Pharma Starts with You

Biomarker Workflow Solutions
A toolbox for successful biomarker
discovery, development, and validation

FROM DISCOVERYTO VALIDATION

Biomarkers provide vital information regarding safety and efficacy during drug
discovery and development. They can also be used to select patients for clinical
trials, to monitor their response, and to monitor a treatment’s efficacy. In addition,
after regulatory approval, biomarkers can be used in the clinic to allow more
personalized treatments.
Biomarkers range from specific molecules such as a protein, gene sequence,or expressed RNA to physiological parameters such as blood pressure or bodytemperature. They may be expressed in a specific location or tissue or found inserum, plasma, and other bodily fluids. Because of this variety most successful
biomarker programs have a broad reach and investigate cellular, proteomic,
and genetic sources in order to find the most valuable biomarkers. Bio-Rad’s
broad product portfolio is able to support biomarker discovery across all ofthese areas and is ideally suited to support even the most comprehensive
biomarker programs.

2 | BIO-RAD LABORATORIES, INC.

FROM DISCOVERY TO VALIDATION

BIO-RAD’S BIOMARKER WORKFLOW:FROM DISCOVERY TO VALIDATIONCELLULAR

ZE5™ CELL ANALYZER

S3e™ CELL SORTERZOE™ CELL IMAGER

V3 WESTERN WORKFLOW ™

BIO-PLEX® 200 SYSTEM

HuCAL® CUSTOM
ANTIBODY TECHNOLOGY

QX200™ DROPLETDIGTAL™ PCR SYSTEM

CFX AUTOMATIONSYSTEM II

PrimePCR™
PANELSBIOPLEX® 2200 SYSTEMQUALITY CONTROLSDIAGNOSTIC ANTIBODIESPROTEOMIC

GEN0MICSOLUTIONSBEYONDDISCOVERYBIO-RAD LABORATORIES, INC. | 3

CELLULAR
BIOMARKERSUNCOVER RARE CELL POPULATIONSZE5™ CELL ANALYZER

S3e™ CELL SORTERZOE™ CELL IMAGER

The ability to uncover novel and rare cell types through multiparameter analysis allows researchers to characterize cell
populations in greater detail and identify subpopulations thatcan serve as markers for disease. Simple and reliable isolationof these cells for downstream assays further supports thecellular biomarker workflow by providing pure populations forgenomic and proteomic analysis.
Our comprehensive and easy-to-use suite of cell analysis
products includes the ZE5 Cell Analyzer, the S3e Cell Sorter,and the ZOE Fluorescent Cell Imager, as well as a wide rangeof flow cytometry and cell imaging reagents. Examine up to
30 parameters simultaneously with the ZE5 Cell Analyzer
to distinguish subtly different cell populations, sort rare andsingle cells with the S3e Cell Sorter, and perform traditional
immunocytochemistry and -histochemistry studies forbiomarkers in live or fixed cells right at your bench with the
ZOE Cell Imager.
In this example we demonstrate the ability of the ZE5 Cell
Analyzer to allow broad yet in-depth analysis of a blood sample
in a single run. A 15-color panel of known leukocyte markers
was used to differentiate subpopulations of normal peripheral
blood mononuclear cells (PBMCs) and those from a patientwith chronic B cell leukemia.

SUCCESS STORY Bio-Rad bulletin 6950 shows how the ZE5 Cell Analyzer
can be used to design and optimize a 21-color panel for quantificationof T cell subsets in stem cell transplant patients.
Visit bio-rad.com/biomarker1 to learn more.

4 | BIO-RAD LABORATORIES, INC.

