COMMERCIALIZATION OF TRANSIENTLY TRANSFECTED CELL LINES FOR

HIGH THROUGHPUT DRUG SCREENING AND PROFILING APPLICATIONS

by

KALPITA DEEPAK MEHTA

Submitted in partial fulfillments of the requirements

for the degree of Master of Science

Thesis Committee:

Christopher Cullis, Ph.D.

Stephen Smith, Ph.D.

James Zull, Ph.D.

Emmitt Jolly, Ph.D.

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

May 2010

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Kalpita Deepak Mehta

candidate for the Master of Science degree *.

(signed) Christopher Cullis Ph.D.

(chair of the committee)

Stephen Smith, Ph.D. _______________________

James Zull, Ph.D._____________________________

Emmitt Jolly Ph.D._____________________________

(date) March 22, 2010

*We also certify that written approval has been obtained for any

proprietary material contained therein.

Dedicated to my parents and family

Table of Contents

List of figures:....................................................................................................................vi

Abstract............................................................................................................................viii

1 Recommendations and Conclusions........................................................................1

2 Introduction..............................................................................................................4

2.1 Advantages of transient transfected over stably expressed cell lines:.........5

3 ChanTest Corp. Overview.......................................................................................9

4 Background and Objective of the validation summary report...............................10

5 Validation results...................................................................................................15

5.1 Specificity of fluorescent signals in transiently transfected HEK cells. .15

5.2 Performance of previously frozen to fresh cells........................................15

5.3 Control of membrane potential by changes in external potassium

concentrations....…………………………………………………………15

5.4 Activation and inactivation of the calcium signal by external potassium. 15

5.5 Assay stability............................................................................................15

5.6 Pharmacological sensitivity and use-dependence......................................15

iv

6 Protocol: Transient transfection cell line construction:.........................................16

6.1 Solutions and chemicals used in FLIPR TETRA TM assay..............................18

6.2 Experimental methods –FLIPR TETRA TM assay...........................................20

7 Discussions-Technical challenges and relative propositions.................................23

7.1 Problems and relevant suggestions............................................................23

8 Commercial conclusions........................................................................................28

8.1 Business proposition..................................................................................35

9 Appendices............................................................................................................37

9.1 Appendix A: Estimated production cost for 2250 vials.............................37

9.2 Appendix B: Globally projected estimation of units sold per year...........38

9.3 Appendix C: Comparison of expenses (stable vs. transient).....................39

10 Bibliography..........................................................................................................40

v

List of figures:

Figure 1: Mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1 in

response to K+ stimulation................................................................................................12

Figure 2: Variability in results...........................................................................................24

Figure 3: Compromise with expression level....................................................................25

Figure 4: Low transfection efficiency................................................................................27

Figure 5: Survey methodology..........................................................................................28

Figure 6: Market potential is identified for biotechnology and pharmaceutical...............30

Figure 7 Demographics area of research...........................................................................30

vi

List of tables

Table 1: Assay Stability ………………………………………………………………..15

Table 2: Hill fit and reference values for selected reference compounds………………15

vii

COMMERCIALIZATION OF TRANSIENTLY TRANSFECTED CELL LINES

FOR HIGH THROUGHPUT DRUG SCREENING AND PROFILING

APPLICATIONS

Abstract

By

KALPITA MEHTA

Drug screening and profiling in the drug discovery process can be carried out either by

using stable transfected or transiently transfected cell lines. Transient expression system

is a more appealing alternative in contrast to stable expression system because the latter is

a very labor intensive, time consuming and expensive. It takes between eight to twelve

weeks to develop a stable cell line as opposed to seven to ten days to develop a transient

cell line. Moreover, given the size and cost of a High Throughput Screening (HTS)

program, researchers cannot afford to perform an assay that has a high batch to batch

variation. To meet these performance criteria, ChanTest has developed transiently

transfected cell lines called Ion Channel EZCellsTM TT. These cell lines have been

validated using voltage gated calcium channels into HEK293 cells (Human Embryonic

Kidney) using scalable electroporation method and run calcium influx assay in FLIPR

(Fluorometric Imaging Plate Reader). This report includes a detailed description of the

validation results of Ion Channel EZCellsTM TT and its commercial feasibility including

market entry and pricing strategies.

viii

1 Recommendations and Conclusions

The conclusions are made based on the data obtained by learning psychographics, market

trends, and challenges faced by the researchers using Ion Channel1 EZCellsTM TT. The

process was adapted to include an understanding of customer needs by conducting a

survey. The survey responses proved very valuable in making recommendations to

support the technical feasibility. The main conclusion from the survey was that:

transiently transfected cell lines are a platform that researchers identify as meeting

performance standards, which is often a bottleneck in the lead discovery process.

The scalable electroporation method2 used has the ability to reduce assay development

time and provide more clinically relevant assays. The power of scalable electroporation

technology is related to the speed, consistency, capacity and throughput efficiency in

sterile and non–toxic environments, which supports the validation of drug targets and

conducting HTS (High Throughput Screening).

Advantages of the technology include:

Transient cell lines eliminate in-house cell line development or costs to purchase

replicating lines. They are designed to eliminate the cell culture and cell line maintenance

costs. The transfected cells with transient expression are packed in vials, each vial

contains 6 million cells resulting in ~15,000 cells per well of 384-well plate derived from

each vial.

