resdnaseq residual dna quantitation...

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The world leader in serving science

ResDNASEQ™ Residual DNA Quantitation System

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Rapid molecular methods for pharmaceutical manufacturing

Product Safety Microbial Identification and Detection

Bacterial ID Fungal ID Virus Detection Mycoplasma

Product Quality Impurity Analysis

Real-Time PCR

Residual DNA Custom HCP Protein A

DNA Sequencing

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Where does testing for residual DNA occur?

Cell Line Development

Cell Culture Purification Formulation

Expression Vector

Development

Transfection Clone

Selection

Cell Banks

Bioreactors

Harvest/

Cell Removal

Harvest

Media

Column

Chromatography

Virus Clearance

Concentration

Bulk Drug

Substance

Finished Batch

of Active Substance

Residual DNA

Quantitation

96-well plate format and automated sample prep enable

design and execution of rigorous process clearance studies

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Why is residual DNA quantitation important?

Biological products produced in cell culture

contain unique impurities, including host cell

proteins and host cell DNA.

Current testing methods lack reliable DNA

recovery methods, assay specificity, sensitivity,

speed and robustness.

Residual host cell DNA quantities contained in

final dosage form must follow regulatory

guidelines established by WHO, EU, FDA, EMEA.

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resDNASEQ™ Residual DNA Quantitation System

First integrated Real-Time qPCR system

for quantitation of residual host cell DNA

Prepare Sample

Kit

• PrepSEQTM

Residual DNA

Sample

Preparation Kit

Kit

• resDNASEQTM

Quantitative

CHO DNA Kit

Kit

• resDNASEQTM

Quantitative

CHO DNA Kit

Instrument

• 7500 Fast Real-Time

PCR System

Software

• AccuSEQTM 2.1 Software

• 21 CFR Part 11 Module

5 hours

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Key features of the resDNASEQ™ Residual DNA Quantitation System

Optimized Sample Prep Quantitative DNA recovery from

complex matrices with high precision

Specific to Target DNA No cross-reactivity to unrelated DNA

Highly Sensitive

LOQ to 1.5 pg/mL for mammalian DNA

LOQ to 15 pg/mL for bacterial/yeast DNA

Rapid Results

In <5 hours

Consistent Results

Optional automation for minimal variation

Support Network

Worldwide technical and validation support and custom capabilities

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Enables consistent recovery and quantitation of high and low MW DNA

DNA

Added

M1 M2 M3 M4 M5

Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I

1 ng 122% 91% 122% 100% 95% 88% 103% 93% 85% 105%

0.1 ng 95% 94% 95% 108% 109% 86% 99% 107% 82% 110%

10 pg 91% 84% 91% 98% 96% 89% 84% 98% 86% 101%

1 pg 88% 82% 88% 87% 91% 79% 85% 82% 66% 101%

0.1 pg 91% 87% 91% 80% 100% 87% 82% 86% 76% 115%

0.5

1

1.5 2

10

kb

Un

-cu

t

Sa

u96

I

Matrix Buffers IgG

M1 Ion Exchange 0.8 M NaCl

20 mM NaPO4 (pH 7.5) 10 mg/ml

M2 Hydrophobic

Interaction

0.75M Ammonium sulfate

50 mM NaPO4 (pH 6)

10 mg/ml

M3 Ion Exchange 0.7 M KPO4 (pH 6) 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

100 mg/ml

Surrogate Matrices: Typical Monoclonal Antibody Purification

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Specific to CHO target DNA

Standard curves generated from analysis of 10-fold dilution series

of CHO standard DNA in the presence or absence of human DNA

TaqManTM assay detects only CHO DNA, unaffected

by the presence of foreign DNA

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Broad dynamic range and high sensitivity

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

6 30 fg

Amplification plots generated from analysis of 10-fold dilution

series of CHO standard DNA

Broad dynamic range leads to high sensitivity

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Consistent recognition and quantitation across a broad range of DNA sizes and CHO strains

10 min sonication

30 min sonication

0 min sonication

1 min sonication

5 min sonication

0.5

1

1.5

10

kb

1. 1kb ladder

2. 0 min (>20 kb)

3. 1 min (>10 -1.5 kb)