CELLULAR BIOMARKERS

15-COLOR CELLULAR BIOMARKER SCREENING PANELFOR CHRONIC B CELL LEUKEMIA

1x10 01x10 0 5x104 10x104 15x104 20x104

CD3–

1x104
1x103
1x102
1x10 0–1x102CD3+
1x10 01x102

1x10 01x10 0 1x101 1x102 1x103 1x104 1x104

1x103

20
0

1x10 0B cells

1x102
1x10 0–1x1021x103 1x104

1x10 0Comp-FL23-A:: CD19 (BUV) 737-A
Comp-FL27-A:: CD20 APC-Vio770-A-A

Comp-FL16-A:: CD56 (BV) 786-A

1x103 1x104 1x105

1x105
1x10

CD19–, CD20–

4

1x103
1x10 0–1x1031x10 01x103 1x104 1x105

Comp-FL23-A:: CD19 (BUV) 737-A

5x104

400

1x104
CD8+
CD4+

1x102
1x10 0–1x102

CD7+

200
100

CD20+ and CD8+

1x104
1x103
1x102
1x10 0–1x102–1x1031x10 01x103 1x104 1x105

Comp-FL27-A:: CD20 APC+Vio770-A

1x105
1x104
Monocytes
1x103

1x103 1x104 1x105

300

1x103

1x105

1x10 0Comp-FL11-A:: CD11b PE-A

0

Comp-FL22-A:: CD4 (BUV) 661-A

Comp-FL26-A:: CD38 APC-R700-A

CD56
low

1x103

10x1041x10 01x10 0 1x101 1x102 1x103 1x104 1x105

Comp-FL03-A:: CD33 FITC-A

40

CD56
high

Comp-FL16-A:: CD56 (BV) 766-A

Count

60

NK cells
1x104

15x10

Myeloid cells

4Comp-FL20-A:: CD45 VioGreen-A

Comp-FL21-A:: CD3 (BUV) 496-A

80

SS02-A::SSC 488_10_A

5x104

FS00-A::FSC 488_10-A

Comp-FL16-A:: CD56 (BV) 766-A

FS00-A::FSC 488_10-A

10x10

4

Count

5x104

15x10

CD14–

1x102
1x10 0–1x102
1x10 0

1x103

1x104

1x105

Comp-FL08-A:: CD14 PE-Cy5-A

1x10 01x103 1x104 1x105

Comp-FL14-A:: CD7 (BV) 650-A
Comp-FL18-A:: Kappa light chain (BV) 421-A

1x10 –3
1x10 0 5x104 10x104 15x104 20x104

10x10

4

4

20x104

CD45+

Comp-FL17-A:: HLADR VioBlue-A

5x104

15x10

SS02-A::SSC 488_10_A

10x10

4

4

20x104Single cells

Comp-FL15-A:: CD8 (BV) 711-A

15x10

4

20x104PBMC

FS00-H::FSC 488_10_H

SS02-A::SSC 488_10_A

20x104

1x105

Q1

Q2

1x104
1x103
1x102
1x10 0Q4–1x102–1x103 1x10 0Q31x103 1x104 1x105

Comp-FL29-A:: Lamda LC APC-A

1x105

Dendritic cells

1x104
1x103
1x102
1x10 0–1x102
1x10 01x103

1x104 1x105

Comp-FL08-A:: CD14 PE-Cy5-A

Differentiating subsets of malignant cells from normal cells using a 15-color panel of known leukocyte markers. We wereable to distinguish and define a broad range of immune cell phenotypes within normal and chronic B-cell leukemic PBMC samples.
Our 15-color panel provides the ability to separate PBMC into base cell populations and includes broad prognostic and diagnostic
markers such as CD7, CD38 kappa, and lambda light chain. Normal and diseased PBMCs (AllCells.com) were thawed and allowed
to recover in culture media at 37°C, 5% CO2 for at least one hour. Single-color antibodies were pooled, mixed gently with one dropof AbC Total Antibody Compensation Beads (Thermo Fisher), and incubated for at least one hour. Unstained controls, normal, anddiseased PBMCs were incubated in blocking buffer for 45 min on ice before staining with the 15-color antibody cocktails for 1 hour.
Cells and beads were spun down, washed once in wash buffer, and then resuspended and analyzed on the ZE5 Cell Analyzer.
Positive and negative bead populations were used to set compensation for fluorochromes. ZE5 Cell Analyzer parameters: 5 lasers
at 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm; fluorescence detectors and dual forward-scatter channels at 405 nm and 488 nm
and a side scatter channel.

