development of droplet digital polymerase chain reaction … · 2020-05-11 · droplet digital pcr...

Ruda Cui1*, Vijetha Bhat1*, Yimeng Zeng1, Caitlin Tripp1, Anandita Seth1, and Young Shin1

1. Research & Development, Lonza Houston Inc., Houston, TX, 77047 * Co-first authors

Lentivirus (LV) is one of the most popular delivery vehicles in cell and gene therapies. The accurate measurement of LV infectious titer is an absolute requisite in the process of manufacturing, purification, andapplication of lentiviral vectors. Conventional assay methods measuring LV titers, such as flow cytometry or quantitative polymerase chain reaction (qPCR), have some major drawbacks. For these assays, one needs tohave a reporter, or a specific antibody for the measurement of infectious titers. In addition, it is necessary to optimize the primers, probes, and standards in the qPCR assay before putting them in, which is a verycumbersome process. Droplet digital polymerase chain reaction (ddPCR) has emerged as a reliable, cutting-edge technology to quantify the absolute copy number of any gene of interest without using a standard curve.The RNA genome of LV is first reverse-transcribed to its cDNA before it integrates into the host chromosome. Therefore, infectious titers of LV can be determined using ddPCR by measuring the integration frequency ofthe transgene into the chromosomes of target cells. However, current methods to determine LV titers by ddPCR are rate-limited due to the tedious process of genomic DNA isolation, which involves extractingchromosomal DNA from a large number of transduced cells. Here, we developed a new, high-throughput method, which enabled us to skip the genomic DNA extraction during sample preparations for ddPCRapplications.

To avoid the tedious DNA extraction process, which commonly involves detergent-mediated cell lysis and column purification of the DNA thereafter, we mechanically disrupted the cells using glass beads. The crudelysates prepared from cells transduced with LV (encoding green fluorescent protein, GFP) were applied directly to the assay. To compare and validate our new approach with conventional methods, DNA was also isolatedfrom transduced cells using a commercially available kit from Qiagen (QIAamp DNA Blood Mini Kit). The primer-probe sets specific to the long terminal repeat (LTR) region of LV and the beta-actin sequence of the hostwere used to amplify target sequences. To calculate infectious titers, we have compared the following three methods among each other:

1. Sample DNA for ddPCR was isolated using a Qiagen kit. The cell number in the corresponding sample was calculated from the copy number of beta-actin in the same sample, based on which the LV titer wascalibrated.

2. Sample DNA for ddPCR was isolated using a Qiagen kit. The cell number in the corresponding sample was calculated from the DNA amount in the same sample, based on which LV titer was calibrated.3. Crude cell lysates were prepared by disrupting cells using glass beads and directly applied to ddPCR. Cell number in the corresponding sample was directly counted before disruption using ViCell, based on which

LV titer was calibrated.The infectious LV titers calculated from the above three different methods were comparable to each other for any given sample, indicating that crude cell lysate prepared by bead beating is sufficient for direct ddPCRapplication. It enabled us to eliminate the time-consuming DNA extraction step during sample preparation in the ddPCR assay. Through this approach, it was possible to handle and process large numbers of samples in arelatively short time with minimal hands-on time. Thus, Lonza has developed a high throughput format of ddPCR assay for the measurement of infectious LV titers, which will greatly support the LV manufacturingprocess in cell and gene therapies.

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Development of Droplet Digital Polymerase Chain Reaction

(ddPCR) Assay for a Measurement of Infectious Titers of

Lentiviral Vectors in a High Throughput Format

Abstract

Development of High-Throughput ddPCR Format

Validation of Bead Beating Method and Process Optimization

Summary

Introduction to Droplet Digital PCR

Average 6.7E7 4.4E7 4.7E7

CV 41% 61% 25%

Flow Diagram Comparing Conventional vs. High-throughput Method

Traditional method

Qiagen-based DNA extraction involving pellet lysis, several rounds of column purification and elution

Cell pellet

Mechanical glass bead lysis using a cell disruptor

Analysis by

ddPCR

Sample PreparationTransduction of HT1080 cells

High-throughput

method

4 X 6 well plates

1 X 24 well plate

Advantages of ddPCR Assay

Absolute measurement of LV titers

Accurate and reproducible measurements

No significant effect from DNA matrix

Measurement of LV infectious titers regardless of GOI sequence

Limitation: Current sample preparations are largely based on the

extraction of chromosomal DNA using a Qiagen DNA blood mini kit.

Chromosomal DNA extraction is labor-intensive and tedious

Longer sample processing times

Fewer samples per batch

Larger volume of cell transduction required

Alternative methods such as chemical lysis of cells leads to

interference in ddPCR measurements and inaccuracies

Validation of LTR Primers and Probe

Using The Known Standard

25002732

2634

0

500

1000

1500

2000

2500

3000

Theoritical In water In DNA solution

Co

pie

s /

mL

Workflow for LV Infectious Titer Measurement

OR 1.E+05

1.E+06

1.E+07

1.E+08

Infe

ctio

us

Tite

r (T

U/m

L)

Average: 1.4E7 TU/mL CV: 17% (n = 25)

Consistent Performance by Flow Cytometry-

based Infectious Titer Assay

Lonza’s reference standard (1004-19) measured on different days

Flow cytometry has a limitation to measure non-GFP GOIs

Comparison of Titers with Other Methods

High throughput method using glass beads produces quite consistent titers.

Lonza’s reference standard titers compared vs. methods using DNA extraction

0.0E+00

4.0E+07

8.0E+07

1.2E+08

1.6E+08

Qiagen - DNA Amount Qiagen - Beta Actin Glass Bead *

Infe

ctio

us

Tite

r (T

U/m

L)

* Performed without calibration using a housekeeping gene

Droplet digital PCR has proved to be an effective and accurate assay in determining the titers of GOIs that are generally not detectable through flow cytometry. Conventional methods of ddPCR needs DNA extraction from transduced cells, which can be time-consuming, labor intensive, and costly. We are currently developing an efficient high-throughput ddPCR method through the mechanical lysis of cells using glass beads in a cell disruptor. Samples processed by glass bead produces consistent and comparable titers compared to the other methods. As a part of further developmental work, we are going to use a housekeeping gene to calibrate the titers obtained from glass bead method.

Thorough agitation is essential for optimal titer output. Suboptimal agitation speeds and times result in incomplete cell lysis and overestimation of titer

Optimization of Cell Lysis Conditions using Cell Disruptor

Effect of agitation speed on cell disruption

4.7E+07

1.3E+08 1.2E+081.1E+08

0.0E+00

4.0E+07

8.0E+07

1.2E+08

1.6E+08

ExpectedAverage

2000 2500 3000

Infe

ctio

us

Tite

r (T

U/m

L)

Cell Disruptor Agitation Speed (rpm)

Duration of agitation: 2 minutes

Time course analysis at max speed

4.7E+07

1.3E+08

1.0E+08 1.1E+08

9.4E+07 9.2E+078.1E+07

5.5E+07

4.2E+073.6E+07

5.6E+07

0.0E+00

4.0E+07

8.0E+07

1.2E+08

1.6E+08

ExpectedAverage

1 1.5 2 2.5 3 4 6 9 12 15

Infe

ctio

us

Tite

r (T

U/m

L)

Time of Agitation (min)

Agitation speed: 3000 rpm

Measure LV titers

Infection on target cell

Incubate (3 days)

Flow cytometry ddPCR

Transduction Unit (TU/mL) = F * C * D / V

F = Fraction of cells with integrated

viral genome

C = Input cell number

D = Dilution factor

V = Input volume of virus inoculum There was no matrix effects from the surrounding DNA