Process Chromatography

Purification of a Recombinant Monoclonal Antibody —a Rituximab Biosimilar — with a Non–Affinity Based
Chromatographic Process
Anton Posch,1 Franziska Kollmann,1 Chelsea Pratt,2 Laura Moriarty,2 and Elizabeth Dreskin2
1
Bio-Rad Laboratories GmbH, Kapellenstraße 12, 85622 Feldkirchen, Germany
2
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Abstract

While Protein A resins are capable of producing high selectivity and purity,
they also have the disadvantage of high cost and the need to use a low pH for
elution, which can cause aggregation and limit resin lifetime. To address theseION
MIXED-MODE
issues, we evaluated the purification of a recombinant monoclonal antibody
EXCHANGE(mAb), a rituximab biosimilar, using two non–Protein A purification schemes.
These were a cation exchange (CEX) resin for capture purification followed by an anion exchange (AEX)
resin for intermediate purification and two mixed-mode media (CHT™ Ceramic Hydroxyapatite XT Media;
hydrophobic cation exchange, Nuvia cPrime Media) for polish purification. Simultaneous purification was
performed with a Protein A process. The CEX–AEX–CHT XT workflow yielded >96% purity and ~86% yieldof rituximab, indicating a more economical process. Host cell protein (HCP) levels were reduced by 4 logs
and host cell DNA (hcDNA) levels by 5 logs after the final purification step. A pharmacokinetic (PK) bridging
assay demonstrated the purified monoclonal antibody behaved in a similar way to a rituximab standard,showing no quantifiable loss of structure or function throughout purification. We therefore propose that a
non–affinity based purification workflow for a rituximab biosimilar provides better process economics andhas the potential to facilitate uncompromised results.
Introduction
Protein A–based chromatographic processes are often used forthe purification of antibodies because Protein A binds selectively
and efficiently to antibodies in complex cell culture supernatants
and removes the majority of product impurities in a single step.
The major disadvantages of chromatographic workflows withProtein A as the capture step include protein aggregation, highcost, poor resin stability, and a long lead time for delivery. An
alternative approach, one that considers process economics,
is the use of non-affinity resins like cation exchange chemistries.
According to Follman and Fahrner (2004), it is feasible to create
three-step purification schemes based on non-affinity resins thatcan compete with three-step chromatographic workflows thatstart with Protein A. The results of this study impressively indicatethat the order of process steps is important for the successful
establishment of a high-performance antibody purification scheme.

Bulletin 7382

Here we have evaluated the performance of well-proven orthogonalresin combinations (Figure 1) for the non-affinity purification of arituximab biosimilar (144 kD), which is a genetically engineered
chimeric murine/human monoclonal antibody directed against
the CD20 antigen found on the surface of normal and malignant
B lymphocytes. A high-capacity cation exchanger (Nuvia S Resin)
was chosen for the initial purification step because of its highcapture performance over a wide range of operating conditions.
Additionally, it is well-known to efficiently remove product-related
contaminants, such as HCPs and monoclonal antibody (mAb)aggregates and fragments. For intermediate purification, the cationexchange step is commonly followed by an anion exchange step,
performed in flow-through mode, for further reduction of remaining
impurities such as hcDNA. In the final polish step, mixed-mode
resins like CHT XT (a ceramic hydroxyapatite) or Nuvia cPrime (ahydrophobic cation exchanger) offer unique selectivity for the finalPurification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Process(PBS) for Protein A chromatography or the pH was adjusted to
6.0 and further diluted 1:1 (v/v) with 1x binding buffer for Nuvia S
chromatography. Buffer exchange of the protein pools between
chromatographic runs was performed with ultrafiltration devices(10 kD cutoff) in a spin column format.

HEK 293 Cell SupernatantOverexpressing rituximab

Capture Purification
UNOsphere SUPrA —Protein A affinity resin

Capture Purification
Nuvia S — strong cationexchange resin

Chromatography

All equipment and resins were from Bio-Rad Laboratories, Inc.
(Table 1). Chromatography was monitored at 280 nm andperformed at room temperature using an NGC 10 MediumPressure Chromatography System. The four-tier chromatographysystem was configured with a sample pump, including sample
inlet valve, two buffer inlet valves, column switching valve,
multi‑wavelength ultraviolet (UV) detector, pH and conductivity
meter, outlet valve, and fraction collector with a benchtop Peltiercooling option. The chromatography operating conditions arepresented in Table 1. The buffers were degassed and filteredthrough 0.45 µm membranes before use. Cell culture supernatant(50 ml) was used for chromatography.

