Application Note

May 1, 2022
Keywords or phrases:
Protein quantitation, titer determination, upstream
development, high precision streptavidin biosensors,
recombinant protein, sax biosensors, GMP, matrix effects

Customized Quantitation of Recombinant
Therapeutic Proteins Using High Precision
Octet® Streptavidin Biosensors (SAX)
Steve Turner 1, Scientist I, Weillei Ma 2, Senior Scientist, Nick Moss 1, Director Instrumentation Technology, John Welsh 1, Senior Manager, R&D andDavid O Apiyo 2, Marketing Applications Manager
1. Pall Life Sciences, City, State
2. Sartorius, Fremont, CA
Correspondence
Email: octet@sartorius.com

Abstract
Selection of high-producing clones during upstream process development and optimization relies on accurate determinationof recombinant protein titers. Traditional technologies such as ELISA and HPLC are well-established but require long assay
times, are labor intensive, and in some cases, are low throughput.
This application note shows that the Octet® systems are a rapid, high throughput, and accurate replacement for traditional
techniques and allow quantitation of recombinant proteins cell line development. Best practices for assay design, data
analysis and dealing with matrix effects are also shown.

Find out more: www.sartorius.com

Introduction
The accurate determination of recombinant protein titer iscritical to the selection of high-producing clones during cellline development and in optimization of bioreactor conditions during production of therapeutic proteins. Concentration measurements for these therapeutic proteins areoften done using enzyme-linked immunosorbent assays(ELISAs), RP-HPLC or coomassie-stained SDS-PAGE gels.
Although these techniques are prevalent, they are encumbered by drawbacks that include long assay times, extensive hands on and in some cases, low throughput. In
addition, techniques such as ELISA tend to exhibit highvariability, resulting in lower accuracy.
Bio-Layer Interferometry (BLI) is an optical analytical technique that utilizes the changing interference pattern ofwhite light shown over a reflective biosensor surface immobilized with a ligand to detect interactions with an analyte insolution. The binding between the ligand and the analyte
produces an increase in optical thickness on the tip of thebiosensor that can be measured as a wavelength shift fromthe reference surface, and is a direct measure of the change
in thickness of the biological layer upon binding events(Figure 1).

Octet® instruments utilize BLI technology and are a suitable replacement for both ELISA and HPLC techniques
for quantitaion of recombinant proteins. Octet® systems
are high-throughput (up to 96 samples can be analyzed ata time), accurate, fast and are relatively easy to use. Moreover, due to their sample plate-based format, Octet®
instruments can be used to accurately quantitate recombinant proteins in their crude form, allowing for rapid analysis of bioreactor samples during cell line development.
The measured wavelength shift is directly proportional tothe concentration of the binding analyte.
This Application Note demonstrates the use of Octet® systems in the quantitation of five biologically-relevant protein-based drug molecules: Insulin, Factor VIII, Factor IX,
Erythropoietin (EPO) and Granulocyte Colony Stimulating
Factor (GCSF). We specifically illustrate the use of Octet®
instruments in monitoring concentration during protein
expression and hence cell line development for these fiveproteins; however, the methods described here can, withproper assay development, be used for other recombinant
proteins. Although only single-analyte step detection
methods typically used for relatively high sample concentrations are discussed, the technique can be modified toinclude signal amplification steps that can be used toincrease assay sensitivity beyond what is reported.

1.0

BLI signal processing
0.8

Biocompatible surface

Relative Intensity

Incident
white
light

0.6

0.4

Bound molecule
0.2

Unbound molecules
have no eﬀect

Figure 1: Relative intensity of light reflection pattern on a biosensor surface using BLI.

2

Wavelength (nm)

Insulin, Factor VIII, Factor IX, GCSF
and EPO
Human Insulin, produced by beta cells of the pancreas, is arelatively small 5.8 kDa protein. It plays a critical regulatoryrole in the control of glucose homeostasis and is involved
in regulating carbohydrate and fat metabolism in the
body. While Insulin plays a regulatory role in the body, Factor VIII and Factor IX are both blood coagulants that act as
anti-hemophilic factors (AHF). A deficiency in these two
proteins can lead to Hemophilia A and B for Factor VIII
(280 kDa) and Factor IX (55 kDa) respectively. In addition
to these three proteins, EPO and GCSF also play criticalroles in the body. EPO is a 34 kDa glycoprotein producedin the adult kidney and in fetal liver. It controls erythropoiesis by stimulating maturation of erythroid precursor cells
in bone marrow. Its interactions with receptors expressedon the cell surface results in increased numbers of red
blood cells1. GCSF (18.7 kDa) is also a glycoprotein but isproduced by a variety of tissues and tumor cell lines that
regulate hematopoiesis and is required for the clonal
growth and maturation of normal bone marrow cell precursors. Commercial forms of GCSF such as filgrastim andbiosimilars are clinically used to induce granulopoiesis inconditions such as myelosuppressive chemotherapy,
acute myeloid leukemia and severe chronic neutropenia2.
Together these five proteins form part of an ever growing
list of proteins used for therapeutic purposes.
In general, recombinant proteins intended for therapeutic
use are often produced in mammalian host cells, mainly
Chinese hamster ovary (CHO) cells. Recombinant human
EPO is now available in sufficient amounts for clinical use in
the treatment of anemia that results from chronic renal failure3. Accurate quantitation techniques for these proteins
are therefore critical in enhancing their rapid development.