BIO-RAD LABORATORIES, INC. | 5

PROTEOMICBIOMARKERSMULTIPLEX YOUR PROTEIN ANALYSIS

V3 WESTERN WORKFLOW ™

BIO-PLEX® 200 SYSTEM

HuCAL® ANTIBODY
GENERATION TECHNOLOGYInterrogating your sample to identify its proteomic profile gives rise
to a veritable feast of potential biomarkers. In addition, monitoring
the posttranslational state of proteins of interest can yield importantbiomarkers.
The V3 Western Workflow is a validated and streamlined workflowthat allows quantitation of protein expression faster and with moreaccuracy than other western blotting procedures. By incorporating theChemiDoc™ MP Imaging System, this workflow allows simultaneous
analysis of up to three proteins via multiplex fluorescent western blotting.
Bio-Plex Multiplex Immunoassay Systems and Assays allow analysisof large numbers of biomarkers in a single experiment. Predesigned
panels for a given indication remove the guesswork from your search
while custom assays allow you to design panels for your uniquemarkers as your biomarker program progresses.
Antibody selection and antibody specificity are crucial to theperformance of your proteomic assays. Our HuCAL custom antibody
technology can generate highly specific custom human antibodies in
only 8 weeks for a variety of applications, including flow cytometry,
ELISA, and western blotting.
For this example we identified proteins whose expression ismodulated by TNF-a in cancer cell lines, we investigated changes
in protein expression in wild type and knock out prostate cancer
cell lines with and without TNF-a stimulation.
SUCCESS STORY Evidence suggests that CXCL10 is a strong predictor of poor
clinical outcomes from HCV treatment. We show that HuCAL Antibodies canquantitatively discriminate between agonist and antagonist forms of CXCL10,which differ by only two amino acids at the N-terminus.
Visit bio-rad.com/biomarker2 to learn more.

6 | BIO-RAD LABORATORIES, INC.

PROTEOMIC BIOMARKERSPROTEIN BIOMARKER DISCOVERY USINGMULTIPLEX IMMUNOASSAYS
A
5,000
Concentration, pg/ml

4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0
ICAM-1B
800
Concentration, pg/ml

700
600
500
400
300
200
100
0
MMP-13

Concentration, pg/ml

C
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
IFN-g

IL-11

IL-28a/IFN-I

IL-34

TWEAK

Concentration, pg/ml

D
220
200
180
160
140
120
100
80
60
40
20
0
NF-kB

SRC

ERK1/2

Measuring protein expression in wild-type PC3 cancer cells and ICAM-1 knock down PC3 cells using Bio-Plex Multiplex Immunoassays.
Cell lysates were collected from each of the four sample types after ICAM-1 induction with TNF-a. We found that A, ICAM-1 knock down is
incomplete; ICAM-1 expression is still induced, albeit at a lower level (Bio-Plex Pro Human Cytokine 21-Plex Panel, catalog #MF005KMII) and that
B, MMP-13 expression is 2-fold higher in the edited PC3 cell line than in wild-type PC3 cells (9-plex Bio-Plex Pro™ Human MMP Panel, catalog#171AM001M). We also observed C, attenuated cytokine induction in the edited PC3 cell line in response to TNF-a (37-plex Bio-Plex Pro Human
Inflammation Panel 1, catalog #171AL001M); and D, found that three cytokines, p-Src, p-NF-kB and p-Erk1/2, were detectable in both wild-type
and edited PC3 cell lines but expression levels remained unchanged upon TNF-a stimulation (Bio-Plex Pro Magnetic Cell Signaling Assays,various catalog numbers). Lysates were treated according to the instructions provided with each Bio-Plex Panel. PC3 ( ); PC3 + TNF-a ( );edited PC3 ( ); edited PC3 + TNF-a ( ).

