Improved Process Economics of HUMIRA Biosimilar
Purification with Ion Exchange and Mixed-Mode Resins
Randy Drevland, Chelsea Pratt, Payal Khandelwal, Laura Moriarty, and Elizabeth Jordan Dreskin
Bio-Rad Laboratories, Inc., 6000 Alfred Noble Drive, Hercules, CA 94547

Biosimilar Purification

Bulletin 7130

Abstract

The global biosimilars market is exploding and is expected to reach US$10.9 billion by 2021.
Biosimilars are significantly cheaper than biologics, owing to their shorter development timeand fewer involved costs. However, further reduction in their development costs is requiredfor broader affordability. Biosimilar purification costs constitute more than half of the total
development costs. A significant number of these costs are due to the expense of Protein
A–based media. Here, we propose an alternate but equally effective approach to biosimilar
purification, by substituting a less expensive ion exchange resin for the affinity capture step. Withan adalimumab (HUMIRA) biosimilar, we show that using a Nuvia S Cation Exchange Resin can
not only help minimize purification costs, but also overcome the limitations of Protein A capture,including low binding capacity and Protein A leaching. In addition, using the IEX-based workflow
in conjunction with a mixed-mode resin polish step can help achieve greater than 99% purityof the biosimilar, which is comparable to a Protein A–based purification. Thus, an IEX-based
process works as a great alternative to traditional biosimilar purifications and leads to improved
process economics, which is an underlying requirement of a biosimilar.
Introduction
The WHO defines biosimilars as “a biotherapeutic productwhich is similar in terms of quality, safety and efficacy to an
already licensed reference biotherapeutic product” (World
Health Organization 2009). The goal of developing a biosimilar
is to have a cheaper but equally efficacious alternative tothe already licensed reference biotherapeutic product, ororiginator biologic, since biologics development involves, on
average, over a decade of time investment and approximately
US$2.6 billion (DiMasi et al. 2016). Biosimilars show promise
as an alternative to originator biologics with cost-saving
predictions reaching US$250 billion in the next ten years(Jacoby et al. 2015). These savings, at least in part, are due tothe fact that the development of a biosimilar takes 7–8 years,
at a cost between US$100 and 250 million (Blackstone andJoseph 2013).
It is noteworthy that over 50% of the total costs requiredfor biosimilar development come from the preclinical phase(Gutierrez 2015). A significant part of the preclinical phase

comprises purifying an active biomolecule and downstream
bioanalytical characterization steps. Protein A purification
platforms are often used for these purification steps. However,there are multiple limitations to using Protein A affinity resins.
They are expensive and have low dynamic binding capacities(DBC), which results in a limited amount of purified sample perunit of resin. Some of the newer Protein A resins with higher
DBCs lead to increased protein aggregation, which also leads
to decreased productivity. Leaching of Protein A from resins
often further complicates the purification process, requiring
additional steps to remove leached material and additional QC
assays to test that the leached material has been removed.
In complete contrast, ion exchange resins are cheap, havehigh DBCs, can be used for more cycles, and can be cleaned
and stored with more aggressive solutions. They therefore
offer a potential for cost saving, provided the end product canbe shown to be similar to the originator.

Improved Process Economics of HUMIRA Biosimilar Purification with Ion Exchange and Mixed-Mode Resins(HCl) and applied to the pre-equilibrated Nuvia S Column at aflow rate of ~120 cm/hr. The column was washed with 5 CV of
buffer A followed by 5 CV of 25 mM sodium acetate (pH 4.75),
75 mM NaCl. The bound biosimilar was eluted with 10 CV of
25 mM sodium acetate (pH 5.0), 250 mM NaCl (buffer B).