1 Ion channel are pore forming membrane proteins that facilitates diffusion of ions across the membranes. Ion channels are important targets of therapeutic agents.

2 Electroporation is a mechanical method used to introduce polar molecules into a host cell through the cell membrane. In this procedure, a large electric pulse temporarily disturbs the phospholipid bilayer, allowing molecules like DNA to pass into the cell (Purves et. al., 2001).

1

A pilot study was conducted by ChanTest to demonstrate the performance standard and

feasibility of the transient transfection3 approach. This study validates these cell lines

using voltage gated calcium channels into HEK293 cells (Human Embryonic Kidney) run

in calcium influx assay in FLIPR (Fluorometric Imaging Plate Reader). The FLIPR

allows rapid assays of cellular signaling processes made feasible by simultaneous kinetics

measurement of cell-based fluorescence changes in a 96- or 384-well format.

This case study involves transfection of an equimolar ratio of the following four ion

channel cDNAs in HEK293 cells:

1. Cav pore-forming alpha subunit (~5-6 kb cDNA)

Cav2.2 (N-type)

2. Modulatory β subunit

3. Modulatory α2δ subunit

4. Inward rectifier potassium channel (Kir2.1) to allow modulation of resting

membrane potential by external K+.

Specific results involving assay stability, performance and specificity of fluorescent

signals in transiently transfected HEK cells are shown later in this report.

ChanTest`s ion channel-expressing EZCellsTM TT are commercially feasible having

benefits over ion channel stable cell lines4 and division- arrested cell lines. Pricing is

3 Transfection is the process of introducing nucleic acids in eukaryotic cells used for non-viral methods. (Biotechniques 2000)4 Stable expression is obtained by integrating the expression vector into the host cell genome or be maintained as an extra-chromosomal element, under conditiond of chronic selection. (Biotechniques 2000)

2

based on the cost per well – (6 million cells per vial result in a 384-well plate, with

~15,000 cells per well).

Pricing: $599 per vial for one vial ($1.56 per well) and discounted pricing is allotted on

the purchase of more than one vial.

An addressable market of $4.5 million was estimated (Appendix A) for the ion channel

EZCellsTM TT. Today, ChanTest Corp. acquires about 12% of the total Pharma/Biotech

ion channel stable cell line market and is expected to grow up to 17% by 2011. Market

entry strategy and financials give a clear picture of the commercialization plan, shown in

detail later in this report.

3

2 Introduction

Drug discovery is one of the most important sectors in pharmaceutical research and

development. It involves High Content Screening (HCS) and analysis, and includes

applications that require sufficient levels of sample throughput along with the study of

complex cellular events and phenotypes

Choice of Targets

Reagent Procurement Assay Development and Validation

Mass Screening HTS implementation

Data Capture, Storage and Analysis

Leads

Reference: (Macarron and Hertzberg 2002)

Reagent appropriation (Reagent type such as cell line) is often a major bottleneck in the

HTS (High Throughput Screening) process. This can delay the early phase of assay

development. Elements of drug performance such as toxicity and specificity can be

established simultaneously using mixed cell types. The ion channel is the most

commonly used target type. Other target types are GPCRs (G-protein coupled receptors),

nuclear hormone, kinases and protease. Time require to develop the test system is much

shorter in transient transfected than stable cell lines.

To meet assay performance standards, reduce assay development time and to obtain fast

results ChanTest has developed transiently transfected Ion Channel EZCellsTM. They have

4

similar expression levels as replicating cell lines. Transient gene expression is achieved

by introducing foreign genes into eukaryotic cells, in particular mammalian cells, by non-

viral methods; this process is called transfection. The gene is introduced with the help of

chemical, lipid or physical methods. However, transient gene expression is temporary and

is predicated on the burst of gene expression between 12 and 72 hours after transfection.

This burst is followed by a rapid deterioration in expression of the transgene because of

cell death or loss of the expression plasmid. Reporter gene5 activity is used to evaluate the

transient expression system.

2.1 Advantages of transient transfected over stably expressed cell lines:

1. Development time:  Developing a stably transfected cell line is a costly, multi-

step process that normally takes at least several months. Transiently transfected cells, on

the other hand, can be created very quickly once the DNA is obtained and engineered into

a mammalian expression vector.  This makes transient transfection ideal when a new

protein target becomes of interest and results are needed quickly.  It is also a good first

step to determine whether a stably expressing cell line is likely to produce good results in

a particular assay.

2. Expression level:  Stably transfected cell lines have only a few copies of the

transfected gene (those that are successfully integrated into the host cell genome).

Transiently transfected cells often have a higher number of copies of the transfected gene

immediately after transfection, which usually results in a higher expression level of the

encoded protein (and higher current amplitudes for the ion channel). For most ion

channels, higher expression is better because higher current amplitude produces better

5 Reporter genes are selectable markers used to determine whether the gene of interest has shown expression in the cell or not. (Bloom, Freyer and Micklos 1996)

5

resolution between signal and noise, which allows the electrophysiologist to calculate

channel block IC50 (half maximal inhibitory concentration) more precisely and reliably.

