Pharma Starts with You

A Biosimilar Workflow:
From Purification to Characterization

FLEXIBLE AND
COST-EFFECTIVE

Biologics are medicines produced using living cells and highly complex manufacturing
processes. As the number of biologics with expired patents increases, biosimilar
medicines promise to be an attractive, cost-effective alternative. To bring a biosimilar
to market, developers are required to demonstrate high similarity to the approved
biological originator product, which includes evidence that demonstrates no clinicallymeaningful differences between the biosimilar and the reference biologic. This isprovided as part of the totality of evidence reported to regulatory agencies.
We present a workflow that guides you through four of the main steps of biosimilar
development: purification, impurity analysis, comparability studies, and bioanalysis.
We designed a three-step, cost-effective purification strategy that includes impurity
analysis for adalimumab, a HUMIRA-like biosimilar. A downstream cell-based
assay was designed to assess comparability and efficacy of the biosimilar. We also
developed a series of immunoassays to measure other characteristics such as
immunogenicity and pharmacokinetics (PK), which are important considerations forthe development of any biologic or biosimilar.

2 | BIO-RAD LABORATORIES, INC.

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2
3
4

FLEXIBLE AND COST-EFFECTIVE

BIO-RAD’S BIOSIMILAR WORKFLOW:FROM PURIFICATION TO CHARACTERIZATIONPURIFICATIONSTAIN-FREE GELSNGC™ CHROMATOGRAPHYSYSTEM, CHROMATOGRAPHY
COLUMNS AND RESINSHOST CELL PROTEINWORKFLOW

QX200™ DROPLET DIGITAL™
PCR SYSTEM AND ddPCR™ KITS
FOR RESIDUALDNA QUANTIFICATIONZE5™ CELL ANALYZER

S3e™ CELL SORTER

HuCAL® CUSTOM
ANTIBODY TECHNOLOGYBIOANALYTICALANTIBODIES

IMPURITIESCOMPARABILITY

CHARACTERIZATION

BIO-RAD LABORATORIES, INC. | 3

PURIFICATIONFLEXIBLE AND COST-EFFECTIVENGC™ CHROMATOGRAPHYSYSTEM

The NGC Chromatography System along with Bio-Rad
chromatography resins enable fast and easy screening of
methods and components when designing a purification
strategy for biosimilars. The modular NGC System allows
addition of new capabilities as you move from discovery toscale up. Software tools for scouting and multidimensional(Multi-D) chromatography automate both discovery and scale-up
processes and improve reproducibility.

RESINS

PLATES

Bio-Rad chromatography resins can be utilized in a variety of
applications, including size exclusion, ion exchange, HIC, mixedmode, and affinity chromatography. Resins are available inmultiple formats, including plates, columns, and in bulk and aredesigned to facilitate small- to commercial-scale purifications.

STAIN-FREE GELS

NOsphereNuvia S

UNOsphere

We describe a novel three-step workflow that uses the NGC
System to purify adalimumab. This workflow reduces the costs
associated with employing a Protein A capture strategy.

CHT

MPC

ution

Nuvia Q

Methacrylate

Nuvia HR-S

CHT

CHT

SUCCESS STORY Lončar et al. describe single-step downstream
processing of an important industrial enzyme by using the mixed-mode
resin Nuvia™ cPrime™. This resin combines hydrophobic interactions
CFTwith cation exchange groups. These interactions can happen either
individually or simultaneously, leading to enhanced selectivity.

UNOsphere
Lončar N et al. (2015). Mixed-mode resins: taking shortcut in downstream processing of
raw-starch digesting a-amylases. Sci Rep 5, 15772.

Ceramic

Hydroxyapatite

4 | BIO-RAD LABORATORIES, INC.

Nuvia cPrime

A challenge for the biosimilar developer is to produce a productat a cost lower than that of the original biologic. Identifying thebest purification strategy and migrating it from lab to processscale requires a reliable stream of workflow components from
instruments to resins and columns. Costs must be controlled bydesigning a purification strategy with the least number of steps
possible while retaining the purity, efficacy, and structure of
the biosimilar.

