Quality Tools and their Application in the Production of Cellular [PDF]

adopted the use of a variety of six sigma tools. This approach ... Batches of cells are usually quality controlled in a

0 downloads 4 Views 611KB Size

Recommend Stories


Films and their production
Learning never exhausts the mind. Leonardo da Vinci

hormesis and cellular quality control
Life is not meant to be easy, my child; but take courage: it can be delightful. George Bernard Shaw

Quality Control in the Production of High-Quality Plastic Parts
When you do things from your soul, you feel a river moving in you, a joy. Rumi

Cellular Hypersensitivity and Cellular Immunity in the Pathogenesis of Tuberculosis
I want to sing like the birds sing, not worrying about who hears or what they think. Rumi

Factors and methods of pig oocyte and embryo quality improvement and their application in
When you do things from your soul, you feel a river moving in you, a joy. Rumi

Extraction of Nickel Nanoparticles from Electroplating Waste and Their Application in Production of
If you want to go quickly, go alone. If you want to go far, go together. African proverb

PdF Tools of the Mind
If you want to go quickly, go alone. If you want to go far, go together. African proverb

The Effectiveness of Lean Manufacturing Tools in Maintaining Quality Control
Don't be satisfied with stories, how things have gone with others. Unfold your own myth. Rumi

economics of the external and the extended orders of markets and politics and their application in
The only limits you see are the ones you impose on yourself. Dr. Wayne Dyer

Idea Transcript


Quality Tools and their Application in the Production of Cellular Reagents for Screening Assays. Sharon Davies, Lynne Smith, Lisa Bailey, Martyn Birch, Rahman Ismail, Peter Tatnell, Michelle Doyle, Liz Price and J.M Kendall* GE Healthcare Healthcare, The Maynard Centre, Whitchurch, Cardiff, CF14 7YT, UK UK. Tel: +44 (0)29 2052 6000; Fax: +44 (0)29 2052 6230; e-mail: [email protected]

5500

2600

4750

2300

4000

2000

3250 2500

Fresh Frozen

1700

800 -5

-4

-3

-2

-1

0

1

2

-5

-4

-3

-2

-1

0

1

2

[Propranolol] log μM

[Isoproterenol] log μM

Figure 2. CHO cells expressing hβ2AR were expanded to 1x1010 and cryopreserved. cAMP production was measured in response to agonist and antagonist. There were no differences in the assay performance of cryopreserved cells compared to fresh cells.

Transiently transfected cells in cryopreserved format are emerging as potential screening reagents (Figure 3). a)

b)

2000

are key reagents in the drug discovery pipeline. We offer a comprehensive stable cell line generation service including molecular biology and functional validation in a variety of assay platforms ( Figure 1). 75.0

Control

62.5

(X)

F grains

Agonist 25.0 12.5 0.0 -3

-2

-1

0

1

2

3

Stable - frozen

400

Transfected - frozen

Figure 4. Multiple plates containing a cAMP 11 point standard curve (n=8) were prepared by one operator. 3 operators read all plates in triplicate on instruments X and Y. Data were analysed with MINITAB software.

Transfected

-4

-3

-2

-1

0

Isoproterenol log μM Isoproterenol

1

Figure 3. Transient transfection of cells (a) Stable and transient HEK cells expressing the hβ2AR were assayed for cAMP following stimulation with agonist in fresh and cryopreserved format. All formats produced similar assay performance and pharmacology. (b) Optimisation of transfection on microcarriers (HEK cells transfected with FP tagged protein).

Log [nM] Agonist

Figure 1. Pharmacological characterisation of a stable cell line generated for an HCA assay.

uHTS size batches of cryopreserved cells have been produced in our dedicated cell culture unit. A variety of culture techniques are used including microcarrier/stirred tank bioreactor and high density plasticware

(Y)

Stable

50.0 37.5

Essential to the production of functionally validated cells is a reliable assay measurement system. Gauge R & R is a six sigma tool that determines the total variation within a measurement system and is able to differentiate between error arising from the operator or from the instrument itself, allowing remedial action to be applied if necessary. For the purpose of this six sigma project, we used a commercially available cAMP luminescence assay kit (which was appropriate for the cell culture optimisation) and measured assay output from a standard curve on 2 different instruments (Figure 4).