Materials and Reagents
All biotinylated antibody reagents were obtained from Life
Technologies and are 13 kDa camelid antibody fragments
specific to each antigen and selective for both recombinant and plasma derived proteins: Part nos 7103362500(insulin), 7103130500 (GCSF), 7103372500 (EPO),
7103002500 (Factor IX) and 7102862500 (Factor VIII).
Recombinant insulin (Part no. cyt-270) and Factor VIII(Part no. pro‑318) were both obtained from ProSpec. EPO(Part no. 100‑64) and GCSF (Part no. 300-23) weresourced from PeproTech and recombinant Factor IX wassourced from Cambridge Bioscience.

EX-CELL® NS0 Serum-Free Medium for NS0 Cells, Dulbecco’s Modified Eagle’s Medium-high glucose, EX-CELL®
CD CHO-3 Medium, Chemically Defined, and EX-CELL®
293 Serum-Free Medium for HEK 293 Cells were obtained
from Sigma. SFX-CHO HyClone and B-PER Reagents wereobtained from ThermoFisher Scientific and CD DG44
Medium was sourced from Life Technologies.

Instruments
Sartorious’ Octet® RH16 and Octet® R8 systems.
Agilent Zorbax 300SB-C18 RP-HPLC column; 5 µm,
4.6 × 250 mm.

MethodThe method discussed here can be applied to all Octet®
instruments. Data is acquired using the advanced quantitation mode in Octet® Analysis Studio Software. High-precision Streptavidin (SAX) Biosensors are first hydrated in therelevant assay matrix prior to immobilization or loading of
biotinylated antibody for each respective antigen. To
achieve optimal assay conditions, antibody loading studies
are first performed and are used to determine optimal antibody loading concentrations. Refer to Sartorious Technical
Note 40: High Precision Streptavidin Biosensor (SAX)
Quantitation and Kinetic Assays for more information. This
process is applicable to any custom quantitation of recombinant proteins using the Octet® platform. Once the optimal antibody loading concentration is determined, theassay is run as stipulated in Table 1.

Antibody Loading on SAX Biosensors
In these studies, commercially available biotinylated
camelid VHH antibody fragments were used. However, antibody biotinylation can be done using reagents that target
the antibody’s primary amino groups. The most commonlyused reagents are NHS-esters of biotin with spacer arms or
linkers that allow for optimal orientation of the antibody
once immobilized onto the biosensor surface. For an
in-depth understanding of the protein biotinylation process, refer to Sartorious Technical Note 6: Biotinylation ofProtein for Immobilization onto Streptavidin Biosensors.
The antibody should be protein carrier-free and should bein an amine-free buffer. For optimal performance it is crucial
to remove all free biotin after the biotinylation reaction,either by dialysis or through the use of a desalting column.
To achieve consistent antibody biotinylation, the number of3

biotins per antibody molecule should be kept at a minimum. An initial antibody load scouting assay (Figure 2),
where the antibody concentration is varied, typically around
100 nM or 15 µg/mL with concentrations above and below
15 µg/mL while the analyte concentration is left constantshould be performed. The goal is to saturate biosensor surface and select the lowest antibody concentration (devoidof assay artifacts) that yields maximum and reproducible
analyte response signals.

Recombinant Protein Concentration Analysis
Protein concentration determination on Octet® systems
requires the development of a standard reference curve. A
standard reference curve is obtained using a purified standard sample of the respective protein. Known concentrations of the purified standard sample are spiked into thematrix of interest and are serially diluted (often two-fold)within the desired concentration range using an analyte
depleted assay matrix.