Nuvia S

A

Dilute Adalimumab CHO Lysate

Nuvia S Resin Purification™

Nuvia Q Purification: Flow-Through Mode

Nuvia cPrime Purification™

™

Purified Adalimumab

BCMW, kD
250 –
150 –
100 –
75 –

1

2

3

4

5

MW, kD

1

2

3

4

5

250 –
150 –
100 –

50 –
37 –

75 –
50 –
37 –

25 –
25 –

Rapid development of a manufacturing process for adalimumab. Screening of resins for downstream purification was carried out using
96-well filter plates containing multiple resins. The final optimized process contained three chromatography steps: A, capture with Nuvia S
Cation Exchange Resin, intermediate purification with Nuvia Q Anion Exchange Resin, and polishing with Nuvia cPrime Mixed-Mode Media.
Adalimumab was harvested from Chinese hamster ovary (CHO) cells and the lysate was adjusted to pH 4.5 with 1 M hydrochloric acid (HCl).
This was then applied to Bio-Rad Nuvia S Resin equilibrated with 25 mM sodium acetate (pH 4.5), 150 mM NaCl (buffer A). The resin waswashed several times with buffer A and adalimumab was eluted with 25 mM sodium acetate (pH 5.0). Eluted adalimumab was concentrated
and the buffer was exchanged into 25 mM HEPES (pH 8.25), 5 mM NaCl (buffer B) using an Amicon Ultra-4 Centrifugal Filter Unit with a 50 kD
molecular weight cutoff. Bio-Rad Nuvia Q Resin, equilibrated in buffer B, was used to purify adalimumab in flow-through mode. Resulting
adalimumab was concentrated and buffer exchanged into 25 mM sodium acetate (pH 5.0), 250 mM NaCl (buffer C) and applied to Bio-Rad
Nuvia cPrime Media equilibrated with buffer C. The media was washed several times before adalimumab was eluted with 25 mM Tris (pH 8.0),
800 mM NaCl. B, resulting protein was analyzed by native gel electrophoresis. C, this plate method was scaled up tenfold to 1 ml columns
and showed comparable results by native gel. Lane 1, Precision Plus Protein™ Unstained Protein Standard; Lane 2, CHO supernatant; Lane 3,
Nuvia S Resin eluate; Lane 4, Nuvia Q Resin eluate; Lane 5, Nuvia cPrime Media eluate.
BIO-RAD LABORATORIES, INC. | 5

PURIFICATIONRAPID PROCESS DEVELOPMENT FOR BIOSIMILAR PURIFICATIONIMPURITIESVALIDATED ABSOLUTE QUANTIFICATION

ChemiDoc™ MP
IMAGING SYSTEM

TRANS-BLOT® TURBO™
TRANSFER SYSTEM

QX200™ DROPLET DIGITAL™
PCR SYSTEM

ddPCR™ SUPERMIX FOR RESIDUALDNA QUANTIFICATION

Expression systems used to generate biosimilars contain many
components that can compromise the safety and efficacy of abiopharmaceutical, even when present at low levels in the final
product. Contaminants such as host cell proteins (HCPs) and hostcell DNA (hcDNA) can be reduced to safe levels by employing arobust purification strategy. To meet regulatory requirements, drug
manufacturers employ sensitive and reliable detection methods.
Antibodies are the most commonly used tool for detecting andassessing HCPs because they can be used for identification,
detection, and quantification. Evaluation and validation of anti-HCP
antibodies are streamlined by Bio-Rad’s HCP validation workflow,which provides fast, precise, and cost-effective evaluation of
antibodies. Highly sensitive, reliable detection on western blots
and subsequent analysis are provided in a workflow that takes lessthan two days.
Quantifying residual hcDNA by Droplet Digital PCR (ddPCR) provides
the most sensitive absolute quantification. Traditionally, qPCR has
been used for hcDNA quantification, but this technique can besusceptible to PCR inhibitors found in complex matrices, such as
biological material, and thus often requires a DNA extraction step.
Bio-Rad’s ddPCR technology eliminates the need for DNA extraction
and allows direct quantification of residual DNA from multiple species
without compromising accuracy, sensitivity, or precision.