There are a few ion channels for which we selected lower-expressing stable clones

because a higher-expressing clone produced such high current amplitudes that saturated

the amplifiers (making it impossible to determine IC50 of channel blockers), but

generally, higher expression is better. The caveat of this argument is that with each cell

division cycle, the expression level in transiently transfected cells decreases:  since the

DNA is not incorporated into the host genome, the same number of copies of the gene

gets diluted with each cell division.  This means that in order for the transiently

transfected cells to be useful, they must be either used very soon after transfection, or

frozen very soon after transfection and then used very soon after thawing.

3. Cell health:  Over-expression of some proteins can be toxic to cells. For ion channel

proteins, constitutive high expression of a number of calcium channels triggers apoptosis

(Cell death).  ChanTest's early attempts to develop stably transfected cell lines that

constitutively expressed members of the calcium channel family failed because no high-

expressing clones grew well in culture.  One way to get around this problem is by using

transiently transfected cells since these cells can be used soon after transfection so that

any negative impact of expression on the rate of cell growth and division is minimized.

Another way to work around this problem is to develop stably transfected cell lines where

expression of the problem gene is inducible. Expression of the transfected gene is turned

off during clone selection, and cell culture stocks are maintained without ever inducing

expression. Only daughter cultures are induced. Both these options (transient transfection

6

and inducible expression) are used successfully at ChanTest, with each approach being

better for certain situations, depending on the ion channel and testing platform involved.

4. Versatility of subunit compositions:  Most, if not all, ion channels exist in the cell

as heteromers; several proteins encoded by different genes come together to form an

active ion channel.  In most cases, a single gene encodes the pore-forming subunit

(through which ions flow across the membrane), and auxiliary subunits modulate gating

of the channel.  These auxiliary subunits can vary depending on tissue type, stage of

development, and other factors, which can result in an associated varying degree of

sensitivity of the channel to stimuli (including drug compounds). In other cases, two or

more genes encode proteins that come together to form the pore structure.  For example,

many potassium channel pore structures are formed by four protein subunits, each

containing six transmembrane domains.  For hERG channels, four copies of the same

gene (hERG) form a homomultimer, (Zhou, Qiuming Gong and January 1998) which acts

as the pore structure (thus, the hERG protein can be referred to as the "pore-forming

subunit" of hERG channels).  For KCNQ3/KCNQ5 channels, two copies of each protein

subunit (KCNQ3 and KCNQ5) form a heteromultimer that acts as the pore structure.

For many ion channels, over-expression of only a single pore-forming subunit is

sufficient to obtain electrophysiological recordings that approximate the response in the

body to a drug compound.  For these ion channels, stably transfected clones are very

useful.  However, for some ion channels, co-expression of multiple subunits is necessary

to obtain meaningful electrophysiological recordings (for example

Cav1.2/beta2/alpha2delta or KCNQ3/KCNQ5).  Some investigators are interested in

7

different variations of subunit compositions (because they exist in the body such as in

different tissue types, different stages of development, etc.).  It would be very costly and

time-consuming to develop a stable cell line for each set of subunit compositions of

interest.  If there is little demand for a particular subunit composition, it may not be worth

the cost of developing a stable cell line with that composition.  Transient transfection

allows us to quickly swap in or out different subunits as required.

8

3 ChanTest Corp. Overview

ChanTest was founded in 1998 by Arthur Brown, to meet the demand for cardiac safety

testing services. This demand stemmed from the identification of the human ether-a-go-

go related gene (hERG), which encodes a potassium channel as the principle target of

concern for drug-induced sudden cardiac death (Brown and Rampa 2000). ChanTest is a

science - driven Ion Channel and GPCR products and services company serving the

Biotech and Pharmaceutical industry. It is recognized as “most trusted ion channel

Services Company” by independent survey conducted by HTStec consultancy. (Comley,

John HTStec 2007)

Services are functional screens for Ion Channel and GPCRs on manual and automated

platforms, comprehensive pre-clinical cardiac risk assessment services, cell culture

services on large-scale cell culture, transfection and cryopreservation along with custom

cell line development. ChanTest’s ion channel portfolio is the most comprehensive,

commercially available library of ion channel - expressing cell lines. Cell lines are

validated for structure, function and pharmacology, and performance is characterized by

manual patch clamp and on one or more automated electrophysiology platforms. Cell

lines are also offered in EZCellsTM format (division arrested or transient transfected) for

evaluation, assay development and screening.

9

4 Background and Objective of the validation summary report

The objective of this study was to:

1. Validate the scalable transfection system for its suitability in creating an HEK cell line

that transiently expresses human N-type calcium channels (Cav2.2)

2. To develop and validate a FLIPR (Fluorometric Imaging Plate Reader) that allows

rapid assays of cellular signaling processes by simultaneous kinetic measurements of

cell-based fluorescence changes in a 96- or 384-well format.

