection and analyzed
from the small scale and large scale EPs yielded consistent titers and exhibited high
Protein A capture assays. The optimized process produced antibody titers of 2.74 g/L at day 17 post EP as using SDS PAGE. VLP production was observed within 48 hours post EP.
viabilities. Viabilities and titers from the electroporated cells were much higher than
a fed batch.
corresponding values for a customer’s optimized PEI process.

Viral Vector Production: Seamless Scalability for Rapid Manufacturing

Consistent Large-scale Lentivirus Manufacturing Using Seamless Lentivirus Scalability - MaxCyte STX to VLX High Titer AAV Production in HEK Cells
Suspension Cells
Transfer
A. plasmid
(GFP)

Rep/Cap
+
4e7 cells 1e9 cells 1.1e10 cells
(OC-400) (CL-2) (VLXD)
( x1e5 TU/mL)

Helper 4e7 cells Plate in T Flasks; assay titers

Virus Titer

24 & 48 hours post EP

C. qPCR Analysis of AAV Titers from Cell Pellets

Production Volume 48 hr Titer Cumulative Productivity B.

Total Cells
Run (mL) (IU/mL) Titer (IU) (IU/cell) Small-scale Large-scale Large-scale
400 µl 10 mL 110 mL
1 2300 6.0 x 109 9.8 x 107 2.2 x 1011 37 STX STX VLX

2 2300 4.8 x 109 8.8 x 107 2.0 x 1011 42 MaxCyte STx MaxCyte VLX
3 2100 7.4 x 109 1.3 x 108 2.7 x 1011 36
99% GFP+
Figure 6. Scale Up of Lentiviral Vector Production from Small-Scale to Large-Scale
Production Using the MaxCyte Platform. Suspension-adapted HEK 293FT cells were
Figure 5. Large-scale Lentiviral Vector Production. Suspension-adapted HEK suspended in MaxCyte EP buffer at 1E8 cells/mL. A mixture of plasmids encoding lentiviral
293FT cells were harvested, resuspended at 1E8 cells/mL, and co-transfected with vector components was added to the cells (0.4µg of DNA/1E6 cells), and cells were
4 plasmids (HIV-based lentivector system) using flow EP. Cells were cultured post transferred to sterile OC-400, CL-2 and VLXD processing assemblies. Cells in the OC-400
EP in 10-L Cellbag in a Wave Bioreactor in a final volume of 2.1 to 2.3 L. 48 hours and CL-2 were transfected by static and flow EP, respectively, using the STx instrument; Figure 7: Production of AAV in HEK Cells. (A). Adherent HEK cells were transfected with three
post EP, media was collected and infectious units measured. Results for three cells in the VLXD were transfected by flow EP on the VLX. Lentiviral titers were measured plasmids encoding AAV vector components (GFP transgene) via static EP using the MaxCyte
independent production runs are shown. These data demonstrate the after 24-48 hrs in culture. Normalized titer data show seamless scalability of the MaxCyte STx. (B). Nearly 100% of the transfected cells exhibited robust transgene expression 48 hours
transfection process.
reproducibility of MaxCyte flow EP enabling large-scale, quality lentivirus post EP. (C). High AAV titers were detected in cell pellets via qPCR analysis.
production. *See Human Gene Therapy (2012) 23:243-249 for detailed methodology.

Summary
MaxCyte Delivery Platform
• MaxCyte’s EP-based delivery platform is fully scalable from 5e5 cells to 2e11 cells allowing for production of milligram to multi-gram quantities of
proteins, viral vectors, and multi-protein complexes such as VLPs.
The MaxCyte STx® and MaxCyte VLX® Transient Transfection
• MaxCyte EP is high performance means of transiently transfecting adherent and suspension cell lines commonly used during vaccine Systems use fully scalable flow electroporation for rapid,
development and production such as CHO, HEK, and insect cells. highly efficient transfection.
• MaxCyte transfection rapidly produces a variety of proteins including antigens, antibodies, lentiviral vectors, and VLPs more efficiently than MaxCyte STx® • High efficiency & high cell viability
chemical transfection methods and baculovirus expression systems. 5e5 Cells in Seconds
Up to 2e10 Cells in <30 min • Broad cell compatibility
• MaxCyte transfection of CHO cells can produce secreted antibody titers >2.5 gram/L with optimization of post transfection culture conditions.
• True scalability requiring no re-optimization
• Insect cells rapidly express recombinant proteins, including VLPs, at high efficiency following MaxCyte EP, eliminating the need for baculovirus
• Closed, computer-controlled instruments
use.
• cGMP-compliant & CE-marked
• MaxCyte EP results in high-performance transfection of suspension-adapted HEK cells allowing for large-scale, highly reproducible production of
• Master file with US FDA & Health Canada
viral vectors.
• Production scale-up from the MaxCyte STx to the MaxCyte VLX is seamless – maintenance of transfection performance without the need for re-
optimization.
MaxCyte VLX®
Up to 2e11 Cells in <30 min

Corresponding Author: James Brady; [email protected] MaxCyte, Inc., Tel: (301) 944-1700 [email protected], www.maxcyte.com
Flow Electroporation, MaxCyte, ExPERT, MaxCyte VLX and MaxCyte STX are trademarks of MaxCyte, Inc. World Vaccine Congress, April 2017 © MaxCyte 2020 All Rights Reserved.