SUCCESS STORY Hussain et al. developed a Droplet Digital PCR based
method for hcDNA detection that increases sensitivity and the linear range
of determination while retaining high precision and accuracy. This method
simplifies residual hcDNA quantification by eliminating DNA extraction,
protease digestion, and the need for DNA standards.
Hussain M et al. (2016). A direct Droplet Digital PCR method for quantification of residual DNA inprotein drugs produced in yeast cells. J Pharm Biomed Anal 10, 128–131.

6 | BIO-RAD LABORATORIES, INC.

A

IMPURITIESMONITORING HOST CELL PROTEIN AND HOST CELL DNACHCP VALIDATION WORKFLOW

16

PREPARE SAMPLE

6.5

PERFORM 2-D ELECTROPHORESIS

HOURS

hcDNA WORKFLOW

30

PREPARE ddPCR REACTION MIX

30

GENERATE DROPLETS

2

PERFORM PCR WITH EvaGreen®
OR HYDROLYSIS PROBES

2.5

READ DROPLETS
AND ANALYZE RESULTSMINUTESHOURS

10MINUTESTRANSFER PROTEINMINUTES4HOURS

IMAGE BLOTAND ANALYZE RESULTS

HOURS

HOURS

HCP ELISA (3 HOURS)
B

DOverall HCP Reduction, %Host Cell dsDNA, pg per mg of adalimumab

Plate Method

Scale-Up

Raw feedstock——

Raw feedstock

2.84 x 1011

After resin 1

93.91

90.56

After resin 1

2.63 x 107

After resin 2

99.93

99.65

After resin 2

12.6

After resin 3

100.0099.99

After resin 3

5.3

HCP and hcDNA analysis. HCP analysis was performed prior to, during, and after three-step purification using an HCP Detection ELISA Kit(Cygnus Technologies) (A). Details of our HCP validation workflow can be found in Bio-Rad bulletin 6493. In both the plate-based purificationand the scale-up column purification more than 90% of HCPs were removed in step 1 of the purification. Up to 99% reduction was found after
step 2 and 99.99% after step 3 in the final purified product, with 6 ppm HCP for the plate purification and 5 ppm HCP for the column scale-up.
(B). To assess residual hcDNA, eluate at each stage of our three-step adalimumab purification protocol was analyzed using the ddPCR Host Cell
Residual DNA Quantitation Kit (C). Residual hcDNA was reduced by almost 100% by using the three-step purification strategy (D).
BIO-RAD LABORATORIES, INC. | 7

COMPARABILITY

CELL-BASED CONFIDENCE

ZE5™ CELL ANALYZER

S3e™ CELL SORTERZOE™ CELL IMAGERANTIBODIES

Biosimilars must show comparability to their originator biologic acrossmultiple factors, including structure, function, and efficacy. Designinga cell-based assay to measure comparability requires an understandingof the underlying mechanism of action of the originator and the biosimilar.
The data that are produced must be precise, accurate, and reproducible
in order to satisfy regulatory requirements for comparability studies.
Cell-based assays are valuable at this stage as they provide multifaceted
data that can inform downstream preclinical and clinical phases ofdevelopment. The ZE5 Cell Analyzer supports this by enabling analysis of
up to 30 parameters from a single sample, greatly expanding the numberof biomarkers that can be monitored by a given cell-based assay. This isimportant when gathering data to provide as part of the totality of evidence
required by regulatory agencies. The ZE5 Cell Analyzer is also well-suited formeasuring rare or transient cell populations due to its fast analysis speed.
If a particular cell type is required for cell-based assays, then the benchtop
S3e Cell Sorter allows sorting of those populations at your bench, without
the need to schedule time at a core facility.
The ZOE Fluorescent Cell Imager lets you easily image cells at your bench,
allowing you to monitor the health and activity of your cells throughout
your experiment.
We designed a cell-based assay that uses the ZE5 Cell Analyzer tomonitor the activity of the purified HUMIRA biosimilar adalimumab.