Intermediate Purification
Nuvia Q — strong anion
exchange resin

Final Polish Purification
Nuvia cPrime —mixed-mode resin

Final Polish Purification
CHT XT — mixed-mode resin

Fig. 1. Different processes using three chromatography steps for the
purification of a rituximab biosimilar.

removal of both product- and process-related impurities. In order
to compete against a Protein A–based chromatography workflow,
it was critical to determine the optimal operating conditions
for the resins in key positions, like Nuvia S and Nuvia cPrime.
Operating conditions were determined by using simple screening
experiments in spin columns. To rate the success of the individual
chromatographic steps, important process performanceparameters, such as mAb purity and yield, were measured.
Antibody integrity was evaluated with an anti-idiotypic antibody
pharmacokinetic bridging assay.

Purification Condition Screening for Nuvia S and cPrime Resins in Spin Columns

The chromatographic conditions for Nuvia S Resin were analyzed
by a simple pH screening experiment (Table 2) whereas for
Nuvia cPrime Resin a more complex approach was appropriate(Table 3). The corresponding step-by-step protocol is describedin bulletin 7128. In brief, the spin columns were loaded with
100 µl chromatography resin and equilibrated with the binding
buffers to be tested. Five hundred microliters of cell supernatant(0.25 µg/µl), buffer exchanged into its respective binding buffer,
were added and incubated with the resin at room temperature for10 min with agitation (end-over-end mixer). Unbound material was
removed by centrifugation for 1 min at 1,000 x g. The resin waswashed two times with binding buffer and eluted twice. For yield
estimations, the resin was stripped with 2% SDS solution to detect
any remaining protein material on the resin. The samples collectedfrom the spin column experiments were qualitatively analyzed by
SDS-PAGE under nonreducing conditions.

Materials and Methods
Biological Material and Sample Preparation

HEK 293 cell culture supernatant containing a rituximab biosimilar(Absolute Antibody, Catalog #Ab00126-10.0) was centrifuged
to remove cellular debris and dialyzed (20 kD cutoff membrane)
overnight in the corresponding binding buffer of Protein A or
Nuvia S Resin (Table 1). Protein concentration after dialysis was
about 0.35 mg/ml containing 6–8% of monoclonal antibody.
Alternatively, cell culture supernatant containing the rituximab
biosimilar was diluted 1:1 (v/v) with phosphate buffered saline

Table 1. Overview of all columns and their operating conditions for the chromatographic purification of rituximab biosimilar.
Column Type

Resin Type

Mode of Operation

Workflow Position

Buffer A

Buffer B

Flow Rate, cm/hr

Bio-Scale Mini
UNOsphere
SUPrA (5 ml)

Protein A

Bind and elute

Capture step

10 mM phosphate,
27 mM KCl, 140 mM
NaCl, pH 7.4

100 mM citrate, pH 3.3;
step elution

240–480

Nuvia S (1 ml)

Strong cationexchanger

Bind and elute

Capture step

25 mM MES, 10 mM
NaCl, pH 6.0

Buffer A with 1 M NaCl;
10 column volumes (CV)gradient to 1 M NaCl or
step elution

240

Nuvia Q (1 ml)

Strong anion
exchanger

FlowthroughIntermediate step

50 mM Tris, pH 8.5

Buffer A with 1 M NaCl;
10 CV gradient to
1 M NaCl or step elution

120

Nuvia cPrime (1 ml)

Hydrophobic
cation exchanger(mixed mode)

Bind and elute

Polishing step

50 mM Tris, pH 8.5

Buffer A with 1 M NaCl;
10 CV gradient to
1 M NaCl

240

CHT XT (1 ml)

Ceramic
hydroxyapatite(mixed mode)

Bind and elute

Polishing step

10 mM phosphate,
pH 7.0Buffer A with 2 M NaCl;
20 CV gradient to
2 M NaCl or step elution

240© 2024 Bio-Rad Laboratories, Inc.

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Purification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Process

For Nuvia cPrime Resin, the effects of buffer pH and conductivity
were evaluated by JMP Software to predict optimal binding andelution conditions.

the assay. Droplets were generated using the Automated Droplet
Generator (Bio‑Rad, #1864101) according to instructions. Following
amplification, droplets were read in a QX200 Droplet Reader(Bio‑Rad, #1864003) and analyzed using QuantaSoft™ Software(Bio‑Rad, #1864011).