In this study, we performed parallel capture of a HUMIRA
biosimilar with a Protein A affinity resin and a next-generation
ultra-high capacity cation exchange (CEX) resin, Nuvia S. The
purification workflow (Figure 1) included the anion exchanger
Nuvia Q for the intermediate polish step and the mixed-mode
media Nuvia cPrime for the final polish purification. Our results
demonstrate that the IEX-based workflow resulted in greaterthan 99% purity of the biosimilar fraction, which is equal tothat expected from the Protein A affinity resin workflow. In
addition, the biosimilar purified from the IEX workflow retained
its structure and function. We also show the comparative
cost analysis of both workflows to demonstrate the improved
process economics of using the IEX-based workflow.

Capture by Protein A Affinity Resin

MabSelect SuRe Protein A Resin (GE Healthcare,#17-5438-01) was packed in a 1 ml column according to the
manufacturer’s instructions and the biosimilar was loaded onto
it. The bound biosimilar was eluted with 10 CV of 100 mM
sodium acetate (pH 3.5), 100 mM NaCl.
Intermediate Polishing by Nuvia Q AEX Resin

The eluate from the capture step was concentrated and buffer
exchanged into 25 mM HEPES (pH 8.25), 5 mM NaCl (bufferC) using an Amicon Ultra-4 Centrifugal Filter Unit (50 kD MW
cutoff) (EMD Millipore, #UFC805024). This sample was applied
on a 1 ml Foresight Nuvia Q Column (Bio-Rad, #732-4721)
equilibrated with 10 CV of buffer C. The flow-through fractions
were collected for further purification.

CHO Cell SupernatantOverexpressing Adalimumab
Sample conditioning
Capture Purification
Protein A Affinity Resinvs.

Capture Purification
Nuvia S
Strong Cation Exchange Resin

Final Polishing by Nuvia cPrime Mixed-Mode Media

Sample conditioning

The pooled flow-through fractions from the previous step wereconcentrated, buffer exchanged with buffer B, and applied toa Foresight Nuvia cPrime Column (Bio-Rad, #732-4722) preequilibrated with 10 CV of the same buffer. The biosimilar was
eluted with 5 CV of 25 mM Tris (pH 8.0), 250 mM NaCl. The
column was then stripped with 10 CV of 25 mM Tris (pH 8.0),
800 mM NaCl to remove all bound impurities.

Intermediate Polish
Purification
Nuvia Q
Strong Anion Exchange Resin
Sample conditioning
Final Polish Purification
Nuvia cPrime
Mixed-Mode Resin

Adalimumab Activity Assays
Pharmacokinetic ELISA Antigen Capture Format

A 96-well black flat-bottom plate was coated with 100 µl of
5 µg/ml TNFα, incubated overnight at 4°C, and then washedfive times with phosphate buffered saline with Tween-20(PBST). Each well was blocked with 300 μl 5% BSA in PBST
for one hour at room temperature. The plate was then washedfive times with PBST, and 100 μl of each sample was addedin triplicate. The plate was incubated for one hour at room
temperature and then washed five times with PBST. Antiadalimumab conjugated with HRP (Bio-Rad, #HCA232P) was
diluted to 2 μg/ml in HISPEC buffer (Bio-Rad, #BUF049) and100 µl of the detection antibody was added to each well. The
plate was incubated for one hour at room temperature andthen washed ten times with PBST. QuantaBlu Fluorogenic
Peroxidase Substrate (100 μl) (Thermo Fisher Scientific,#15169) was added to each well and the fluorescence wasmeasured after 30 minutes. The concentration of purifiedadalimumab was calculated from a standard curve ofadalimumab prepared in 10% human serum in PBST.

Fig. 1. Adalimumab biosimilar purification workflow.

Materials and Methods
GeneralAn adalimumab biosimilar produced in Chinese hamster ovary(CHO) cells was purchased (Syd Labs, catalog #C003P).
Protein fractions were analyzed by SDS-PAGE using Criterion
Tris-HCl 8–16% Linear Gradient Gels (Bio-Rad Laboratories,#3450037) stained with Bio-Safe Coomassie Stain (Bio-Rad,#1610786). HCP clearance was determined using the CHOHCP ELISA Kit (Cygnus Technologies, #F550) and host cellDNA (hcDNA) was quantified using the ddPCR CHO Residual
DNA Quantification Kit (Bio-Rad, #17000031). Preliminarychromatography experiments to compare both the workflows
were conducted in 96-well plates (Pall Corporation, #8119).
Larger scale purifications were conducted using 1 ml columns
filled with the appropriate resins.
Capture by Nuvia S CEX Resin