SUCCESS STORY The S3e Cell Analyzer was used to sort fluorescently labeledtransfected cells prior to seeding and development into a 3-D cell culture early lungmodel. This lung model may be used to test the effectiveness of potential treatments,
such as chemotherapy and small and large molecule drugs, against lung disease.
Chen YW et al. (2017). A three-dimensional model of human lung development and disease from pluripotent
stem cells. Nat Cell Biol 19, 542–549.

8 | BIO-RAD LABORATORIES, INC.

COMPARABILITY

CELL-BASED ASSAY VALIDATION OF IN VITRO BIOSIMILAR ACTIVITY

A
800

Dead cells

Live cells

Count

600

400

200

0
10 0

101

102
103
104
FL24-A :: VivaFix 353-442-A

105

B
Dead cells

800

Count

600

Live cells

400

200

0
10 0

101

102
103
104
FL24-A :: VivaFix 353-442-A

105

Measuring live vs. dead cells using the VivaFix™ Cell Viability Assay. A, comparison of cells treated with TNF-a only ( ) and cells treated with TNF-aand the anti-TNF-a antibody adalimumab purified from lysate ( ). B, comparison of cells treated with TNF-a only ( ) and cells treated with TNF-a andcontrol anti-TNF-a antibody (purchased control adalimumab) ( ). Both adalimumab biosimilars show a protective effect on cells after treatment with
TNF-a. To carry out these experiments, murine L929 cells were plated on growth media (MEM, 2 mM glutamine, 10% FBS) at 37°C, 5% CO2. Anti-TNF-aantibody (either control adalimumab or purified adalimumab) was prepared in serum-free media and added to cells at a final concentration of 300 ng/ml.
Following a 2 hour incubation with the antibody, TNF-a was added at a final concentration of 5 ng/ml and the cells were further incubated for 36 hours
at 37°C, 5% CO2. VivaFix Viability Assays fluoresce more brightly in dead cells than in live cells. In live cells they bind only to cell surface primary amines.
In dead cells the compromised cell membrane allows additional binding to intracellular primary amines. Cells were stained using VivaFix Viability Assays
according to manufacturer’s instructions. Treated cells were washed with PBS, treated with trypsin, neutralized, and cell suspensions were added tomicrocentrifuge tubes. Cells were counted, spun at 300 rcf, and then resuspended in PBS. VivaFix 353/442 Viability Assay was reconstituted in DMSO
and added to each treated sample. One sample was left untreated and unstained for instrument setup. Samples were incubated at RT in the dark before
washing in 1 ml PBS. Samples were resuspended in PBS at 2 x 106 cells per ml. Cell viability was assessed using Bio-Rad VivaFix 353/442 Cell Viability
Assay and the ZE5 Cell Analyzer. VivaFix Dye was excited with the 355 nm laser and detected in the 447/60 nm channel. Data were analyzed using FlowJo
10 Software. 5 ng/ml TNF-a ( ); 5 ng/ml TNF-a + 300 ng/ml purified adalimumab ( ); 5 ng/ml TNF-a + 300 ng/ml control adalimumab ( ).
BIO-RAD LABORATORIES, INC. | 9