3. To assess the commercial feasibility of transient transfected cell lines.

Specifications of Cav2.2/beta3/alpha2delta1 Calcium Channel cells +Kir2.1

Synonyms: N-type calcium channel; alpha1B

Host cell: HEK293, transiently transfected

Gene name CACNA1B/CACNB3/CACNA2D1 (Ca2+ channel); KCNJ2 (Kir2.1)

Mycoplasma status: Negative (MycoAlert Kit)

Packaging: Three vials of cryopreserved, transiently transfected cells, 2x106 cells/vial (6x106 cells total)

Growth media: DMEM/F12; 10% FBS6

Growth characteristics: cells remain viable 1–2 days post-thaw

Expression: Adequate expression for at least 36 hrs post-thaw

Storage recommendation: frozen under liquid nitrogen

Recommended assay: FLIPR(Fluorometric Imaging Plate Reader)

Recommended plating density (96-well format): 35,000-38,000 cells/well.

Background

HEK293 cells- Human Embryonic Kidney Cells

6 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)

10

The most commonly used cell types for monitoring conduction using the Fluorometric

Imaging Plate Reader (FLIPR) assay are HEK and CHO (Chinese Hamster Ovary) cell

types. In the current experiment, we use HEK293 cells that are transfected using target

gene(s) using scalable electroporation method followed by calcium influx assay in

FLIPR. HEK293 cells were generated by transformation of Human Embryonic Kidney

cell cultures with sheared adenovirus 5 DNA. HEK cells are significant because they

natively have a relative depolarized membrane potential (ChanTest 2009), which should

inactivate and subsequently reduce availability of calcium channels for stimulation,

which is a desired effect for the experiment.

The N-type Calcium channel (Cav2.2)

Calcium channels are selective to calcium ions and allow entry of calcium ions into the

cell. Voltage gated calcium channels function to regulate gene expression and mediate

cell death. Biophysical and pharmacological criteria are used distinguish various types of

voltage gated Ca channels, labeled L, T, N, P, Q and R. In the current study, the potential

therapeutic targets are inflammatory and neuropathic pain, the N-type calcium channel is

more pharmacological sensitive. The N-type calcium channel is predominantly expressed

in the nervous system where it is a main contributor to synaptic transmission. Incoming

action potentials invade the synaptic terminal and calcium influx through N-type

channels activates neurotransmitter exocytosis (secretion from the cell). In the spinal

cord, N-type calcium channels play a prominent role in the pain-sensing pathway. In Ca 2+

channels, the principal 1 subunits can co-assemble with 2, , and possibly subunits

with profound effects on pharmacology (Mould J 2004). There are four different major

forms of Calcium channel subunits (1-4). There is some evidence that points to 3 as

11

being the predominant subunit that co-assembles with the principal Cav2.2 subunit

(Nampkung 1998). A number of industrial research groups also seem to favor 3 for co-

assembly with Cav2.2 (Benjamin, et al. 2006). We, therefore, choose to express Cav2.2

together with 2 and 3 in response to an inward rectifier.

Figure 1: Mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1 in

response to K+ stimulation.

Reference: (Benjamin, et al. 2006)

In figure 1 panel A: mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1

in response to K+ stimulation. The blue trace represents the control signal (n = 8) and the

red trace the mean response in wells that were treated with the specific antagonist -

Conotoxin GVIA (n = 4). Figure 2 panel B: mean fluorescent signal in untransfected

HEK293 cells in response to the same experimental conditions that were used in A.

A B

12

Together, the experiments in A and B show that the fluorescence signal in the transiently

transfected cells is due to expression of Cav2.2/3/21.

Cotransfection with an inward rectifying potassium channel (Kir2.1)

In addition, Kir2.1, an inward rectifier potassium channel was also co-transfected along

with the calcium channel genes. Inward rectifiers conduct inward potassium current up to

relatively depolarized membrane potentials, which stabilizes the membrane potential at

more negative values. HEK cells natively have a relative depolarized membrane potential

(ChanTest, unpublished) which should inactivate and subsequently reduce availability of

calcium channels for stimulation. By co-transfecting the inward rectifier, the membrane

potential can be kept hyperpolarized and calcium channel availability should be greatly

enhanced. (Xia M 2004 Apr 1).

Further, co-expressing the inward rectifier permits graded control of the membrane

potential that can be utilized for identifying use-dependent interactions of

pharmacological compounds with Cav2.2 and therefore, targeting of specific pathological

states of Cav2.2 activity (Winquist RJ 2005). During high frequency action potential

firing of neurons, N-type calcium channels in the synaptic terminals are thought to

accumulate in the inactivated state. A drug that preferentially targets the inactivated state,

should theoretically limit high frequency synaptic activity associated with certain

pathologies such as neuropathic pain while normal synaptic activity should be unaffected.

In the FLIPR assay, preincubating cells transfected with Cav2.2 with elevated K+

concentrations induces calcium channel inactivation and therefore simulates these

pathological conditions.

13

14

5 Validation results

(Removed due to proprietary information)

5.1 Specificity of fluorescent signals in transiently transfected HEK cells

5.2 Performance of previously frozen to fresh cells

5.3 Control of membrane potential by changes in external potassium

concentrations

5.4 Activation and inactivation of the calcium signal by external potassium

5.5 Assay stability

5.6 Pharmacological sensitivity and use-dependence

15

6 Protocol: Transient transfection cell line construction:

1) Grow un-transfected CHO and HEK cells in 37°C incubator with 5% CO2 and

95% humidity, keep cell density in about 70-80% (number of cells per unit volume ~2

billion cells/plate for large scale transfection), pass them though the buffer at least twice a

week. (use Ham’s/F12 for CHO cells and DMEM/F12 for HEK cells, supplemented with

10% FBS and 1% pen-strep)

2) A day before transfection, plate cells in dishes/flasks/chambers (with appropriate

size and number in order to have enough cells (~2 billion cells/plate) according to

transfection scale, and let the cells reach about 70-80% confluent at harvesting point.