Table 2. pH screening experiment in spin columns of Nuvia S Resin for the
purification of a rituximab biosimilar. Buffer molarity: 25 mM. Condition #3 wasapplied in chromatography (capture step).
Binding

A pharmacokinetic bridging enzyme-linked immunoabsorbent
assay (ELISA) was carried out according to the provided protocol:
bio-rad-antibodies.com/protocol-pk-bridging-elisa-rituximabantibodies.html.

ElutionCondition

pH

NaCl, mM

pH

NaCl, mM

1

4.5

10

4.5

1,000

2

5.5

10

5.5

1,000

3

6

10

6

1,000

4

6.5

10

6.5

1,000

5

7

10

7

1,000

Electrophoresis

Samples from the purification condition screening in spin columns
and the chromatographic purification steps were monitored for
purity and yield by SDS-PAGE with Criterion TGX Stain-Free
Precast Gels (4–20% midi gels; Bio-Rad, #5678093). HEK 293
cell culture supernatant containing the rituximab biosimilar
was characterized with two-dimensional (2-D) electrophoresis(Posch et al. 2013). In brief, after sample preparation according
to Ambort et al. (2008), 100 µg protein were loaded onto
11 cm ReadyStrip IPG Strips, pH 3–10 (Bio-Rad, #1632014)
and separated according to charge for 40 kVh. For the second
dimension, Any kD Criterion TGX Stain-Free Precast Gels
(Bio-Rad, #5678121) were used. SDS-PAGE gels and 2-D gelswere UV activated after the run for 2.5 and 5 min, respectively.
The corresponding Stain-Free images were recorded with a
ChemiDoc™ MP Imaging System (Bio-Rad, #12003154). After
imaging, the gels were counterstained with Oriole Fluorescent Gel
Stain (Bio-Rad, #1610495), followed by QC Colloidal Coomassie
Stain (Bio-Rad, #1610803). Gel analysis was performed with
Image Lab Software 6.01 and PDQuest 2-D Analysis Softwarefrom Bio-Rad Laboratories.

Analytical Assays

Protein concentrations were measured with the DC Protein Assay
Kit I (Bio-Rad, catalog #5000111).
Antibody aggregate detection was performed using size exclusion
chromatography (SEC) with a prepacked high-resolution ENrich
SEC 650 Size Exclusion Column (10 x 300 mm, 24 ml; Bio-Rad,#7801650) at a flow rate of 1 ml/min on the NGC Chromatography
System. Filtered PBS was used as the equilibration buffer andpeaks were recorded at 215 nm. Two hundred microliters of sample(adjusted for protein amount) were injected into the chromatographysystem and aggregates were defined as the front eluting peakof the SEC profile. Fractions (1 ml) were collected and analyzed
with SDS-PAGE under nonreducing conditions.
HCP levels were analyzed with the HEK 293 HCP ELISA Kit(Cygnus Technologies, #F650R). The lower limit of detectionof this kit is about 1–2 ng/ml.
Levels of hcDNA in chromatographic fractions were measured
with Droplet Digital™ PCR (ddPCR™). Reactions were made using
samples diluted 1:25 to 1:200 in TE Buffer and ddPCR Supermix
for Residual DNA Quantification (Bio-Rad, #1864038). A proprietary
probe assay for a multicopy human gene was used. A standard
curve using BamH1-digested human genomic DNA was generated
to verify that the samples were analyzed in the linear range ofResults and Discussion
Two-Dimensional Electrophoresis of HEK 293 Cell Culture Supernatant Containing
the Rituximab Biosimilar

Two-dimensional electrophoresis is a high-resolution technique forthe characterization of complex protein mixtures. The technique isusually performed under denaturing and reducing conditions, thus
only protein subunits can be displayed on a 2-D gel. Figure 2 shows

Table 3. A wide range custom design for spin column experiments with Nuvia cPrime Resin for the purification of a rituximab biosimilar. Includes data
analysis with SDS-PAGE. Buffer molarity: 50 mM. Condition #9 was applied in chromatography (final polish).
Binding