Chromatographic purification was conducted using the NGC
Chromatography System (Bio-Rad, #7880001). A 1 ml ForesightNuvia S Column (Bio-Rad, #732-4720) was equilibrated with10 column volumes (CV) of 25 mM sodium acetate (pH 4.5),5 mM NaCl (buffer A). The CHO cell supernatant containing thebiosimilar was adjusted to pH 4.5 with 1 M hydrochloric acid© 2018 Bio-Rad Laboratories, Inc.

2

Bulletin 7130

Improved Process Economics of HUMIRA Biosimilar Purification with Ion Exchange and Mixed-Mode Resins

Results and Discussion

Pharmacokinetic Bridging ELISA

An anti-adalimumab capture antibody (Bio-Rad, #HCA202;
AbD18654) was diluted to 1 μg/ml in PBS, and 100 μl of theantibody was added to each well of a 96-well black
flat-bottom plate. The plate was incubated overnight at 4°C
and then washed five times with PBST. Each well was thenblocked with 300 μl 5% BSA in PBST. The plate was incubatedfor one hour at room temperature and then washed five times
with PBST. Each sample (100 μl) was added in triplicate andincubated for one hour at room temperature. The plate wasthen washed five times with PBST. The HRP-conjugateddetection antibody HCA204P (Bio-Rad, #AbD18655_hIgG1)
was diluted to 2 μg/ml in HISPEC buffer and 100 μl of theantibody was added to each well. The plate was incubatedfor one hour at room temperature and then washed ten times
with PBST. QuantaBlu Substrate (100 μl) was added to eachwell and the fluorescence measured after 30 minutes. The
concentration of purified adalimumab was calculated from astandard curve of adalimumab prepared in 10% human serum
in PBST.

Plate-Based Comparison of Capture with Protein A vs. Nuvia S Resin

Biological Assay to Assess Adalimumab Blocking TNFa

Initial proof of concept studies with parallel scalable
purification processes using Protein A affinity or CEX capture
purification, performed in 96-well plates, showed better
removal of the host cell proteins (HCPs) in a single stepwith Protein A relative to Nuvia S (Table 1). However, theIEX process produced material that was equally pure after
the polish step. This small-scale study partly supports our
hypothesis of improving process economics by replacing
Protein A resin with an IEX media. This improvement comes
not only in terms of the actual difference in resin price, but also
the far greater DBC of the Nuvia S Resin (Table 2).
Table 1. Comparison of HCP clearance on 96-well filter plates
between Nuvia S Resin and a Protein A resin.

Murine L929 cells were plated in growth media (MEM, 2 mM
glutamine, 10% FBS) and kept at 37°C in 5% CO2. Anti-TNFα
antibody (either control adalimumab or adalimumab preparedfrom lysate) was prepared in serum-free media and added
to cells at a final concentration of 300 ng/ml. Following a twohour incubation with the antibodies, TNFα was added at a final
concentration of 5 ng/ml, and the cells were incubated for afurther 36 hours at 37°C in 5% CO2.

HCP, ppm
Nuvia S Capture

HCP, ppm
Protein A Capture

CHO cell supernatant

72,00090,300Capture purification

6,800

980

Nuvia Q intermediate polishpurification

250

20

Nuvia cPrime final polishpurification

5

20

Table 2. Comparison of cost and binding capacity between Nuvia S
Resin and a Protein A resin.
Nuvia S Resin

VivaFix Cell Viability Staining

MabSelect SuRe
Protein A Resin

Technique

Strong cationexchange
chromatography

Affinity
chromatography

Binding capacity≥110 mg/ml~35 mg/ml

Cost (25 ml)*$110

$2,791* Pricing mentioned is valid for 2018.