A
6

Binding (nm)

5

4

3

A1
B1
C1
D1
E1

2

1

0
0

50

100

150

200

B

250
Time (sec)

300

350

400

450

60

80
100
Time (sec)

C
4.0

2.4

2.0
3.0

Binding (nm)Binding (nm)

1.6

2.0

1.2

0.8

1.0

0.4
0
0

20

40

60
Time (sec)

80

100

120

0

0

20

40

120

140

160

Figure 2: Antibody load scouting. Biotinylated anti-insulin loading was screened at 20, 10, 5 and 2.5 µg/mL. (A) shows raw data obtained when 5 µg/mLof recombinant insulin was used to probe for binding at the different antibody concentrations. Note: NSB was not observed when insulin was bound tobiosensor surface with no antibody loaded (curve E1) (B) - antibody and (C) - antigen show the respective aligned response signals at 20 µg/mL (darkblue), 10 µg/mL (red), 5 µg/mL (blue) and 2.5 µg/mL (green). No discernible difference in signals was observed at the top three concentrations
implying that 5 µg/mL is optimal for anti-insulin antibody loading. Similar studies were performed for the other recombinant proteins with 5 µg/mL
deemed optimal in all cases.

4

A biosensor blocking step using biocytin that blocks nonreacted streptavidin molecules or using BSA in the binding
matrix/buffer may be necessary post antibody capture toprevent non-specific binding (NSB). In addition, the use ofdetergents in the sample diluent to prevent potential
non-specific hydrophobic interactions is recommended
especially when dealing with glycoproteins. NSB refers toeither the binding of the analyte to a biosensor surface notcoated with the specific antibody or to observation of anappreciable binding of the analyte-depleted matrix to theantibody-coated biosensor surface and should be investigated and eliminated for each assay during development. If
blocking is performed, a short baseline step should be established in the analyte sample matrix prior to sample detection.

6. Under available assays, select Standard Assay.
7. Under assay parameters, insert cells using the Insert
command. Use the pull down arrow to select assay
steps in this order: Buffer, Capture antibody, 2nd buffer,
Sample. Input the appropriate assay step times and theshaking speed per Table 1. Note that each buffer column can be used only once within the same assay whenthe method is developed in the advanced quantitation
mode. Click OK.
8. Input sample plate information (example sample plateis shown in Figure 3).
9. The software will automatically assign the required
number of biosensors.
10. Review the method and click Go.

Data Analysis
Octet Analysis Studio Software allows users to selectbetween one of two binding rate analysis models: an initialslope of binding rate analysis and an end-point R-equilibrium analysis. The initial slope option should always be
used when the analyte binding step is a simple single step
post antibody loading. If the assay is modified to allow for a
signal amplification step such as what would be used in ahigher sensitivity assay, the detection step should beallowed to reach equilibrium, and the R-equilibrium binding rate model used to analyze the data. The analyzed data
can be fit using one of several models; however, careshould be taken to ensure that accurate analyte concentration is determined off the linear portion of the standard
curve. The unknown analytes concentration is extrapolated off the generated standard curve.
®

Assay Protocol
To start the Octet® instrument, first turn on the computer
followed by the instrument. Wait at least 1 hour for the lamp
to warm up before running an experiment.
1.
2.

3.
4.

5.

Hydrate SAX Biosensors in assay matrix for at least
10 minutes.
Load samples onto the sample plate using 200 µL
per well for 96-well microplates and 80 µL(Greiner microplates, Part no. 655209) or 40 µL
per well (Sartorious 384-well, black, tilted-bottom
polypropylene microplates, Part no. 18-5076) whenusing 384-well microplates.
Open Octet® Analysis Studio Software to design
the method.
In the Experiment Wizard window, select the New
Quantitation - Advanced Quantitation method
option. Next, click the green forward arrow.
In the plate definition window, click Modify to input
assay parameters.

1

2

3

4

5

A

B

C

2

B

B

C

2

C

B

C

2

D

B

C

2

E

B

C

2

F

B

C

2

G

B

C

2

H

B

C

2

6

7

8

9

10

11

12

Figure 3: 96-well plate design for Advanced Quantitation on the Octet®
system showing standard samples (black), buffer (B), capture antibody(C), 2nd buffer (2) and unknown concentration samples (yellow). At least
two replicates are required for the standard sample.