3) Wash culture vessels with HBSS7, and trypsinize for 1-5 minutes at room

temperature, then resuspend cells in complete medium, count cells using hemacytometer.

4) Spin down cells at 1000 g for 5 minutes at room temperature, and wash cell pellet

once with transfection buffer (washing volume is determined by final cell suspension

volume, at least 10 times as much as final volume)

5) Spin down cells again at 1000 g for 5 minutes, and resuspend cells in certain

amount of transfection buffer (final cell suspension volume is calculated based on the

total cell number harvested) to make 1*108 cells/mL cell suspension. (final cell

suspension – ready for transfection)

6) Mix target plasmid DNAs (if the target has more than one subunit, mix different

subunits at equal molar ration) and GFP8 plasmid DNA at 10:1 ratio

7 Hank`s Buffered Salt Solution-HBSS8 Green florescent protein-GFP

16

7) Choose a transfection unit (OC-100, OC-400, CL-2, OC-100 holds 100ul, OC-400

holds 400 ul and CL-2 holds 50 ml) according to transfection scale. Mix DNA and cells

together (DNA final concentration, 100ug/ml for HEK and 200ug/ml for CHO)

8) Select appropriate programs on transfection machine, after electroporation,

transfer cells into dish/plates, and let cells recover for 20 minutes at 37°C with 5% CO 2

and 95% humidity.

9) Plate cells into dishes/flasks incubate overnight at 37°C in a CO2 incubator and let

GFP (Green fluorescent protein) and target genes express.

10) Monitor transfection efficiency under fluorescent microscope or using FACS

(Flourescent Activated Cell Sorting) sorter.

11) Collect GFP positive populations by sorting on FACS machine, if needed

12) Culture GFP positive populations for EP assays, or freeze cells for later use and

sale (freezing solution=90% FBS9+10% DMSO10).

CHO cells were transiently transfected with cDNA for Cav2.2 1, 2δ, and 3 subunits

in addition to Kir2.1 cDNA using the scalable electroporation system. Cells were

immediately frozen in liquid nitrogen for later use.

For use in this study, cells were removed from storage, quick-thawed in a 37°C water

bath, and then transferred to a conical tube also containing 30 mL cell culture medium -

(Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12)

supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 g/mL

streptomycin sulfate and 500 g/mL G418). The cell suspension will be centrifuged at

1,500 rpm for 2 minutes and the supernatant will be discarded. Cell will be re-suspended

9 Fetal Bovine Serum- FBS

10 Dimethyl sulfoxide-DMSO

17

in 30 mL of fresh medium and 20 L of this suspension will be mixed with Trypan blue

to determine cell viability and density. Cells will be counted immediately using a

Hemacytometer. Cells will be diluted to a density of 35,000 to 38,000 cells/well of a 384-

well FLIPR assay plate (Type: BD Biocoat Poly-D-Lysine Multiwell Cell Culture Plate)

depending on measured viability and density.

Using a multichannel pipettor, cells will be plated into the wells of the multi well plate.

The day before a scheduled FLIPR experiment, cells will be plated onto a Polylysin-

coated 384-well assay plates (BD Biosciences) using a multichannel pipettor (Titertek

Multidrop) for overnight growth.

6.1 Solutions and chemicals used in FLIPR TETRA TM assay

Formulations

All chemicals used in solution preparation were purchased from Sigma-Aldrich (St.

Louis, MO) unless otherwise noted and were of ACS reagent grade purity or higher.

Stock solutions of test and control articles were either prepared in dimethyl sulfoxide

(DMSO) or deionised water and stored frozen. Test article (drug sample from sponsor)

and positive control concentrations were prepared fresh daily by diluting stock solutions

into appropriate buffer solutions. Solutions used in this study contained 0.01% Pluronic

F-127. The composition of HEPES-buffered physiological saline (HB-PS), 10; Glucose,

10; pH adjusted to 7.4 with NaOH (prepared weekly and refrigerated until use. These

formulations were then loaded in glass- lined 96-well compound plate of FLIPRTETRA

instrument (Molecular Devices Corporation, Union City CA).

Dye Solution

18

Calcium 4 Assay Kit (Molecular Devices)

Assay buffers

Name: Preincubation buffer (0.5K/0Ca HB-PS)

Storage Conditions: Refrigerated

Composition (in mM): NaCl, 140.2; KCl, 0.5; CaCl2,0; MgCl2, 1;

HEBES, 10; Glucose, 10; pH adjusted to 7.4

with NaOH

Calcium 4 No Wash Kit (Molecular

Devices Corp.)

0.5 K+ was included to achieve sufficient

current through the inward rectifier and

therefore a more negative membrane potential

and sufficient availability of calcium channels

for stimulation.

Stimulation buffer (135K/10.8Ca HB-PS)

Source: ChanTest Corp.