VivaFix Cell Viability Dyes fluoresce more brightly in deadcells than live ones. In live cells, they bind only to cell surface
primary amines. In dead cells, the compromised membraneallows additional binding to intracellular primary amines.
Staining with VivaFix Dyes was performed according to the
manufacturer’s instructions. Briefly, TNFα-treated cells were
washed with PBS, trypsinized, and neutralized, and cellsuspensions were added to microcentrifuge tubes. The cells
were counted and centrifuged at 300 RCF. The supernatant
was removed and the cells were resuspended in PBS.
VivaFix 353/442 (Bio-Rad, #1351111) was reconstituted withDMSO and added to each treated sample. One sample wasleft untreated and unstained to help with instrument setup.
Samples were incubated for 30 min at room temperaturein the dark before washing in 1 ml PBS. Samples were
resuspended in PBS to a concentration of 2 x 106 cells/ml.
Cell viability was detected using the ZE5 Cell Analyzer(Bio-Rad, #12004279) using the 355 nm laser in the 447/60
channel. Data were analyzed using FlowJo 10 Software.

© 2018 Bio-Rad Laboratories, Inc.

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Comparable Purity of Biosimilar Antibody Purified with IEX vs.
Protein A Resins

100
80
60
40
20
0

2,000
1,600
1,200
800
400
0
0

10

20
30
Column volume = 1.01 ml

λ 3 (280 nm), mAU

B

5

10

C
λ 3 (280 nm), mAU

50

1,000
800
600
400
200
0
0

Nuvia cPrime Mixed-Mode Resin was used for final polishpurification. It is a hydrophobic cation exchange resin with afunctional ligand that can interact with biomolecules not only
by electrostatic and hydrophobic interactions, but also by
simultaneous hydrogen bonding (bulletin 6242). Nuvia cPrime
is tolerant to high salt concentrations in the feedstream due toits hydrophobicity, allowing the loading of the partially purified
flow-through sample from the Nuvia Q Column after only one
buffer exchange to decrease the pH. The bound biosimilar
was eluted by a buffer with slightly higher pH. Both the binding
and elution conditions used in this chromatographic step
were gentle, which is crucial for maintaining the integrity of theantibody. At the same time, the purification was effective inthat a highly pure adalimumab biosimilar was obtained (Figure2C). Good separation of the monomeric biosimilar antibody
from aggregates and other impurities was seen. The impurities
were eluted using a buffer with high conductivity. Samples
from each step of purification were run on an SDS-PAGE gel
to confirm the increase in purity (Figure 3).

40

15
20
25
Column volume = 1.01 ml

30

10

20

30
40
Column volume = 1.01 ml

50

100
80
60
40
20
0

35

1,000
800
600
400
200
0
0

100
80
60
40
20
0

Conductivity, mS/cm

The plate-based IEX capture was repeated in 1 ml columns toestimate the levels of purity achieved at a larger scale.
Nuvia S is a high-capacity CEX resin (bulletin 5987). The hcDNA
contaminants present in the CHO cell extract do not bind to
the resin, appearing instead in the flowthrough as shown by the
255 nm absorbance trace (Figure 2A) resulting in a reductionof ~99% hcDNA and ~94% HCP (Table 3). The hcDNA wascleared to below detection level by the intermediate polishpurification with Nuvia Q Resin (bulletin 6129). The primary goalof this purification step was to maximize the recovery of thetarget biosimilar in the flow-through fraction while keeping thehighest amount of contaminants bound to the Nuvia Q Column(Figure 2B). We simultaneously saw a decrease of over 99% in
the HCP content.