4. 5 min (8 -1 kb)

5. 10 min (7 – 0.6 kb)

6. 30 min (6 – 0.5 kb)

DNA Sonication

Standard curves with identical detection across all CHO DNA strains

Standard curves generated from 10-fold dilution series

of CHO standard DNA of varying molecular weight

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Time-to-results: Typically less than 5 hours

PrepSEQTM Residual

DNA Sample

Preparation Kit

Sample

Preparation

resDNASEQTM

Quantitative CHO

DNA Kit

Assay Setup

Applied

BiosystemsTM 7500

FAST Real-time

PCR System

Real-time PCR

AccuSEQTM

Real-time PCR

Software

Results

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Worldwide support network

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Residual Host Cell DNA Assay Portfolio

Covers the majority of host cell lines

commonly used in biopharmaceutical

manufacturing

• CHO

• E.coli

• NS0

• Vero

• MDCK

• Human

• Pichia pastoris

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resDNASEQ™ Residual DNA Quantitation System

Optimized Sample prep Quantitative DNA recovery from complex matrices with high precision

Specific to Target DNA No cross-reactivity to unrelated DNA

Highly Sensitive LOQ to 1.5 pg/mL for mammalian DNA

LOQ to 15 pg/mL for bacterial/yeast DNA

Rapid Results In <5 hours

Consistent Results Optional automation for minimal variation

Support Network Worldwide technical and validation support and custom capabilities

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Appendix

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Human

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Broad dynamic range, high sensitivity

Amplification Plot (ΔRn vs. Cycle)

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

Human Dynamic Range: 14-28

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Linearity and PCR efficiency

Standard curve generated from 10-fold dilution series of

human standard DNA

PCR

Efficiency

R²

98.9% 0.999

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High Specificity: No Cross-Reactivity with Unrelated DNA

DNA Ct

CHO 29.27

E.coli 31.20

MDCK 31.51

NS0 31.29

Pichia 30.38

Human (3ng) 13.59**

Assay cross-reactivity tested in the presence of high concentration

(10ng) of non-target DNA

Assay is specific to human DNA

target and is unaffected by the

presence of high concentration

of unrelated DNA

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Assay performance evaluation

• Surrogate sample matrices typical in biopharma purification tested

• Known amounts of human standard DNA added to six test samples

• Samples prepared using PrepSEQ™ Residual DNA Sample Preparation Kit

• Samples analyzed using resDNASEQ™ human Residual DNA Quantitation Kit

• Results calculated using analysis software

• % DNA recovery calculated

• Consistency of results demonstrated by low % coefficient of variation

Experimental Design

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Assay performance evaluation

• Six samples spiked with 100 picograms, 10 picograms or 1 picogram Human standard DNA

• Automated Sample Preparation using PrepSEQ® Residual DNA Sample Preparation on the MagMAX™ Express-96 System

Test

Sample 100 pg Spike 10 pg Spike 1 pg Spike

Buffer

pg DNA

Recovered % CV

pg DNA

Recovered % CV

pg DNA

Recovered % CV

Assay 1 75.2

11%

7

7%

0.65

5%

Assay 2 65.4 6.2 0.69

Assay 3 61.9 6.1 0.63

Assay 4 58.3 6.3 0.63

Assay 5 55 5.9 0.66

Assay 6 60 7.1 0.72

Average 62.63 6.43 0.66

From Typical Antibody Purification In-Process Samples

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NS0

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Broad dynamic range, high sensitivity


series of NS0 standard DNA

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

6 30 fg

NS0 Dynamic Range: 17-33

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Linearity and PCR Efficiency

PCR efficiency 97.9%

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Consistent quantitation high molecular weight DNA and fragmented DNA Standard curves generated from analysis of 10-fold dilution series

of high molecular weight and fragmented NS0 standard DNA

Consistent recognition and quantitation across

a broad range of DNA fragment sizes

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High specificity

Standard curves generated from analysis of 10-fold dilution series

of NS0 standard DNA in the presence or absence of human DNA

The TaqManTM assay detects only NS0 DNA,

unaffected by the presence of foreign DNA

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Assay Performance Evaluation