CHARACTERIZATIONCUSTOMIZED AND SPECIFIC

HuCAL® ANTIBODY
GENERATION TECHNOLOGYBIOANALYTICALANTIBODIESAs part of the drug development program, the biosimilar is compared to thereference product to ensure that there are no clinically meaningful differencesin terms of safety, efficacy, and immunogenicity, including the titre and reactivityof anti-drug antibodies (ADA) produced in vivo. The results form part of thetotality of evidence required by regulatory agencies. These data must be robust;
therefore the reagents used must meet high standards.
Bio-Rad’s antibiotherapeutic antibody reagents for ADA and PK assay
development are selected in vitro from the synthetic Human Combinatorial
Antibody Libraries (HuCAL). Guided selection strategies enable the generation of
fully human, highly specific inhibitory and noninhibitory anti-idiotypic antibodies
and specialized drug-target complex binders. These different types of antibodies
enable development of PK assays to detect free or total drug, or drug bound to its
target. Additionally, the inhibitory antibodies can be used as reference standards in
an ADA assay, replacing the need to source antibodies raised in animals.
Bio-Rad provides an expanding portfolio of antibodies against marketed
monoclonal antibody drugs and a custom service for additional specificities.
Our QC process incorporates several relevant published best practices, helping
you with critical reagent life cycle management and characterization and themaintenance of optimal assay performance over time.
Here we designed a series of ELISA experiments to show the precision of ourantibodies in PK and ADA assays.

SUCCESS STORY Bio-Rad’s Characterization of Critical Reagents for Ligand Binding
Assays article provides recommendations and best practices used by bioanalyticallaboratories in the development of ligand binding assays for the successful
characterization of critical reagents in drug development and manufacturing.
Visit bio-rad.com/biosimilar1 to learn more.

10 | BIO-RAD LABORATORIES, INC.

CHARACTERIZATIONPK AND ADA BIOANALYSIS FOR BIOSIMILAR DEVELOPMENTA.

Drug Target
Anti-IDFab Antibody

Drug

TYPE 1

TYPE 2TYPE 3

Anti-idiotypic (Anti-ID) antibody■■

Anti-idiotypic antibody■■

Drug-target complex specific■■Paratope-specific■■Paratope-specific■■Not inhibitory■■

Inhibitory■■

Not inhibitory■■

Detects bound drug exclusively■■

Neutralizing■■■■

Detects free drug■■ etects total drug (free, partiallyDbound, fully boundB. Capture ELISA

C. Bridging ELISA
100,000

Fluorescent signal

Fluorescent signal

100,000

10,000

1,000

100

10,000

1,000

100
0.1

1
10
100
1,000
Adalimumab, ng/ml in 10% human serum

10,000

0.1

1
10
100
1,000
Adalimumab, ng/ml in 10% human serum

10,000Multiple PK assay orientations for the biosimilar adalimumab using highly specific anti-idiotypic antibodies. A, three main types of antibody bioassay
orientation are shown. B, data from PK Antigen Capture ELISA. A 96-well plate was coated with 100 µl of 5 µg/ml TNF-a overnight at 4°C and washed five times
with PBST. Each well was blocked with 300 μl 5% BSA in PBST for 1 hour at room temperature (RT). The plate was washed five times with PBST and 100 μl ofeach sample was added in triplicate. The plate was incubated for 1 hour at RT followed by washing with PBST. Anti-adalimumab conjugated with HRP (catalog#HCA232P; clone AbD20349_hlgG1) was diluted to 2 μg/ml in HISPEC buffer (catalog #BUF049) and 100 µl of the detection antibody was added to each well.
The plate was incubated at RT for 1 hour before being washed with PBST. One hundred microliters of QuantaBlu (Thermo Fisher Scientific) was added to each welland the fluorescence was measured after 30 minutes. The concentration of purified adalimumab was calculated from a standard curve of adalimumab preparedin 10% human serum in PBST. C, data from PK bridging ELISA. The anti-adalimumab capture antibody (catalog #HCA202; clone AbD18654) was diluted to 1 μg/ml
in PBS. One hundred microliters of the antibody was added to each well of a 96-well plate and incubated overnight at 4°C. The plate was washed with PBST
followed by blocking with 300 µl 5% BSA in PBST. The plate was incubated at RT for 1 hour before washing with PBST. One hundred microliters of each sample
were added in triplicate and incubated for 1 hour at RT. The plate was washed with PBST. The HRP conjugated detection antibody (catalog #HCA204P; clone
AbD18655_hIgG1) was diluted to 2 μg/ml in HISPEC buffer and 100 μl of the antibody was added to each well. The plate was incubated at RT 1 hour and thenwashed with PBST. QuantaBlu (100 µl) was added to each well and fluorescence was measured after 30 minutes. The concentration of purified adalimumab was
calculated from a standard curve of adalimumab prepared in 10% human serum in PBST. purified adalimumab ( ); adalimumab control ( ).
BIO-RAD LABORATORIES, INC. | 11