Conductivity, mS/cm

λ 3 (280 nm), mAU

A

60

Conductivity, mS/cm

Improved Process Economics of HUMIRA Biosimilar Purification with Ion Exchange and Mixed-Mode Resins

Fig. 2. Chromatograms from purification of the biosimilar. A, capture
purification with Nuvia S Resin. The biosimilar obtained from fractions 36 to
44 was subjected to further purification and purity analysis. B, intermediate
polish purification with Nuvia Q Resin. The column flow-through fractions from
1 to 12 were subjected to further purification and purity analysis. C, final polishpurification on Nuvia cPrime Resin. The biosimilar eluted from column fractions
33–37 was collected and subjected to purity analysis. λ 2 (255 nm) ( );λ 3 (280 nm) ( ); conductivity ( ); pH ( ).
Table 3. HCP and hcDNA clearance with 1 ml Foresight Columns.
Overall
hcDNA, Overall
HCP, ppm Reduction, % pg/µl
Reduction, %SampleCHO supernatant (crude) 170,000–

26,000–Nuvia S Cation Exchange 10,300(capture)

93.91

300

98.86

Nuvia Q Anion Exchange 120(intermediate polish)

99.93

0

100

Nuvia cPrime
Mixed-Mode (final polish)

6

99.99

–

–

1

4

2

3

5

kD
250
150
100
75
50
37
25

Fig. 3. SDS-PAGE analysis of the chromatographic fractions following
purification on Foresight Columns. Lane 1, Precision Plus Protein UnstainedProtein Standard; lane 2, CHO supernatant; lane 3, Nuvia S sample; lane 4,
Nuvia Q sample; lane 5, Nuvia cPrime sample.

© 2018 Bio-Rad Laboratories, Inc.

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Improved Process Economics of HUMIRA Biosimilar Purification with Ion Exchange and Mixed-Mode Resins

C

Functional Testing of Purified Biosimilar Binding to the TNFα Ligand Using
ELISA and Cell-Based Assays

The integrity of the purified biosimilar antibody was tested
using in vitro ELISA and cell-based assays. Part of theextensive characterization that is required by regulatory
bodies in filings for biosimilar submissions is to determine theactivity of the biosimilar molecule. An in vitro antigen capture
ELISA assay showed comparable binding of both the purifiedadalimumab and the control adalimumab to the TNFα antigen(Figure 4A). The bridging ELISA showed the same trend ofbinding of both types of adalimumab to an anti-adalimumab
antibody (Figure 4B), thereby confirming that the purified
HUMIRA biosimilar shows comparable binding patterns to thecontrol antibody. Cell-based assays measuring the ability ofthe biosimilar to protect cells after the addition of TNFα werecarried out. The cell viability seen with the purified adalimumab
after addition of TNFα was similar to the one achieved withthe control (Figures 4C and 4D), further confirming that the
purification protocol did not compromise the functionalityof adalimumab.

800

600

400

200

0
10 0

101

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

105

D

A
100,000

Dead cells

800

600

10,000Count

Fluorescent signal

Dead cells

Live cells

1,000Live cells

400

200
100
0.1

1

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

10,000B

0100,000

10 0

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

105

Fig. 4. Functional testing of purified adalimumab. A, pharmacokinetic
antigen capture ELISA; comparison of capture of TNFα by the adalimumab
standard ( ) and the purified adalimumab ( ) in the 96-well plate. B,
pharmacokinetic bridging ELISA; comparison of anti-adalimumab binding by theadalimumab standard ( ) and the purified adalimumab ( ) in the 96-well plate.
Both adalimumabs show similar binding trends to TNFα and the anti-adalimumab
antibody. C, comparison of cells treated with TNFα only ( ) and cells treatedwith TNFα plus adalimumab purified from lysate (PFL) ( ). D, comparison of cells
treated with TNFα only ( ) vs. cells treated with TNFα and control anti-TNFα
antibody (purchased purified adalimumab) ( ). Both adalimumab biosimilars
show a protective effect on the cells after treatment with TNFα.

10,000
Fluorescent signal

101

1,000

100

0.1

1

10

100

1,000

10,000Adalimumab, ng/ml in 10% human serum© 2018 Bio-Rad Laboratories, Inc.

