APPLICATION NOTEAutomate Express Nucleic Acid Extraction System
AutoMate Express
Nucleic Acid Extraction System
Introduction
Biopharmaceuticals produced in live-cell systems such
as CHO cells need to be tested for contaminants and
impurities that originate from the process or from the
host cell line. Testing for bacterial and viral contaminants
such as mycoplasmas, mouse minute virus (MMV), and
vesiviruses is performed during the upstream cell culture
process, whereas host cell–derived impurities such as
residual DNA need to be tested for as part of downstream
process characterization. The availability of rapid and
accurate testing methods is key to enabling in-process
testing during manufacturing, and the results from those
tests can then be used to make fact-based, go or no-go
forward processing decisions.
The Applied Biosystems™ AutoMate Express™ Nucleic Acid
Extraction System is an easy-to-use benchtop instrument
that enables hands-free automation of the nucleic acid
extraction process for contaminant and impurity testing.
The instrument uses the established Applied Biosystems™
PrepSEQ™ nucleic acid extraction chemistry for quantitative
recovery of DNA and RNA from diverse samples types,
including cell culture, raw material (i.e., serum), and inprocess downstream purification samples such as column
purification or bulk drug substance (BDS) samples.
AutoMate Express Nucleic Acid Extraction System
• Time-saving, cost-effective DNA extraction for
mycoplasma, MMV, vesivirus, and residual DNA
quantitation
• Quantitative and reproducible recovery of DNA from
complex samples
• Out-of-the-box automation with prefilled PrepSEQ™
express cartridges and ready-to-run protocols
• Closed system minimizes the risk of contamination
• Ideal for in-process testing of contaminant and impurities
Three-in-one extraction of cell culture contaminants
The AutoMate Express™ platform can be used to perform
nucleic acid extraction of the common contaminants for
mammalian cell culture manufacturing, i.e., mycoplasmas,
MMV, and vesiviruses. Flexible protocols are available to
isolate DNA and RNA from a single organism, or a 3-in-1
surveillance panel protocol can simultaneously extract
mycoplasma, MMV, and vesivirus nucleic acid from a
single sample. Figures 1 and 2 demonstrate recovery
of DNA from spikes of DNA, or from cells or viruses of
mycoplasmas and MMV into typical cell culture matrices,
extracted using the AutoMate Express™ system.
A
B
31
Ave Ct from 10 µL assay input
Ave Ct from 10 µL assay input
28
26
24
22
20
PBS RC
PBS
FCS RC
FCS
CM RC
CM
CCM RC
29
27
25
23
21
CCM
PBS RC
PBS
FCS RC
FCS
CM RC
CM
CCM RC
CCM
Figure 1. Detection of (A) mycoplasma DNA and (B) MMV DNA spiked into PBS (blue), fetal calf serum (black), CHO medium (gray), and CHO–
conditioned medium (orange). For each matrix, a recovery control (RC) was used to assess extraction efficiency.
A
B
31
29
Ave Ct from 10 µL assay input
Ave Ct from 10 µL assay input
31
27
25
23
21
29
27
25
23
21
19
2,000 copies
1,000 copies
500 copies
PBS
CM
CCM
2,000 copies
1,000 copies
MMV Pos Control
500 copies
PBS
FCS
CM
MMV (ATCC VR1346) 15TCID50 units/200
CCM
L
Figure 2. Detection of DNA from (A) M. arginini and (B) MMV spiked into PBS (blue), fetal calf serum (black), CHO medium (gray), and CHO–
conditioned medium (orange). For each plate, control reactions were run with 500, 1,000, and 2,000 copies of the mycoplasma discriminatory positive
control or MMV positive control in the real-time PCR assay (green).
PrepSEQ™ Express kits enable quantitative and consistent
recovery of DNA from challenging matrices, including
samples that have high protein concentration, low pH,
or high salt. Figure 3 shows recovery of CHO and Vero
cell line–derived genomic DNA spiked into five diverse
matrices. The data demonstrate the quantitative recovery
and reproducibility of the system.
A
B
9.0
8.0
8.0
7.0
7.0
6.0
6.0
DNA recovery (pg)
DNA recovery (pg)
Quantitative recovery for residual DNA applications
The AutoMate Express system is also designed for highly
efficient recovery of host cell residual DNA from complex
samples typically recovered during biopharmaceutical
purification processes. When used together with the
AutoMate Express system, the Applied Biosystems™
5.0
4.0
3.0
5.0
4.0
3.0
2.0
2.0
1.0
1.0
0.0
0.0
Matrix 1
Matrix 2
Matrix 3
Matrix 4
Matrix 5
Matrix 1
Matrix 2
Matrix 3
Matrix 4
Matrix 5
C
Matrix
Buffer
IgG
M1
Ion exchange
0.8 M NaCl,
20 mM NaPO4 (pH 7.5)
10 mg/mL
M2
Hydrophobic interaction
0.75 M ammonium sulfate,
50 mM NaPO4 (pH 6.0)
10 mg/mL
M3
Ion exchange
0.7 M KPO4 (pH 6.0)
10 mg/mL
M4
Protein A
100 mM sodium citrate (pH 3.0)
10 mg/mL
M5
Purified drug substance
3% mannitol,
2% sucrose,
10 mM L-arginine,
0.01% Tween™ 20
50 mg/mL
Figure 3. Recovery from 10 pg of (A) CHO genomic DNA and (B) Vero genomic DNA spiked into five different matrices. Panel (C) shows the
composition of each of the five matrices.
Closed system with walk-away automation
The AutoMate Express platform automatically performs all
steps for efficient nucleic acid extraction from contaminants
and impurities in a closed system. The system uses
PrepSEQ Express kits that contain sealed cartridges
with extraction reagents, and plastics, including tips, and
sample, lysis, and elution tubes. The workflow (Figure 4)
1
Centrifuge sample tubes to
pellet cells or debris
2
Load tubes and cartridges
ino the instrument
has been designed for a completely hands-free operation
enabling reproducible extraction efficiencies and reduced
time-to-results. The small footprint of the instrument
enables easy integration into an existing lab setup. Up to
13 samples can be processed in under an hour, and the
eluates from the extraction can be used directly in real-time
PCR-Seq analytics assays.
3
Start the protocol
4
Use the eluate for
real-time PCR
Figure 4. One-hour workflow for the AutoMate Express system.
Ordering information
Product
Cat. No.
AutoMate Express Nucleic Acid Extraction System
4467754
PrepSEQ Express Nucleic Acid Extraction Kit
4466351
AutoMate Express IQOQ, SVC
4470470
Mycoplasma Detection Assay, with discriminatory positive control (does not include protocol and quick reference card)
4460623
Mycoplasma Detection Assay, with discriminatory positive control (includes protocol and quick reference card)
4460625
resDNASEQ CHO Quantitative DNA kit (does not include protocol and quick reference card)
4402085
resDNASEQ CHO Quantitative DNA kit (includes protocol and quick reference card)
4442731
resDNASEQ E. coli Quantitative DNA kit (does not include protocol and quick reference card)
4458435
resDNASEQ NS0 Quantitative DNA kit (does not include protocol and quick reference card)
4458441
resDNASEQ Vero Quantitative DNA kit (does not include protocol and quick reference card)
4458444
resDNASEQ Pichia pastoris Quantitative DNA kit (does not include protocol and quick reference card)
4464336
resDNASEQ MDCK Quantitative DNA kit (does not include protocol and quick reference card)
4464335
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