BIO-RAD LABORATORIES, INC. | 7

GENOMIC
BIOMARKERSDISCOVER MORE

CFX REAL-TIME PCR
DETECTION SYSTEM

PrimePCR™ PANELSQX200™ DROPLET DIGITAL™
PCR SYSTEM

CFX AUTOMATION SYSTEM II

Both gene expression and the presence of specific genetic sequences
are used as biomarkers for many diseases, including cancer. Treatments
can even be tailored to the individual based on these genomic biomarkers.
Typically, large-scale gene expression studies are conducted to identify
genes that are up- or downregulated in a particular indication or disease.
These studies rely on the ability of the researchers to design exploratory
and confirmatory panels as well as on powerful software tools to analyze
resulting data and identify potential biomarkers.
CFX Real-Time PCR Detection Systems along with custom-built orpredesigned pathway or disease state PrimePCR Panels allow you tomeasure multiple genes from multiple samples at once, be they liquidbiopsy, tissue, or cell-based materials. Coupled with the CFX Automation
System II, up to 48 plates can be read unattended. If increased sensitivity
is required, such as for the detection and quantification of CTCs and rare
cancer mutations, the QX200 Droplet Digital PCR (ddPCR™) System is
an ideal choice, as it allows detection of rare, low-abundance genetic
biomarkers. Both systems are provided with IQ/OQ workflows and toolsthat enable 21 CFR Part 11 compliance.
In this example gene expression patterns from prostate cancer samples
across four different stages were analyzed by quantitative PCR. Assay
design allowed the reproducibility of the multiplex assay to be comparedto singleplex analysis. After hierarchical clustering analysis and one-way
ANOVA, potential candidate biomarkers were selected for inclusion in afive-color PrimePCR Multiplex Assay using CFX Maestro™ Software.
SUCCESS STORY Prospective studies have used ddPCR technology to quantify driverand resistance mutations associated with tumors such as melanoma and havereported positive predictive values of up to 100%, turnaround times of 2–3 days, anda significant increase in sensitivity. ddPCR assays for metastatic melanoma have beenfound to be 42% more sensitive than traditional LDH assays (Chang et al. 2016).
Chang et al. (2016). Sensitivity of plasma BRAF mutant and NRAS mutant cell-free DNA assays to detect
metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression. Molecular
Oncology 10, 157–165.

8 | BIO-RAD LABORATORIES, INC.