Spike Concentration % Recovery % CV

60 ng/mL 89% 8%

6 ng/mL 85% 8%

600 pg/mL 82% 17%

• Test sample matrix: 100 mg/mL IgG, Mannitol, Sucrose, L-Arginine, Tween 20 (pH 8.0)

• MagMax Express-based Sample Preparation

NS0


High and reproducible recovery was

obtained across cell lines tested

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MDCK

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Broad dynamic range and sensitivity


series of MDCK standard DNA

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

6 30 fg

MDCK Dynamic Range: 13-30

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Linearity and PCR efficiency

Standard curve generated from 10-fold dilution series

of MDCK standard DNA

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Consistent Quantitation with High molecular weight DNA and fragmented DNA



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No cross reactivity with human DNA

No cross reactivity with as high as

100 ngs of human DNA

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Pichia pastoris

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Broad dynamic range and high sensitivity


series of Pichia pastoris standard DNA

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

Pichia Dynamic Range: 20-34

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Standard curve generated from 10-fold dilution series

of Pichia pastoris standard DNA

LOQ = 15 pg/mL PCR Efficiency = 103% R2 = 1

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High specificity

Standard curves generated from analysis of 10-fold dilution series of

Pichia pastoris standard DNA in the presence or absence of human DNA

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Consistent Quantitation

Standard curves generated from analysis of 10-fold

dilution series of Pichia pastoris standard

DNA of varying molecular weight

High molecular weight DNA and fragmented DNA

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High Recovery and reproducibility using PrepSEQTM Sample Preparation

Spike % Recovery %CV

100 pg 82.4 4.8

10 pg 85.1 1.1

1 pg 84.2 7.4

Results

DNA spiked into surrogate matrix which

contained 3% Mannitol, 2% Sucrose, 10

mM L-Arginine, 0.01% Tween 20 and 50

mg/mL IgG

• Surrogate sample matrices typical in

biopharma purification tested

• 100 pg, 10 pg and 1 pg of Pichia pastoris

standard DNA added to triplicate test samples

• Samples prepared using PrepSEQ™ Residual

DNA Sample Preparation Kit

• Samples analyzed using resDNASEQ™ Pichia

pastoris Residual DNA Quantitation Kit

• Results calculated using analysis software

• % DNA recovery calculated

• Consistency of results demonstrated

by low % coefficient of variation

Experimental Design

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Specificity of the Pichia pastoris assay

Life Technologies Proprietary & Confidential

DNA Ct Input

CHO(JKL) Und 10 ng

Vero Und 10 ng

Hamster Und 10 ng

Chicken Und 10 ng

Pig Und 10 ng

S. cerevisiae Und 10 ng

Bovine Und 10 ng

Mouse Und 10 ng

Rat Und 10 ng

Dog Und 10 ng

Human Und 10 ng

E. coli Und 10 ng

Pichia (KM71) 31.7 10 pg

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E. coli

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Broad Dynamic Range, High Sensitivity

Amplification plots generated from analysis of 10-fold

dilution series of E.coli standard DNA

DNA per rxn

1 3 ng

2 300 pg

3 30 pg

4 3 pg

5 300 fg

E. coli Dynamic Range: 19-32.5

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Standard Curve

PCR efficiency 104%

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Consistent Quantitation High molecular weight DNA and fragmented DNA Standard curves generated from analysis of 10-fold dilution

series of E.coli standard DNA of varying molecular weight



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High Specificity: No cross reactivity with human DNA Standard curves generated from analysis of 10-fold dilution series

of E.coli standard DNA in the presence or absence of human DNA

The TaqManTM assay detects only E.coli DNA,

unaffected by the presence of foreign DNA

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Assay Performance Evaluation

Host Cell % Recovery % CV

CHO 78% 4%

MDCK 92% 3%

NS0 89% 5%

Pichia 78% 6%

• Test sample matrix: 100 mg/mL IgG, Mannitol, Sucrose, L-Arginine, Tween 20 (pH 8.0)

• Triplicate samples spiked with 100 picograms, of standard DNA

• Automate ExpressTM instrument-based Sample Preparation

Multiple Cell Lines


Consistent high level

DNA recovery

and quantitation

across all spike

concentrations tested

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resdnaseq residual dna quantitation...

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