1200 800

Cell membrane preparations from stable cell lines are frequently used for binding assays. We have therefore developed proprietary scaleable methods to enable production of these key reagents. (Table 1).

Culture Method

Membrane prep

5.00

Data

4.00

LCL MEAN 3.00

UCL

2.00

1600

IOD

Stable cell lines

6.00

U SPEC

Gauge R & R study

1100

1000

In order to develop a robust framework for the production and validation of the Cell Factory reagents, we have adopted the use of a variety of six sigma tools. This approach ensures reproducibility and quality in the manufacture of different complex biological products.

L SPEC

1400

1750

Application of Six Sigma.

Value

Batches of cells are usually quality controlled in a functional assay and performance benchmarked against fresh cells or customer specifications (Figure 2).

IOD

The use of mammalian cell based assays in compound screening continues to grow. Over 50% of all screening assays (HTS and HCA) now utilise cells or cell derived reagents. Adoption of assays employing cryopreserved cells and other cell based screening reagents in a ‘ready to go format’ is becoming increasingly common practice since it obviates the issues of batch variation, scheduling of cell production and capacity management arising from using ‘fresh’ cells. To address these growing demands Cell Factory provides a number of assay ready cellular reagents and services.

IOD

Introduction

Protein (pg/cell)

Optimum SPA membrane (μg/well)

Low density plastiware

Manual

22

10

High density plastic

Mechanical

29

2.5

Microcarrier/Bioreactor

Mechanical

28

2.5

Table 1. Scaled preparation of membranes from cells cultured on microcarriers and high density plastic-ware illustrating comparable protein yield and assay performance.

The variation observed when different operators each measure the same plates repeatedly on different instruments is shown in Figure 4. Repeatability is the variation due to the instrument, whilst reproducibility is variation due to the operator. Gauge R&R is the sum of these totals, and part-to-part is the variation revealed by the measurement system between the parts measured, i.e. the plates in this instance. Instrument X displays a total variation in the measurement system (Gauge R&R) of ~ 64%, which far exceeds the variation, observed between the plates. In contrast, instrument Y shows only ~ 11% total variation, which is acceptable (less than 10% variation is considered an excellent measurement system). Therefore instrument Y was selected as the instrument of choice for all future measurements.

Process capability Confident that our measurement system Y was adequate for our needs, we then established current process baselines for a cell line in the appropriate functional validation assay. Analysis of the data with this tool enables us to set control limits for the process and to monitor the impact of any potential changes applied to our reagent production (Figure 5).

1.00

0.00 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Sample #

Figure 5. Process capability. Multiple operators (n=5) each set up a number of assay plates (n=4) with cryopreserved cells in agonist dose response format. The assay readout (cAMP) was read once on instrument Y (selected in the Gauge R & R). Data (agonist EC50s) were analysed with MINITAB software. The process capability experiments were set up to incorporate all potential variables (e.g. operators, assay day, assay kit, etc.). Analysis of the data (Figure 5) shows only random variations with no clustering or trending. We can therefore conclude that the process is performing normally, with no external special cause variation impacting on our system. Any future assay data obtained following process improvements should fit within the control limits for this process, indicating no adverse impact on the overall performance.

StdOrder RunOrder CenterPt 2 1 1 6 2 1 1 3 1 7 4 1 8 5 1 3 6 1 4 7 1 5 8 1

Blocks 1 1 1 1 1 1 1 1

Condition A Condition B Condition C 1 -1 -1 1 -1 1 -1 -1 -1 -1 1 1 1 1 1 -1 1 -1 1 1 -1 -1 -1 1

Figure 7. Full factorial DoE required to analyse the effects of changing 3 conditions – A, B &C. Cells were cultured following the DoE, cryopreserved and assayed for the production of cAMP in response to agonist. (a)

(b)

Process Improvement DoE (Design of Experiments) enables assessment of the potential impact of changing multiple conditions simultaneously in a multiparameter process. An example factorial design (Figure 6) illustrates that ‘full’ is the preferred option as it covers all potential interactions. Factors 2 3 4 5 6 7 8 4 Full III 8 Full IV III III III Runs 16 Full V IV IV IV 32 Full VI IV IV 64 Full VII V 128 Full VIII