GENOMIC BIOMARKERSGENETIC BIOMARKER DISCOVERY USINGHIGH THROUGHPUT REAL-TIME PCR
A

B
Cluster 1

ANOVA Results

Cluster 2
Cluster 3

D11
F10

D10

F12
D4

D12
F9

E3

D5
F8
E12

F1
E11
F2
E10
C3
D7
D6
D9
C11
E1
E5
C10
TRAF2
TRAF6
IKBKG
PRKCZ
TRAF3
CHUK
NGFR
TNFRSF11A
NGF
CD40LGRIPK1IKBKBNFKBIE

TargetP Value

TNFSF130.022585

TNFSF40.011183

TNFRSF1A

0.177595

TRAF2

0.017002

Tukey HSD, P ValueTNFSF13B
NFKB2CD40
BCL2L1NFKBIATNFRSF17
BCL2
BCL2A1NFKB1TNFSF12TRADD
IRAK1SQSTM1

C

Contrast

TNFSF13

TNFSF4

TRAF2

Normal – Stage II

0.013433

0.018559

0.230739

Normal – Stage III

0.344713

0.927900

0.675594

Stage II – Stage III

0.367151

0.048767

0.013329

D
104

CY5.5

102

TEX 615FAM

HEX

Relative Normalized
Expression

RFU

CY5
103

*

101

*

*

10 0
10 –1

102
0

10

20
Cycles30

TNFRSF1A

TNFSF13

TNFSF4

TRAF2

TargetDiscovering prostate cancer biomarkers using gene expression analysis. A, CFX Maestro Software for CFX Real-Time PCR Instruments allows
data to be analyzed in clustergram by sample, by target, or both (subset of data shown); normal, ( ); stage II, ( ); stage III, ( ); stage IV, ( ).; B, Oneway ANOVA of gene expression in biological groups (stages of prostate cancer). The ANOVA P values indicate that there was a statistically significant
difference among the means, which was further supported by the Tukey HSD test. Statistically significant differences are noted with P values in red. Basedon ANOVA of all targets, TNFSF4 and TRAF2 may be useful in distinguishing stage II from stage III prostate cancer, while TNFSF13 and TNFSF4 may be
useful in distinguishing prostate cancer from normal cells; C, Validation of five-target multiplex assay composed of five unique PrimePCR Probe Assays.
Multiplex ( ) versus singleplex ( ) traces; targets: TRAF2 (FAM), TNFSF13 (HEX), TNFSF4 (TEX 615), HPRT1 (CY5), TNFRSF1A (CY5.5). Preamplified
cDNA from normal prostate tissue shown; D, Normalized gene expression by biological group (stages of prostate cancer) was averaged. Error bars are
95% confidence intervals. Number of samples: 16 normal, 20 stage II, 22 stage III. Based on these results, TNFRSF1A does not seem to be a useful biomarker
for prostate cancer progression. Normal ( ); stage II ( ); stage III ( ); *, significant difference compared to the signal from the normal sample.

BIO-RAD LABORATORIES, INC. | 9

SOLUTIONS BEYONDDISCOVERYTOOLS TO TAKE TO THE CLINICBIOPLEX® 2200 SYSTEM

Our broad range of solutions for gene, protein, and cellular biomarker discovery
allows you to uncover markers for your specific needs. Each biomarker’s
proposed downstream application defines the extent to which it must be
validated before it can move forward, to the clinic and beyond. Validation data
include selectivity, specificity, accuracy, precision, reproducibility, and analytestability. Whatever your validation pathway or downstream need, you must haveaccess to a reliable stream of assay components and quality controls.

DIAGNOSTIC ANTIBODIES

Bio-Rad is the market-leading manufacturer of high-quality antigens foruse in biomarker and clinical diagnostic test development. Our cell culture
antigens offer an uninterrupted, unlimited supply of consistent, stable, highquality reagents. To accompany our antigens, we have developed monoclonal
antibodies for use in the development of immunohistochemistry, western
blotting, and other assay types. If a novel antibody is needed, our HuCAL®
custom antibody generation service can generate a human antibody within
8 weeks using an animal-free platform.

HuCAL® ANTIBODY
GENERATION TECHNOLOGY

Additionally, we provide human-based controls covering a wide variety ofanalytes at subtherapeutic, therapeutic, and elevated levels to monitor the
precision of testing procedures in the clinic. If you are looking to incorporate
testing for known biomarkers into a clinical trial, you can take advantage of ourclinical diagnostic offerings, including ELISA- and HPLC-based instruments,
and clinical diagnostic assays and kits that are registered for IVD use.

QUALITY CONTROLS

Bio-Rad partners with you on your biomarker journey, providing solutions fromearly discovery to the clinic and beyond, and meeting your needs for securityof supply of critical materials. Now your journey is complete.
SUCCESS STORY Not all biomarker HbA1c tests are created equal. For over 40 years
Bio-Rad has delivered gold standard HPLC technology to give customers like
Biomed 21 accurate results plus variant detection. HbA1c methods that do not detect
hemoglobin variants might lead to misdiagnosis and unnecessary or delayed patient
treatment. Hear from Bernard Desprats about the D-100™ Smart HPLC System.
Visit bio-rad.com/biomarker3 to watch the video.

10 | BIO-RAD LABORATORIES, INC.