Table 1: Octet® Advanced Quantitation assay method step setup for thecustomized quantitation of recombinant proteins
Step

Step typeTime (s)Shaker (RPM)

1

Buffer

180

1000

2

Capture Antibody

300

1000

3

2nd Buffer

180

1000

4

Sample300

1000

Recombinant Protein Quantitation
The evaluation of recombinant therapeutic protein quantitation on the Octet® system suggests varying limit of quantitation (LOQ) and a concentration dynamic range withvarying media conditions (Figures 4–8, Table 2). All samples
investigated exhibited better sensitivity in Sartorious’ 1X
Kinetics Buffer implying that for low sample concentrations,
it is best to dilute off potential sample matrix effects. However, in cases where sensitivity is lost due to matrix effects, awider concentration dynamic range can be detected.
5Insulin
1.3

1X KB

3

1X KB

1.2
1.1

2.5

1.0

2.0

Binding (nm)Binding (nm)

0.9

1.5

0.8
0.7
0.6
0.5
0.4

1.0

0.3
0.5

Standard
Fitting curve
Unknown & control

0.2
0.1

0

0

40

80

120

160

200

240

0

280

0

5

10

15

20

Time (sec)25
30 35 40 45
Concentration (µg/mL)

50

55

60

65

0.7
DMEM

DMEM
0.6

2.0

0.5

Binding (nm)Binding (nm)

1.5

1.0

0.4

0.3

0.2
0.5

Standard
Fitting curve
Unknown & control

0.1

0

0
0

40

80

120

160
Time (sec)

200

240

0

280

0.50
SFX-CHO

2.5

5

10

15

20

25 30 35 40 45
Concentration (µg/mL)

50

55

60

65

SFX-CHO

0.45
0.40
0.35

Binding (nm)Binding (nm)

2.0

1.5

1.0

0.30
0.25
0.20
0.15
0.10

0.5

Standard
Fitting curve
Unknown & control

0.05
0

0
0

20

80

60
Time (sec)

80

100

120

0

5

10

15

20

25 30 35 40 45
Concentration (µg/mL)

50

55

60

Figure 4: Binding curves (left) for the quantitation of recombinant insulin in three different media types with the corresponding standard curves (right).
Standard curves were generated using initial slope binding rate analysis. Data was fit using a 5-PL Dose response curve fitting equation.

6

Factor IX
2.5
1X KB

1X KB
0.14

2.0

0.10

1.5

Binding (nm)Binding (nm)

0.12

1.0

0.08
0.06
0.04

0.5

Standard
Fitting curve
Unknown & control

0.02

0
0

20

40

60

80

100

0

115

0

2

4

Time (sec)

2.5

DMEM

6
8
10
Concentration (µg/mL)

12

14

16

DMEM

0.08
0.07

2.0

1.5

Binding (nm)Binding (nm)

0.06

1.0

0.05
0.04
0.03
0.02

0.5

Standard
Fitting curve
Unknown & control

0.01
0

0
0

10

20

30

40

50

60

70

80

90

100

110

0

2

4

Time (sec)6810Concentration (µg/mL)

12

14

16

3.0
SFX-CHO

SFX-CHO

0.14

2.5

0.12

0.10
Binding (nm)Binding (nm)

2.0

1.5

0.08
0.06

1.0

0.04

Standard
Fitting curve
Unknown & control

0.5

0.02
0
0

10

20

30

40

50

60

Time (sec)

70

80

90

100

110

0

0

2

4

6
8
10
Concentration (µg/mL)

12

14

16

Figure 5: Binding curves (left) for the quantitation of recombinant Factor IX in three different media types with the corresponding standard curves(right). Standard curves were generated using initial slope binding rate analysis. Data was fit using a 5-PL Dose response curve fitting equation.

7

GCSF
0.8

1X KB

0.7

3

0.6

Binding (nm)2.5

Binding (nm)

1X KB

2.0

1.5

0.5
0.4
0.3

1.0

0.2

0.5

0

Standard
Fitting curve
Unknown & control

0.1

0

10

20

30

40

50

60

70

80

90

100

0

110

0

5

10

Time (sec)15
20Concentration (µg/mL)

25

30

2.5
DMEM

DMEM

0.50
0.45

2.0

0.40

Binding (nm)Binding (nm)

0.35
1.5

1.0

0.30
0.25
0.20
0.15
0.10

0.5

Standard
Fitting curve
Unknown & control

0.05
0

0
0

10

20

30

40

50

60

70

80

90

100

0

110

5

10

Time (sec)15
20Concentration (µg/mL)

25

30

1.2
SFX-CHO

SFX-CHO

0.18

1.0

0.16
0.14
0.12
Binding (nm)Binding (nm)

0.8

0.6

0.10
0.08
0.06

0.4

0.04
0.2

Standard
Fitting curve
Unknown & control

0.02
0

0
0

10

20

30

40

50

60
Time (sec)

70

80

90

100

110

0

5

10

15
20
Concentration (µg/mL)

25

30

Figure 6: Binding curves for the quantitation of recombinant GCSF in three different media types with the corresponding standard curves (right).
Standard curves were generated using initial slope binding rate analysis. Data was fit using a 5-PL Dose response curve fitting equation.