Storage Conditions: Refrigerated

Composition (in mM): NaCl, 0; KCl, 135; CaCl2, 10.8; MgCl2, 1; HEBES, 10; Glucose, 0; pH adjusted to 7.4 with NaOH

Rationale: 135 mM K+ is close to the maximum possible potassium concentration that can be achieved in a buffer that also includes 10 mM HEPES and 10.8 mM CaCl2, and 1 mM MgCl2 and that does not violate a quasi-physiological osmolarity of around 300m osmoles. A maximum K+ concentration is desirable to produce maximum stimulation of the expressed calcium channels.

19

Test article carrier

Name: Dimethyl sulfoxide (DMSO)

Source: Sigma-Aldrich

Storage Conditions: Room temperature

Rationale for Selection: DMSO aids in the dissolution of test articles.

1% of DMSO was included in the wells.

Consumables

Type: Compound plate

Cell plates (poly-D-lysine coated)

FLIPR pipette tips

Automated liquid handler tips

6.2 Experimental methods –FLIPR TETRA TM assay.

Fluid Addition Periods

On the experimental day, after removal of the culture medium on a plate washer (Titertek

MAP-C), 10 L/well of dye solution was added to the cells using a Multidrop (Titertek

multidrop) bulk dispensing system. Cells were then incubated for 30 minutes at 37°C and

95% O2/5% CO2. In antagonist assays, 5 µL/well of antagonist compound dissolved in

dye incubation buffer were added to the existing dye solution in the plates using the

pipettor onboard the FLIPR. Plates were then preincubated in dye solution plus test

compound for an additional 30 minutes at ambient temperature, before 25 µL/well of

stimulation buffer addition activated the IC and the fluorescent response was measured.

20

The stimulation buffer contained test article or control article concentrations that were

equal to the test or control article concentrations in the wells. The test compound

preincubation period was omitted in buffer assays and buffers of varying compositions

were added after 60 minutes in dye solution.

Data acquisition:

Fluorescence changes due to ion channel activation and modulation by test articles were

recorded by the FLIPR instrument (FLIPRTETRA, MDS, and Sunnyvale, CA) displayed

with the FLIPR Screenworks software. Data were only recorded during the stimulus

addition period, but not during the 30 minute-long preincubation period that preceded the

stimulus addition. At the beginning of the stimulus addition period, a 30 second baseline

period was recorded which then was followed by stimulus buffer addition to activate the

ion channels. During the first 60 seconds of activation, data were acquired at the

maximum rate (1 sample/second) and for the remaining 5 minutes, data were sampled

every 5 seconds.

Data Analysis:

Data were stored on the ChanTest computer network (and backed-up nightly) for off-line

analysis. Data acquisition was performed via the Screenworks software that is supplied

with the FLIPR System (Molecular Devices, Union City, Ca) and data were analyzed

using Screen works build-in functions for corrections and data reductions, Microsoft

Excel 2003 (Microsoft Corp., Redmond, WA), and Igor Pro 5.0 (Wavemetrics, Inc., Lake

Oswego, OR). Using Igor’s built-in fitting function, concentration-response data were

fitted to a Hill equation of the following form:

21

RESPONSE=Base+ Max−Base

1+( xhalfx )

rate

Where Base is the response at low concentrations of test article, Max is the maximum

response at high concentrations, x half is the EC50, or IC50 (half-maximal inhibitory

concentration of the drug), the concentration of test article producing either half-maximal

activation or inhibition, and rate is the Hill coefficient. Non-linear least squares fits were

made assuming a simple one-to-one binding model. If appropriate fits were weighted by

the standard deviation. No assumptions about the fit parameters were made and the fit

parameters were determined by the algorithm. In addition, the z and z’ factors11 for the

assay will be calculated (Zhang, Chung and Oldenburg 1999).

11 Z` factor is a measure of statistical effect size, defined in terms of the means and standard deviation of both positive and negative controls. (Zhang, Chung and Oldenburg 1999)

22

7 Discussions-Technical challenges and relative propositions

Once the validation report was summarized, based on the advantages of transiently

transfected cell lines over stably transfected cell lines, few problems with transient

transfected cell lines are demonstrated along with the possible suggestions.

7.1 Problems and relevant suggestions

The following are the identified problems and corresponding suggestions for overcoming

those problems seen in transient gene expression system.

1. Problem: Variability in results

Suggestion: We could work on the scale of 1 to 5 billion cells at a time, and the

transfected cells would be frozen and saved in liquid nitrogen for assays, the cell numbers

from one single transfection are more than enough for finishing multi-assays. Therefore,

the variations are very limited. Another method to eliminate batch to batch variation in

results could be minimized for larger screening projects by placing orders from same

batch. Cells are stored at −150 degrees Celsius to ensure consistent performance. Figure 7

shows calcium influx measured with FLIPRTETRA® in HEK293 cells transiently transfected

with Cav1.2, beta2, alpha2delta, and Kir2.1. When calcium influx with FLIPR in HEK

293 measured cells, Panel A before 24 hours post transfection and after 48 hours post

transfection have similar signal strength (RFLU at 24 hours 3500 and 6500 after 48

hours). In fact, there is enhanced signal seen in panel A, 48 hours post transfection which

means that there is no degradation in the signal strength and hence, there is no variation

in cell qualitatively.