SOLUTIONS BEYOND DISCOVERYSOLUTIONS BEYOND DISCOVERY

8 WeekDevelopment

Animal FreeHuCAL
CUSTOM ANTIBODY
GENERATION

Acute Phase
Proteins

Antigens

CRITICAL RAW
MATERIALS

Bulk Options

Diagnostic
Antibodies

Fill Sizes

Urine

CUSTOMPRODUCTS

Reagents

QUALITY
CONTROL

Delivery

Securityof Supply

Serum

QC Data
Management

WORLD CLASSSERVICEAND SUPPORTPhone

On SiteRemoteTroubleshooting

Email

BIO-RAD LABORATORIES, INC. | 11

WHY BIO-RAD?
Bio-Rad has over 60 years of experience supporting life
science and clinical diagnostics. We are the leader in digital
PCR, transfection, and in gene and protein expression analysis
technologies. Our innovative products are available as standalone solutions or combined into workflows that are optimized
to minimize downtime and to provide the right answer the firsttime, every time. Our Biomarker Workflow Solutions are one ofLife Science RUO*

many Bio-Rad workflows designed to shorten discovery time andand increase success rates for novel therapeutics. As part of ourcommitment to your research, and to increase the success rates
for therapeutics and biosimilars, we provide:
Products and reagents that are reliable and validated
Responsive, trusted global service and technical support teams■■ Technical expertise across multiple workflows and applications■■■■

Discovery

Development

Validation

S3e Cell Sorter

•

•

•

ZE5 Cell Analyzer

•

•

•

ZOE Fluorescent Cell Imager

•

•

•

TC20 Cell Counter

•

•

•

Antibodies for Flow Cytometry and Immunohistochemistry

•

•

•

QX200 Droplet Digital PCR System

•

•

•

PrimePCR Assays for qPCR and ddPCR

•

•

•

CFX Real-Time PCR Detection Systems

•

•

•

qPCR Reagents and Consumables

•

•

•

Bio-Plex Multiplex Immunoassay Systems

•

•

•

Bio-Plex Multiplex Immunoassays

•

•

•

Stain-Free Mini and Midi Protein Gels

•

•

•

Trans-Blot® Turbo™ Transfer System

•

•

•

ChemiDoc MP Imaging System

•

•

•

PrecisionAb™ Validated Western Blotting Antibodies

•

•

•

Protein Sample Preparation Beads and Kits

•

•

•

HuCAL Generated Custom Antibodies

•

•

•

Cells

Genes

Proteins* Products listed in this table are for research use only.

In addition to an array of RUO products for use in your biomarker program, Bio-Rad has a broad selection of products that already meet
regulatory requirements and can also be incorporated.1 These products cover many applications: diabetes testing2; autoimmune testing2;
immunohematology3; blood virus testing5, and quality controls.5
Availability differs depending on country and/or region. 2 U.S. FDA 510(k) clearance. 3 U.S. FDA 510(k) clearance and U.S. FDA licensed products. 4 U.S. FDA approved andU.S. FDA licensed products. 5 U.S. FDA 510(k) clearance or exemption.
1

Visit bio-rad.com/web/biomarker4 to learn more about how our products align with your biomarker program requirements.
AbC is a trademark of Thermo Fisher Scientific. FAM is a trademark of Applera
Corporation. Cy is a trademark of GE Healthcare Group. Texas Red is a trademarkof Invitrogen Corporation. TEX is a trademark of DNA Technologies, Inc.
The Bio-Plex Suspension Array System includes fluorescently labeled microspheres andinstrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.
Bio-Rad’s thermal cyclers and real-time thermal cyclers are covered by one or moreof the following U.S. patents or their foreign counterparts owned by Eppendorf AG:
U.S. Patent Numbers 6,767,512 and 7,074,367.

The QX200 Droplet Digital PCR System is covered by claims of U.S. patents, and/or
pending U.S. and non-U.S. patent applications owned by or under license to Bio-Rad
Laboratories, Inc. Purchase of the product includes a limited, non-transferable rightunder such intellectual property for use of the product for internal research purposes
only. No rights are granted for diagnostic uses. No rights are granted for use of
the product for commercial applications of any kind, including but not limited to
manufacturing, quality control, or commercial services, such as contract services orfee for services. Information concerning a license for such uses can be obtained from
Bio-Rad Laboratories. It is the responsibility of the purchaser/end user to acquire any
additional intellectual property rights that may be required.

Bio-RadLaboratories, Inc.
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