9

10 11 12 13 14 15

III IV IV VI

III IV IV V

III IV IV V

III IV IV IV

III IV IV IV

III IV IV IV

III IV IV IV

Figure 6. Typical factorial design. Available factorial designs, dependant on number of runs and number of factors under investigation. We wanted to optimise the culture of a cell line and change 3 conditions (A, B and C) simultaneously. We therefore applied a full factorial DoE (Figure 7). This enabled a complete assessment to be conducted to compare existing conditions (‘1’) against changed conditions (‘-1’) and the potential impact on the process or cell functionality of changing one or multiple conditions simultaneously. Assay data from the DoE were analysed (Figure 8). The first order interactions (Figure 8a) suggest optimal conditions are ‘1’ (existing) for A and B and ‘-1’ (changed) for C. The second order interactions (Figure 8b) show the

© 2008 General Electric Company - All rights reserved. GE, imagination at work and GE Monogram are trademarks of General Electric Company. GE Healthcare and GE Healthcare Biosciences are trademarks of GE Healthcare companies. All goods and services are sold subject to terms and conditions of sale of the GE Healthcare company which supplies them. A copy of these terms and conditions are available on request. GE Healthcare reserves the right, subject to any regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions. Rights to use this product, as configured, are limited to internal use for screening, development and discovery of therapeutic products; NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rights are conveyed. The IN Cell Analyzer 1000 is the subject of US patent application number 10/514925, together with other pending family members, in the name of GE Healthcare Niagara, Inc. The IN Cell Analyzer 1000 and associated analysis modules are sold under use licenses from Cellomics Inc. under US patent numbers US 5989835, 6416959, 6573039, 6620591, 6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993, 7060445, 7085765, 7117098 ; Canadian patent numbers CA 282658, 2328194, 2362117, 2381334; Australia patent number AU 730100; European patent numbersEP 0983498, 1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568, 3576491, 3683591 and other pending and foreign patent applications. HitHunter is a trademark of DiscoveRx Corporation. DiscoveRx Inc grants to the purchaser of this product an end-user license under United States and equivalent patents and patent applications in other countries owned or licensed exclusively to DiscoveRx (including US patent numbers 4956274, 5244785, 5444161, 5604091, 5643734 and equivalent patents and patent applications in other countries) and non-excluisively licensed (including US patent numbers 5851771, 5538847, 5326882, 5145772, 4978614 and 4934569 and equivalent patents and patent applications in other countries) to use this product solely in the fields of drug discovery and regulatory qualifications of therapeutic drugs and not for any other uses, including but not limited to, other research uses, or use in human clinical, animal, or food diagnostics. Graphpad prism is a registered trademark of GraphPad Software, Inc. All third party trademarks are the property of their respective owners. *To whom all correspondence should be addressed. This poster was presented at the 14th Annual Conference of the Society for Biomolecular Sciences, St. Louis, USA (6 - 11 April 2008).

impact that a combination of changed conditions has on the functionality of the cells. Although the second order interactions indicate BC/AC impact on the process, statistical analysis of the data shows these to be negligible and therefore only first order interactions would be considered. Clearly only conditions with no or beneficial effect on the process/functionality would be considered. These would be implemented for full characterisation and assessment in the process capability.

Figure 8. Shows the effect on cell line functionality generated by (a) changing individual conditions (first order interactions) and (b) changing multiple conditions simultaneously (second order interactions) in the culture process.

Summary • Cell Factory is a custom service providing reagents for all aspects of cellular screening assays.

• •

These services include production and full functional validation of key reagents such as stable and transient cell lines, screen size batches of cryopreserved cells and scaled membrane preparations. We employ a number of six sigma tools to ensure robust production optimisation and manufacture of these complex biological reagents.

Smile Life

When life gives you a hundred reasons to cry, show life that you have a thousand reasons to smile

Get in touch

© Copyright 2015 - 2024 PDFFOX.COM - All rights reserved.