WHY BIO-RAD?
Bio-Rad has over 60 years of experience supporting life science
research 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 stand-alone
solutions or combined into workflows that are optimized to minimize
downtime and to provide the right answer the first time, every time.
The Biosimilar Workflow is only one of many Bio-Rad workflows

designed to shorten discovery and development time lines
for therapeutics.
As part of our commitment to your research, 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
Purification

Impurities

Comparability

Characterization

S3e Cell Sorter••ZOE Fluorescent Cell Imager••

ZE5 Cell Analyzer••

TC20™ Automated Cell Counter••

Antibodies for Flow Cytometry and Immunohistochemistry

•

•

•

•

Cells

Genes
QX200 Droplet Digital PCR System•

C1000 Touch™ Thermal Cycler•

PrimePCR™ ddPCR Assays Residual DNA••

PrimePCR ddPCR Assays••

CFX384 Touch™ Real-Time PCR Detection System••

PrimePCR Assays••

qPCR Reagents and Consumables

•

•

•

•

Proteins
Bio-Plex® Multiplex Systems and Immunoassays
NGC Chromatography Systems•

Chromatography Resins•

Stain-Free Mini and Midi Protein Gels

•

•

•

Trans-Blot Turbo Transfer System

•

•

•

ChemiDoc MP Touch Imaging System

•

•

•

PrecisionAb™ Validated Western Blotting Antibodies

•

•

•

•

•

•

•

ReadyPrep™ 2-D Cleanup Kit•

ReadyPrep 2-D Starter Kit•

PROTEAN® i12™ IEF Systems and Consumables•

Criterion™ Dodeca™ Cell•

Criterion™ TGX™ Precast Gels•

SYPRO Ruby Protein Gel Stain•

HuCAL Generated Custom Antibodies•

Anti-idiotypic Antibodies

Visit bio-rad.com/biosimilarinfo to learn more about our solutions to support your biosimilar research and development.
HUMIRA is a trademark of AbbVie Biotechnology Ltd. QuantaBlu, Ruby, and SYPRO are trademarks of Thermo Fisher
Scientific. EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents
containing EvaGreen Dye for use in real-time PCR, for research purposes only. FlowJo is a trademark of FlowJo, LLC.
TGX Stain-Free Precast Gels are covered by U.S. Patent Numbers 7,569,130 and 8,007,646. Bio-Rad Laboratories, Inc.
is licensed by Life Technologies Corporation to sell SYPRO products for research use only under U.S. Patent Number
5,616,502.
Bio-Rad’s thermal cyclers and real-time thermal cyclers are covered by one or more of the following U.S. patents ortheir foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.

Precision Plus Protein Standards are sold under license from Life Technologies Corporation, Carlsbad, CA for use onlyby the buyer of the product. The buyer is not authorized to sell or resell this product or its components.
The QX200 Droplet Digital PCR System is covered by claims of U.S. patents, and/or pending U.S. and non-U.S. patentapplications owned by or under license to Bio-Rad Laboratories, Inc. Purchase of the product includes a limited,
non-transferable right under 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.
The Bio-Plex Suspension Array System includes fluorescently labeled microspheres and instrumentation licensed to
Bio-Rad Laboratories, Inc. by the Luminex Corporation.

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