ElutionCondition

pH

NaCl, mM

Binding Target, %pH

NaCl, mM

Target Yield, %Target Purity, %

1

5.5

0

96

5.5

0

0

N/A

2

5.5

0

99

8.5

0

0

N/A

3

5.5

400

68

5.5

1,000

45

38

4

5.5

400

70

8.5

1,000

42

32

5

7

200

1

7

500

0

N/A

6

7

200

1

7

500

0

N/A

7

7

200

1

7

500

0

N/A

8

8.5

0

99

5.5

1,000

82

65

9

8.5

0

98

8.5

1,000

91

68

10

8.5

400

0

5.5

0

0

N/A

11

8.5

400

0

8.5

0

0

N/A

12

8.5

400

0

8.5

0

0

N/A

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Purification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Processa typical 2-D gel (pH range 3–10) of cell supernatant containing
rituximab, which has a theoretical pI of about 8.6. Light and heavy
chains of rituximab were localized on the 2-D map by comparing
against cell supernatant sample without rituximab (data not shown).
Image analysis was performed with PDQuest Software and the pI
values of the heavy and light chains of rituximab were calculated to
be about 8.9 and 8.5, respectively. These values are consistent withthe theoretical values of 8.7 and 8.4. In general, pI information on
target proteins is helpful when developing chromatographic methods
for the separation of molecules on the basis of their charge, as in ion
exchange chromatography.

Semi-quantitative 2-D gel analysis is also capable of providing
information about the relative abundance of a protein of interest in
a complex sample. Here, rituximab biosimilar expression in the cellsupernatant was exceptionally low (8% of the total protein content)
compared to an industrial mAb-producing cell line. To produce thisantibody at production scale, additional upstream work to increase
expression would have been required.
Purification Condition Screening
Nuvia S Strong Cation Exchange Resin

The purpose of this simple pH screening experiment in spincolumns was to find chromatographic conditions with good bindingof the rituximab biosimilar to the resin and a significant amount of
HCPs in the flow-through fractions. The only variable changed was
the pH of the binding buffer (pH 4.5–7). The protein samples wereanalyzed by nonreducing SDS-PAGE (Figure 3), and qualitative gelanalysis revealed strong binding of rituximab to the resin between
pH 4.5 and about 6.5. At pH 7, binding of the rituximab biosimilar
to the resin became negligible and most of the target protein
could be found in the flow-through sample. The highest amountof contaminating proteins in the flow-through samples was
detected at pH 6 and 6.5. Consequently, all chromatographic runs
with Nuvia S Resin as capture step were performed with 25 mM
MES, 10 mM NaCl, pH 6.0. Working at pH 6.0 also offers morestability in handling higher conductivity conditions of the starting
sample compared to higher pH values.

kD

50

25

pH 3

pH 10

Fig. 2. 2-D electrophoresis of supernatant using a Stain-Free gel. HEK 293
supernatant expressing rituximab biosimilar was analyzed by first separating proteinsaccording to their pI using an 11 cm IPG strip followed by a size separation with
SDS‑PAGE. Heavy and light chains are circled in red. Rituximab biosimilar content
was about 8%.

Nuvia cPrime Resin

Mixed-mode chromatography is becoming an important toolfor downstream process purification, owing to the fact that themedia have a large design space for binding and elution, allowing
for the development of highly robust methods in a commercial
manufacturing setting. However, the behavior of an antibody
during purification by mixed-mode chromatography is often
not predictable due to the joint action of multiple functionalities
present on the ligands. To account for the specific characteristics
of mixed-mode media, including their high salt tolerance, a wide
range custom design was used to elucidate initial binding andelution conditions for the rituximab biosimilar on Nuvia cPrime
Resin. The samples of the spin column experiments were analyzed

The 2-D map shown in Figure 2 provides an excellent overview
of the nature of contaminating proteins. In total, about 550
contaminating protein spots were detected and the majority ofthem were located in the pH range between 3.5 and 8. Rituximab,
in contrast to the majority of contaminating proteins, showed
positive net charge characteristics. Therefore, it is possible tosuccessfully apply cation exchange chromatography as a capture
step in a three-step purification scheme while a significant amountof HCP remains unbound.
kD

S FT

W

E

S

FT W

E

S

FT W

E

S

FT W

E

S

FT W

E

250
R

100
50

Binding25

10
Condition #1

Condition #2

Condition #3

Condition #4

Condition #5

ElutionCondition

pH

NaCl, mM

pH

NaCl, mM

1

4.5

10

4.5

1,000

2

5.5

10

5.5

1,000

3

6

10

6

1,000

4

6.5

10

6.5

1,000

5

7

10

7

1,000

Fig. 3. Screening experiment using pH. Nuvia S Resin in spin columns was used to find chromatographic conditions with good binding of the rituximab biosimilar(R) to the resin and a significant amount of HCPs in the flow-through fraction. Samples were evaluated by SDS-PAGE with Stain-Free technology under nonreducing
conditions. S, HEK 293 supernatant containing rituximab biosimilar; FT, flowthrough; W, wash fraction in binding buffer; E, eluate.

