Droplet Digital PCR

Measure HEK293 Residual DNA Size
without a Reference Curve
Human gene therapy products are created in immortalized cell lines, most commonly
HEK293. Residual DNA from immortalized cell lines can harbor tumorigenic genetic
sequences or retroviral sequences that can cause infection. The U.S. Food and Drug
Administration (FDA) recommends that manufacturers degrade any residual DNA
to below the size of a functional gene, defined as ~200 base pairs. Manufacturers
require an inexpensive, accurate, and reliable way of measuring the size of residualDNA, which Droplet Digital PCR (ddPCR) technology can provide.
The Vericheck ddPCR HEK293 Res DNA Size Kit is the first HEK293-specificsizing solution based on Droplet Digital PCR, designed and validated to meet
regulatory guidance requirements and capable of measuring HEK293 size without
the use of reference curves.
The Vericheck ddPCR HEK293 Res DNA Size Kit provides:
High specificity and sensitivity■■■

Low cross-reactivity and low false-positive rate
 99.99% specificity to HEK293 DNA when tested against Chinese hamster ovary,>
Escherichia coli, Vero, and normal human DNALimit of blank (LOB) of 0 with limit of detection (LOD) of 2 pg/well

Extraction-free protocol without use of standard curves■

Broad range of sample types includes:– In-process samples: cell lysates, cell culture media, sonicated samples,and adeno-associated virus vectors– P urified final product sample: phosphate buffered saline with human serum
albumin using an extraction-free workflow

Visit bio-rad.com/ddPCR-Vericheck-HEK-Size for more information.

Measure HEK293 Residual DNA Size without a Reference Curve

Autothresholding with regulatory compliant software■Provides median size of residual DNA fragments with confidence intervals■

Positive control–based autothresholding with regulatory-compliant 1.2 version of QX Manager or QX ONE Software■

U.S. FDA 21 CFR Part 11 compliant, offering audit trails with tracked protocol changes
10,0008,000

Channel 1 amplitude

FAM- and HEXpositive droplets

FAM-positive
droplets (short andlong fragments)

6,000

4,000

HEX-positive
droplets (≥200 bpfragments)

Double-negative
droplets
2,000

00

1,000

2,000
Channel 2 amplitude

3,000

4,000

2-D amplitude plot for the Vericheck ddPCR HEK293 Res DNA Size Kit. The plot shows HEK293 short and long fragment signals in channel 1 (FAM) and fragments≥200 base pairs (bp) signal in channel 2 (HEX). The cluster in gray is double-negative for HEK293 DNA, the blue cluster is single-positive for HEK293 short and long fragments,
the green cluster is single-positive for fragments ≥200 bp, and the orange cluster is double-positive for both HEK293 DNA sizes.
B
10,00010,000

8,000

8,000

Channel 1 amplitude

Channel 1 amplitude

A

6,000

4,000

2,000

6,000

4,000

2,000

0001,000

2,000
Channel 2 amplitude

3,000

4,0000

1,000

2,000
Channel 2 amplitude

3,000

4,000

2-D plots showing fragmentation of HEK293 DNA. (A) In unfragmented HEK293 DNA, the two amplicons will have approximately equal signal in the FAM and HEXchannels, appearing as double-positives. DNA fragments with a median size of >2,000 bp can be detected. (B) As DNA breaks into smaller fragments, the ≥200 bp HEX
channel will lose signal more rapidly than the short and long FAM channel, shifting the 2-D plot toward the single-positive FAM cluster with 110 bp median fragment size.

© 2022 Bio-Rad Laboratories, Inc.

2Bulletin 3305

Measure HEK293 Residual DNA Size without a Reference Curve

B
8,000

8,000

7,000

7,000

6,000

6,000
Channel 1 amplitude

Channel 1 amplitude

A

5,000
4,000
3,000

5,000
4,000
3,000

2,000

2,000

1,000

1,000

0

1,575

2,458

00500

1,000

1,500
2,000 2,500
Channel 2 amplitude

3,000

3,500

4,000

0500

1,000

1,500
2,000 2,500
Channel 2 amplitude

3,000

3,500

4,000

2-D plots showing positive control–based autothresholding. Positive control–based autothresholding allows for accurate thresholding of an entire plate simultaneously.
Accurate thresholds are applied to HEK293 samples with concentrations spanning the ddPCR dynamic range. The 2-D plots above show the same well before (A) and after (B)positive control–based autothresholding with tilt correction.

Visit bio-rad.com/ddPCR-Vericheck-HEK-Size for more information.
BIO-RAD, DDPCR, DROPLET DIGITAL PCR, and QX ONE are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owner.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the “Products”) from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc. Standard Terms and Conditions ofSale, which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for you to distribute orresell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for the development or commercialization of diagnostic assays for use with the Products
without a license from Bio-Rad. The Products and/or their use are covered by U.S. and foreign patents and/or pending patent applications owned by or under license to Bio-Rad Laboratories, Inc. See
https://www.bio-rad.com/en-us/trademarks.

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