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Figure 2: Variability in results

Reference: (Oestreich 2009)

24

2. Problem: Compromise with expression level

Suggestion: By adjusting DNA concentrations to control expression level

expression level could be maintained. As ion channel and other cell surface proteins are

shown to contribute to cell toxicity, the well-expressed cells might die out during the

course of stable cell line development, and cause stable cell line instability while

culturing. In certain cases, transient transfection is the way to reach high-level expression

for some gene targets. (Figure 8) shows that transiently and stably transfected CHO cells

exhibit comparable levels of assay performance. The curves are super imposed. Hence no

compromise is seen with expression level.

Figure 3: Compromise with expression level

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Reference (Maxcyte; Brady, James 2009)

3. Problem: Consistency

Suggestion: Since the Maxcyte STXTM transfection system uses electroporation,

the only two variables that have major impacts on transfection efficiency are the DNA

concentration and cell density. So, as long as these two factors are well controlled,

consistency should not be a concern.

4. Problem: Cell toxicity

Suggestion: The Maxcyte STXTM electroporation buffer is very close to

physiological conditions, so it is not stressful to any of the types of cells so far we have

used, and no toxic effects have been detected in control group (with buffer and

electroporation, but without DNA).

5. Problem: Low transfection efficiency

Suggestion: This is not a problem transfection system used by ChanTest since the

transfection efficiency with GFP DNA as control in HEK, CHO and 3T3 cells is usually

above 90%.

Figure 9 shows transiently transfected cells perform comparably to stable cells in

2XFYVE- eGFP assay.

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Figure 4: Low transfection efficiency

Reference (Maxcyte; Brady, James 2009)

27

8 Commercial conclusions

Survey methodology

Figure 5: Survey methodology

Survey conducted under the supervision of (Brown, Keck and Smith 2009)

A web- based survey was prepared using Qualtrics.com professional software.

This survey is conducted to meet the needs of survey sponsors, ChanTest Corp;

who is interested in understanding customer needs for Ion Channel and GPCR cell lines,

especially transiently transfected cell lines and division arrested cell lines used in HTS

cell based assays.  A questionnaire was prepared to know what the customers’ want and

how we could find the best fit for them.

The main objectives of this study:

28

a) Experimenter’s specific requirement for cell lines

b) To know experimenter’s specific cell type and species preference in their protocol.

c) Cell lines market trends and demographics.

d) To learn customers’ profile.

The questionnaire consists of 10 questions related to cell lines and 5 questions on

demographics. A total of 15 multiple choice questions and two open ended two are open

ended questions.

The contacts with majority of respondents were by email, mainly by using a

professional website called linkedin.com. Another reminder email was sent in two weeks

as a final call.

The data collection phase of the questionnaire was limited to 4-8 weeks.

The number of respondents from contacts was challenging to achieve. The total

number respondents are 80 out of 7000 contact. Some of them emailed writing their

views instead of taking the survey.

There are 74 surveys started and 70 completed.

Data was analyzed on line using www.Qualtrics.com web analysis tools to sort

and filter specific group responses and to download results. Data was subsequently

processed and presented graphically using Microsoft Excel.

The survey is completely anonymous and no personal identifying information was

collected without permission.

The link to the survey:

https://academictrial.qualtrics.com/SE?SID=SV_3Co3XlDXO6yzFfm&SVID=Prod

29

Survey results

Responses received are from the recipients of different background globally.

Huge market potential is identified for biotechnology and pharmaceutical companies as

illustrated in figure 11

Figure 6: Market potential is identified for biotechnology and pharmaceutical

According to the demographics area of research, 72.34% comes from drug discovery

group, mainly used for HTS applications, secondary screening and profiling. (Figure 12)

Figure 7 Demographics area of research

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• Looking at the market trend, 30-40 % of respondents plan to use ion channel /

GPCR cell lines in their HTS cell based assays for screening drug candidates libraries and

significant number (60%) of responses from biotech companies have agreed that transient

gene expression system (that ChanTest offers) could cost effective and time saving

alternative to stable expression system.

• Top choice from respondents for Ion channel cell lines from responses is

ChanTest Corp. among the other competitive businesses such as Scottish Biomedical,

Millipore, Invitrogen.

• ChanTest Corp. has the largest offering ion channel validated cell lines of about

70 whereas Millipore has about 35 , Bsys about 10, Invitrogen and Scottish Biomedical

has a small group focus on ion channel.

Industry overview12

Biotechnology CRO products and services is 17.5% of R&D expense. (IBIS

World 2009). Products sold are usually a broad range from cell lines to media and service

industry carries a huge portion of this. This expense trends slightly downwards since the

number of Ion channel screening labs reduced due to recession in 2008 through 2009.

Major market segment in Research and Development is private sector industry,

63.7%. Of which 29.9% is Federal Government and 25.5% is non-manufacturing

industries. (IBIS World Report 2009)

Leading businesses in the CRO biotechnology industry belongs to drug discovery.