© 2024 Bio-Rad Laboratories, Inc.

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Purification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Process

on nonreducing SDS-PAGE and quantitatively evaluated forbinding capacity and yield (Table 3). Then purity calculations were
performed as well and the obtained datasets were used to predictbinding and elution conditions (Figure 4). The results suggest thatthe rituximab biosimilar employs very different modes of action
with Nuvia cPrime Resin under the conditions explored. Althoughmore extensive design of experiments could be performed toperfect and determine edge of failure of the chromatographic
buffer conditions, buffer condition #9 represents an acceptable
compromise concerning yield and purity of the rituximab biosimilar.

Performance Comparison of Four Different Three-Step Chromatographic Workflows

A purification process composed of Protein A chromatography
as the robust capture step followed by two additional purification
steps is typical. For the subsequent intermediate and polishingsteps, various resin combinations have been described in literature.
Among those resins are, for example, cation exchange, anion
exchange, mixed-mode ion exchange, ceramic hydroxyapatite,and hydrophobic interaction.
Here we were interested in whether the two non–affinity based
processes illustrated in Figure 1 could achieve a similar levelof purification compared to a purification scheme starting withProtein A. In the two non–affinity based workflows, Protein A
was replaced by Nuvia S Resin as the binding step. Nuvia S isan ultra-high capacity, innovative cation exchange resin built on
the industry proven UNOsphere base matrix technology. Nuvia Q
Anion Exchange Resin was applied for intermediate polishingin flow‑through mode across all workflows. Final polishing was
performed with one of two mixed-mode media, either Nuvia cPrimeor CHT XT.

APredicted binding
<0.10

400
Binding concentration of NaCl, mM

<0.20
<0.30
300

<0.40
<0.50
<0.60

200

<0.70
<0.80

Host cell protein levels were measured in all sample pools andserved as the main indicator of target protein purity (Table 4). In
addition to the HEK 293 HCP ELISA, the protein fractions of all
workflows were analyzed by SDS-PAGE with Stain-Free technology
to monitor the success and progress of each purification step
(Figures 5 and 6), detect contaminating proteins, and measure both
purity and yield. The antibody capture step with Protein A affinity
chromatography followed by anion exchange chromatography
removed more than 99% HEK 293 HCPs. Following the final
polishing step, performed with either Nuvia cPrime Resin or CHT XTMedia, the host cell protein level was determined to be below thedetection limit of the HEK 293 HCP ELISA. SDS-PAGE analysis of
the respective sample pools confirmed the high-purity results of the
HEK 293 HCP ELISA, since no distinct contaminating protein bands
were observed in either final sample.

<0.90

100

≥0.90
0
5.0

5.5

6.0

6.5

7.0
7.5
Binding pH

8.0

8.5

9.0

B
1,200

Predicted yield<0.10

Elution concentration of NaCl, mM

1,000

<0.20
<0.30

800

<0.40
<0.50

600

<0.60
≥0.60

400

In both non–affinity based chromatographic workflows,
Nuvia S Resin showed acceptable binding affinity of rituximab
at pH 6, as indicated by the lane comparison of the feedstream
and the flow-through and eluate fractions in Figures 5 and 6.
The combination of Nuvia S and Nuvia Q Resins was able toremove a significant portion of contaminating HEK 293 proteinsand diminished the HCP content to about 450 ppm. For the final
polishing step, the sequence of purification of having Nuvia Q Resinas the intermediate step followed by Nuvia cPrime Resin as
the polish step was attractive because no buffer exchange is

200
0

5.0

5.5

6.0

6.5

7.0
7.5
Elution pH

8.0

8.5

9.0

Fig. 4. Predicted purification conditions for a rituximab biosimilar using
Nuvia cPrime Resin. A, effect of buffer pH and concentration of NaCl on binding
capacity; B, effect of buffer pH and concentration of NaCl on yield.