5

Bulletin 7130

Improved Process Economics Achieved with the IEX-Based Scalable Process

As shown in Figure 5, we decreased the purification costs of
the adalimumab biosimilar using a Nuvia S–based purification
workflow vs. a Protein A–based one. Even with the cost ofan added conditioning step, the Nuvia S workflow improves
process economics by reducing resin costs by multiple fold.
Numerous further improvements to the process economicsof this purification can be made by optimizing the amountof buffer utilized, minimizing feed conditioning, and enabling
direct loading between steps.
100 cycles use
Nuvia S: $110/25 ml
MabSelect SuRe: $2,791/25 ml

1 g mAb

UF/DF conditioning = $50

Nuvia S

Protein A

Cost of resin for
1 g mAb = $40
Cost of buffer for
100 cycles = $12.50
Based on 25 ml
Nuvia S Resinand 110 mg/ml DBC

Cost of resin for
1 g mAb = $3,126
Cost of buffer for
100 cycles = $37.50
Based on 25 ml
MabSelect SuRe Resinand 35 mg/ml DBC

Nuvia Q
Cost of resin = $40
Based on 25 ml
Nuvia Q Resin and 170 mg/ml DBC

Cost ofthree-step mAb
purification with
Nuvia S
$303

Cost oftwo-step mAb
purification withProtein A$3,204

Nuvia cPrime
Cost of resin = $160
Based on 25 ml Nuvia
cPrime Resin and
40 mg/ml DBC

Fig. 5. Cost cutting by replacing the Protein A resin with Nuvia S Resinto improve process economics during biosimilar purification. Pricing
mentioned is valid for 2018.

Conclusions
In summary, we show that an IEX and mixed-mode
resin−based workflow can deliver a highly pure and active
adalimumab biosimilar, equal to that achieved using apurification workflow involving Protein A. The two IEX resins
used, Nuvia S and Nuvia Q, purify the target biosimilar
efficiently, removing product-related impurities. Use of the
Nuvia S cation exchanger for capture led to better processeconomics, not only by reducing cost but also by enhancing
the process speed, a result of the significantly higher binding
capacity of the resin. This alternative approach overcomesthe issue of purifying any leached Protein A and the need for
subsequent bioanalytical assays to monitor for this processimpurity. Nuvia S Resin is chemically and mechanically stable
and can be used across 100+ purification cycles. In the final
purification step, the multiple interaction modes offered by
Nuvia cPrime ensured the removal of both product- andprocess-related impurities. Since all three resins could be
operated at high flow rates with low backpressure, thisincreased the productivity and thus improved the processeconomics of the workflow. Therefore, an IEX-based
purification platform in general, and Nuvia Resin−based
purification platforms specifically, have the potential tocontribute to faster (to market) and cheaper biosimilars.
References
Blackstone EA and Joseph PF, Jr (2013). The economics of biosimilars. Am
Health Drug Benefits 6, 469–478.
DiMasi JA et al. (2016). Innovation in the pharmaceutical industry: New
estimates of R&D costs. J Health Econ 47, 20–33.
Gutierrez BJ (2015). Financial analysis of biosimilar development candidates:
A case study on the US biosimilar business. dash.harvard.edu/bitstream/
handle/1/24078352/GUTIERREZ-THESIS-2015.pdf, accessed
September 10, 2018.
Jacoby R et al. (2015). Winning with biosimilars. Opportunities in global
markets. deloitte.com/content/dam/Deloitte/us/Documents/life-scienceshealth-care/us-lshc-biosimilars-whitepaper-final.pdf, accessed
September 10, 2018.
World Health Organization (2009). Expert committee on biologicalstandardization. Guidelines on evaluation of similar biotherapeutic products(SBPs). who.int/biologicals/areas/biological_therapeutics/BIOTHERAPEUTICS_
FOR_WEB_22APRIL2010.pdf, accessed September 10, 2018.
Bio-Rad and ddPCR are trademarks of Bio-Rad Laboratories, Inc in certain
jurisdictions. Precision Plus Protein Standards are sold under license from
Life Technologies Corporation, Carlsbad, CA for use only by the buyer ofthe product. The buyer is not authorized to sell or resell this product or
its components. All trademarks used herein are the property of theirrespective owner.

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