12 NAICS code for Chan Test Corp: 54171101 –Research and Development in Biotechnology.SIC code for Chan Test Corp: 873101 – Laboratories- Research and Development

31

Available labs for Ion Channel cell lines sales (Stable, transients, Division

arrested) is estimated about 430 in number. (Appendix B)

Operational/management trends within the industry: MDS predicts that there will

be a drop in net revenues by 28% in CRO market for 2010, which will account for

discontinued operations of about $50 million (–Pharma, Outsourcing 2010). This is due

to economic pressures, enhancing merger activity and reduced access to capital.

ChanTest identifies major customer group with pharma/biotech company for its

Ion Channel transient gene system as drug discovery group (HTS applications),

secondary Screening and profiling.

Market entry strategy for ChanTest`s EZCellsTM TT

1. Creating Awareness: Ion Channel EZCells TM TT is commercially new to the market.

Hence, educating researchers about its benefits is very important. Suggestions include

conducting webinars, distributing flyers, posting advertisements on popular e-journals

and conducting survey.

1. Strategic partnership with instrument makers like Nanion and Sophion who

develop scalable transfection system

2. Convert replicating cell line market into EZCellsTM TT (for assumptions see

Appendix A and B)

32

Market Overview: Focus and demographics:

United States and Europe are the biggest market in the world for the use of Ion

Channel Cell lines in drug discovery. The total European cell-based assays market is

expected to be valued at greater than $380 million by 2011. (Frost and sullivan 2004)

Japan is also a key market with upcoming markets forecasted in India. The

outsourcing sector is expanding at the rate of 43% per annum in India. It is expected that

ten drugs discovered worldwide in the next few years will come from India. (NickTaylor

2009). $2.46 billion in 2010 is accounted only for India’s contract manufacturing. Report

released from KPMG. (Taylor 2008)

Psychographics:

51% researchers who use cell-based assays perform transfection. (Biocompare

2007)

Trends: Customer behavior from survey suggests that researchers are always

looking for less time consuming and fast transfection gene system (GEN 2008).

Major players involved with producing ion channel / GPCR cell lines are mainly

ChanTest, Millipore, Perkin Elmer and Invitrogen, Bsys and Scottish Biomedical.

Companies involved in producing reagents, kits, tools are Applied Biosystems

(Foster, California), Ambion (Austin, Texas), Bio Rad Laboratories Inc. (Hercules,

California), Qiagen Inc. (Europe) and Stratagene Corp. (California).

Consumable costs per data point have ranged from $1 to $4- fluxionbio.com.

(Lexis Nexis 2009) FLIPR assay pricing per point is about $10, FASTPatch costs about

$100-$150 and Quattro $25 - $100 depending on the channel of choice.

33

Cell formats arranged based on extent of usage by customers from most widely

used to the least: stable transfected cell lines > transient transfected > cryopreserved >

division arrested.

Based on number of large pharma-biotech companies, small pharma-biotech companies

and academics involved in ion channel activities(testing) and the estimated budget

expenses made on ion channel cell lines correspondingly, in house and outsourced; global

estimate of market size for the ion channel -expressing stable cell lines is made.

The total market estimate for the global Pharma & Biotech market for ion channel

testing in 2009 was estimated to $106M for in house consumables, $45M for outsourced

testing at fee-for-service providers and $76M for capital expense purchases on

instruments. Where appropriate these markets were broken down, segmented and CAGR

estimates for 2011 made. (HTStec, 2009)

Break down of these 2009 consumable budget for ion channel testing is shown in

the figure below (HTStec 2009). 19% of this is indicated as cell lines and cell media

budget. This account to $16 M of global pharma/ biotech in house ion channel cell lines

and cell culture media.

Estimation for the global pharma/ biotech market size for ion channel expressing

stable or replicating cell line. Cell formats arranged based on extent of usage by

customers from most widely used to the least: stable transfected cell lines > transient

transfected > cryopreserved > division arrested. Therefore, global pharma/ biotech in

house consumable budget market for ion channel stable cell lines estimated is $ 10 M of

the $16 M from global in house ion channel cell lines and cell culture media.

34

Pricing proposition:

Pricing is based on per well cost and number of cells in each vial-transiently transfected

ion channel cells.

6 million cells per vial; plate at ~15,000 cells per well of 384-well plate

All price per well based on 384-well plate

$599 per vial for one vial

(Removed due to proprietary information)

Pricing is still in debate, with suggestions for increasing it to $1000-$1500/vial based on

benefit per cost and competitive pricing over stable cell lines.(Appendix C)

8.1 Business proposition

ChanTest Corp. acquires 12% of total pharma/biotech market for ion channel stable cell

line and this is expected to grow up to 17% in 2011. The analyses reveal that the

addressable market for Ion Channel EZCellsTM TT is $4.5 M at proposed pricing of

$1000/vial. This implies that the required unit sales should be about 2250 vials per year

(Appendix A). The target drug discovery market for Ion Channel EZCellsTM TT are

secondary screening and profiling applications with large market potential in biotech and

pharmaceutical companies and academics.

35

9 Appendices

9.1 Appendix A: Estimated production cost for 2250 vials

36

9.2 Appendix B: Globally projected estimation of units sold per year.

37

9.3 Appendix C: Comparison of expenses (stable vs. transient)

The table below compares the cost of using EZCellsTM TT against replicating cell lines,

depending on the expenses incurred during users’ research

38

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