Table 4. HCP removal by different workflows for rituximab biosimilar purification.
→

CaptureIntermediate

Cell supernatantProtein A eluate pool

Nuvia Q flow-through pool

Steps
Protein A workflow
HCP, ppm
Nuvia S workflow

1,100,000
Cell supernatant© 2024 Bio-Rad Laboratories, Inc.

Polish
Nuvia cPrime eluate poolor

CHT XT eluate pool

200

60

<2

<2

40,000

450

250

20

Nuvia S eluate pool

Nuvia Q flow-through pool

Nuvia cPrime eluate pool

5or

CHT XT eluate pool

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Purification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Process

1

kD

2

3

4

5

6

7

2

3

4

5

6

7

250

100
50

25

Lane

Sample1Marker2

Cell culture feed3

Flow-through pool

4

Protein A/Nuvia S eluate pool

5

Nuvia Q flow-through pool

6

Nuvia Q eluate pool

7

Nuvia cPrime eluate pool

10
Protein A–Nuvia Q–Nuvia cPrime

Nuvia S–Nuvia Q–Nuvia cPrime

Fig. 5. SDS-PAGE analysis of the chromatographic samples from Protein A– or Nuvia S–Nuvia Q–Nuvia cPrime workflow.
kD

1

2

3

4

5

6

7

2

3

4

5

6

7

250

100

50

25

Lane

Sample1Marker2

Cell culture feed3

Flow-through pool

4

Protein A/Nuvia S eluate pool

5

Nuvia Q flow-through pool

6

Nuvia Q eluate pool

7

CHT XT eluate pool

10
Protein A–Nuvia Q–CHT XT

Nuvia S–Nuvia Q–CHT XT

Fig. 6. SDS-PAGE analysis of the chromatographic samples from Protein A– or Nuvia S–Nuvia Q–CHT XT workflow.

necessary between the two chromatographic steps. However,a few distinct protein bands are still visible after Nuvia cPrime
chromatography, yielding a final HCP value of 250 ppm. On theother hand, the final polishing step for rituximab with CHT XTMedia seems to be more effective than Nuvia cPrime Resin, with
HCP levels as low as 20 ppm. Typical chromatograms of theworkflow Nuvia S–Nuvia Q–CHT XT are displayed in Figures 7A–C.
The purity of rituximab was evaluated for the latter workflow by
quantitative SDS‑PAGE image analysis and was in the range of
96–98%. In addition, hcDNA clearance was measured with Droplet
Digital PCR in two chromatographic workflows (Protein A– or Nuvia
S–Nuvia Q–CHT XT) and the corresponding levels were reducedby 5 logs (Table 5).

Qualitative, Quantitative, and Functional Assays
Quantitation of Rituximab with Stain-Free Technology

For yield and recovery calculations of antibody concentrationsin the cell culture feed and in sample pools generated during
chromatography, a Protein A or G high-performance liquid
chromatography (HPLC) assay is usually applied. Column selection
depends on binding affinity of Protein A and G to different humanand mouse IgG subclasses. Those assays have a cycle time of
2–4 minutes and are capable of quantitating mAbs in the rangeof 0.01–5 mg/ml. Here, we have evaluated the applicabilityof SDS-PAGE in combination with Stain-Free technology for
antibody concentration measurements as a low-cost alternative to
Protein A/G HPLC assays. Stain-Free technology employs in-gel
chemistry available in precast SDS-PAGE gels. The gel formulation
incorporates a trihalo compound, which, when exposed to UV
irradiation, catalyzes a covalent reaction to tryptophan residues.
The resulting activated protein fluoresces under UV excitation andcan be readily detected within the gel by suitable imaging systems.

In the course of this project, process performance parameters,such as antibody aggregates and antibody integrity, were measured.
However, chromatographic workflows with Nuvia cPrime Resin were
not considered for further analysis.

© 2024 Bio-Rad Laboratories, Inc.

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Purification of a Recombinant Monoclonal Antibody — a Rituximab Biosimilar — with a Non–Affinity Based Chromatographic Process

Sensitivity and linearity of the Stain-Free approach was evaluatedby running a dilution series of a commercially available rituximab
biosimilar (Protein A purified) under nonreducing conditions. Bandintensities were automatically calculated with the Auto-Analysis toolin Image Lab Software and the corresponding calibration curve,
ranging from 125 to 4,000 ng per lane, is shown in Figure 8. The
graph indicates good linearity between the two sample loading
extremes with a coefficient of determination of 0.99.

5,000

160

4,500

140

4,000

120

λ280, mAU

3,500

100

3,000
2,500

80

2,000

60

1,500

40

1,000

Conductivity, mS/cm

A

20

500

A

0

0
0

20

40

60
80
100
120
Column volume = 1.01 ml

140

160

180

Lane
Number

180

350

160

300

140
120

250

100

200

80

150

60

100

40

50

20

0

0
0

5

10

15
20
25
Column volume = 1.01 ml

30

150

200
150

100

100
50

50

500

5

250

6

125

R2 = 0.99

1.2 x 108
1.0 x 108
8.0 x 107
6.0 x 107
4.0 x 107
2.0 x 107

Conductivity, mS/cm

λ280, mAU

250

1,000

4

1.4 x 108

250
200

2,000

3

1.6 x 108

C

300

4,000

2

B

35

350

1

Relative fluorescence units

400

Conductivity, mS/cm

λ280, mAU

B

Rituximab,
ng/lane

0
0

500

1,000

1,500
2,000
2,500
3,000
Rituximab biosimilar, ng/lane

3,500

4,000

Fig. 8. Electrophoretic Stain-Free assay for the semiquantitative analysis ofa rituximab biosimilar in chromatographic samples. A, SDS-PAGE of rituximab
biosimilar; B, intensity of Stain-Free vs. rituximab biosimilar, ng/lane.

0

0
0

5

10

15
20
25
Column volume = 1.01 ml

30

35

Fig. 7. Chromatograms from the purification of a rituximab biosimilar.
A, capture step with Nuvia S Resin; B, intermediate polish purification with
Nuvia Q Resin; C, final polish purification with CHT XT Media.
Table 5. hcDNA removal by the two major workflows for rituximab biosimilar purification.
SampleProtein A Workflow hcDNA Removal, %Nuvia S Workflow hcDNA Removal, %Capture eluate pool

99.97

75.06

Nuvia Q flow-through pool

100.0099.53

CHT XT eluate pool

100.00100.00

Table 6. Step and process yields calculated for the two major workflows for rituximab biosimilar purification.
Yield, %Chromatographic Process

CaptureIntermediate

PolishOverallProtein A —> Nuvia Q —> CHT XT

97

98

95

90

Nuvia S —> Nuvia Q —> CHT XT

93

98

95

86

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Yields were calculated for all individual chromatography steps
and indicated that the step yield for Protein A (97%) is slightly
higher compared to that for Nuvia S Resin (93%). Nuvia Q Resin(98%) showed almost no protein losses and the step performance
for CHT XT Media was 95%. Thus, we observed an overall yieldof 86% for the non-affinity process while the yield for the control
Protein A process was 90% (Table 6). Possible protein losses
between individual NGC runs due to buffer exchange were notconsidered in those calculations and may reduce overall yield by
5% in each step. Theoretical recovery of those spin columns is
90–95%, according to the technical specifications.

aggregates are often associated with the overproduction of mAbs.
In addition, the strong acidic elution conditions needed for Protein
A chromatography can promote unwanted oligomerization. In thisstudy, possible rituximab aggregates were analyzed in samples
obtained after the capture steps with Protein A or Nuvia S Resinand after the final polishing step with CHT XT Media. Figure 9
provides an overview of the corresponding SEC profiles, whichwere evaluated by quantitative SDS-PAGE analysis. Aggregate
measurement data after Protein A purification (Figure 9A, red arrows)
show 5.5% aggregate levels while data after Nuvia S purification(Figure 9A, blue arrows) show only 1%. However, in both purification
workflows, CHT XT Media reduced the final aggregate content toless than 0.7% (Figure 9B, green arrows, Table 7).

Rituximab Aggregate Analysis With Size Exclusion Chromatography

Aggregate detection and removal is an essential componentof mAb purification (Xu et al. 2012). Elevated levels of antibody
A

B
1,800

1,800

1,600

1,600

1,400

1,400
1,200
λ215, mAU

λ215, mAU

1,200
1,000

1,000

800

800

600

600

400

400

200

200

0

0
8

8

9

9

10

10

11

12
13
Fraction number

SEC fraction
11
12

13

14

14

15

15

16

8

8

kD

9

9

10

10

11

12
13
Fraction number

SEC fraction
11
12

13

14

14

15

16

15

kD

150

150

Fig. 9. Rituximab aggregate analysis with SEC and SDS-PAGE with Stain-Free technology. Arrows indicate samples used to evaluate aggregate levels as shown in
Table 7. A, SEC after Nuvia S (—), SEC after Protein A (—); B, SEC after Nuvia S‑Nuvia Q–CHT XT (—), SEC after Protein A–Nuvia Q–CHT XT (—).

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Table 7. Aggregate percentages for the two major workflows for rituximab biosimilar purification.
Aggregate, %Chromatographic Process

Postcapture

Final

Protein A —> Nuvia Q —> CHT XT

5.5

0.5

Nuvia S —> Nuvia Q —> CHT XT

1

0.7

PK Bridging ELISAAA PK bridging assay can be used for quantitating the therapeuticprotein in biological matrices such as serum. The drug binds to twosites simultaneously, with one binding site to an immobilized antiidiotypic antibody and with the other binding site to a labeled second
anti-idiotypic antibody. Here, the in vitro assay was used as anindicator of the antibody’s integrity/intactness after chromatographic
purification compared to that of a rituximab biosimilar standard.
As can be seen in Figure 10, the two purified mAbs obtained after
Nuvia S–Nuvia Q–CHT and Protein A–Nuvia Q–CHT behaved in asimilar way to the standard antibody, showing no recognizable lossof structure or function during purification.

45,00040,000

Fluorescence signal

35,00030,00025,00020,00015,00010,00010,000
5,000
0
0.1

1

10
Rituximab, ng/ml

100

1,000

0.1

1

10
Rituximab, ng/ml

100

1,000

0.1

1

10
Rituximab, ng/ml

100

1,000

B
45,00040,000

Fluorescence signal

35,00030,00025,00020,00015,00010,00010,0005,000
0C
45,00040,000

Fluorescence signal

35,00030,00025,00020,00015,00010,00010,0005,000
0Fig. 10. PK bridging ELISA of standard and purified rituximab biosimilar.
A, rituximab standard; B, Protein A–Nuvia Q–CHT XT workflow; C, Nuvia S–
Nuvia Q–CHT XT workflow.

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Conclusions
The development of an efficient non-affinity purification process forbiosimilar production is challenging. But, when successful, it confers
benefits such as improved process economics, superior resistanceto sodium hydroxide cleaning, and eliminated ligand leaching. Here,
we show that a workflow combining two ion exchange resins witha mixed-mode resin can deliver similar results to a Protein A–based
workflow in terms of purity, recovery, and viability of rituximab
biosimilar monoclonal antibody. Little process development wasnecessary to establish operating conditions for maximum HCP
protein clearance. A cell culture supernatant with low rituximab
biosimilar titer (and thus an exceptionally high HCP contaminationlevel) was applied in this study.

References

The results of the non-affinity process composed of the combination
Nuvia S–Nuvia Q–CHT XT are very encouraging for applications
where the antibody titer relative to host cell proteins is much more
pronounced. Therefore, due to the reduction in manufacturing costs
they offer, non-affinity processes should be considered for purifying
antibody-mediated therapeutics, specifically biosimilars.

BIO-RAD, CHEMIDOC, CHT, DDPCR, DROPLET DIGITAL, DROPLET DIGITAL
PCR, and QUANTASOFT are trademarks of Bio-Rad Laboratories, Inc. in certain
jurisdictions. All trademarks used herein are the property of their respective owner.
© 2024 Bio-Rad Laboratories, Inc.

Ambort D et al. (2008). Sample preparation of culture medium from Madin-Darby
canine kidney cells. Methods Mol Biol 425, 113–130.
Follman DK and Fahrner RL (2004). Factorial screening of antibody purification
processes using three chromatography steps without protein A. J Chromatogr A 1024,
79–85.
Posch A et al. (2013). 2D-ToGo workflow: Increasing feasibility and reproducibility of
2-dimensional gel electrophoresis. Arch Physiol Biochem 119, 108–113.
Xu Z et al. (2012). Process development for robust removal of aggregates using cationexchange chromatography in monoclonal antibody purification with implementation ofquality by design. Prep Biochem Biotechnol 42, 183–202.

Visit bio-rad.com/Resins for more informationand to request samples.

TGX Stain-Free Precast Gels are covered by U.S. Patent Numbers 7,569,130
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