8

EPO
2.5

Citrate buﬀer, pH 4.0

Citrate buﬀer, pH 4.0

0.35

2.0

0.30

Binding (nm)Binding (nm)

0.25
1.5

1.0

0.20
0.15
0.10

Standard
Fitting curve
Unknown & control

0.5
0.05

0

0

20

40

60

80

100

0

115

0

5

10

15

20

Time (sec)

0.018

DMEM
0.6

0.016

0.5

0.014

45

50

55

60

65

DMEM

0.012
Binding (nm)0.4
Binding (nm)

25 30 35 40
Concentration (µg/mL)

0.3
0.2

0.010
0.008
0.006
0.004

0.1

Standard
Fitting curve
Unknown & control

0.002
0
0
0

10

20

30

40

50

60

70

80

90

100

110

0

10

20

30

40

Time (sec)

50 60 70
80 90
Concentration (µg/mL)

100

110

120

2.5
SFX-CHO

2.0

0.010

1.5

Binding (nm)Binding (nm)SFX-CHP

0.012

1.0

0.008

0.006

0.004

0.5

Standard
Fitting curve
Unknown & control

0.002

0
0
0

20

40

60
Time (sec)

80

100

110

0

5

10

15

20

25 30 35 40
Concentration (µg/mL)

45

50

55

60

Figure 7: Binding curves (left) for the quantitation of recombinant EPO in three different media types with the corresponding standard curves (right).
Standard curves were generated using initial slope binding rate analysis. EPO binding assay is pH-sensitive with the lowest LOQ obtained in 20 mM
Citrate buffer, 250 mM NaCl, pH 4.0. Data was fit using a 5-PL Dose response curve fitting equation.
9

Factor VIII
0.20

1X KB

1X KB

0.18
4.0
0.16

Binding (nm)Binding (nm)

0.14
3.0

2.0

0.12
0.10
0.08
0.06

1.0

Standard
Fitting curve
Unknown & control

0.04
0.02

0

0

10

20

30

40

50

60

70

80

90

100

0

115

0

5

10

15

20

Time (sec)

DMEM

50

55

60

65

DMEM

5.0

0.30

0.25

Binding (nm)4.0
Binding (nm)

25 30 35 40 45
Concentration (µg/mL)

3.0

0.20

0.15

2.0
0.10

1.0

Standard
Fitting curve
Unknown & control

0.05

0
0

10

20

30

40

50

60

70

80

90

100

0

110

0

5

10

15

Time (sec)

20

25

30

35

40

45

Concentration (µg/mL)

50

55

60

65

Figure 8: Binding curves (left) for the quantitation of recombinant Factor VIII in two different media types with the corresponding standard curves(right). Standard curves were generated using initial slope binding rate analysis. Data was fit using a 5-PL Dose response equation.

Table 2: Dynamic range for recombinant therapeutic protein quantitation in different media types. n.a = not applicable; n.d = not determined
Insulin

Factor VIII

Factor IX

GCSF

EPO

1X KB, µg/mL

0.78 – 50

0.78 – 50

0.19 – 12.5

0.39 – 25

n.d

DMEM Media, µg/mL

0.78 – 50

0.78 – 50

3.13 ≥ 12.5

3.13 – 25

n.d

SFX-CHO Media, µg/mL

0.78 – 50

n.d0.19 – 12.5

3.13 – 25

12.5 ≥ 50

n.a

n.a

n.a

n.a

1.56 – 50

20 mM Citrate buffer, 250 mM NaCl,
pH 4.0, µg/mL

10Matrix Effect Analysis
While non-specific interactions can result in false positives
during a binding analysis, matrix interference by cell culture media components can have the opposite effect;quenched signal response despite positive binding
between the immobilized antibody and the antigen. To
investigate whether quantitation could be performed on
upstream samples with minimum pre-processing, and to
determine the minimum dilution required for a successful
quantitation analysis where cell culture media inference isobserved, a matrix effect study was performed for eachprotein using several different common cell culture media.
A binding rate probe at constant sample concentration foreach sample (6.25, 6.25, 3.13, 1.56 and 1.56 µg/mL (for InsuInsulin

lin, Factor VIII, GCSF, Factor IX and EPO respectively) was
performed with the rate of binding used to predict thepotential matrix effect. For all samples except EPO, biosensor equilibration, pre-load baseline and antibody loading were performed in 1X Kinetics Buffer. For EPO, thesesteps were performed in 10X Kinetics Buffer to minimize
NSB. The subsequent post-antibody loading baseline andthe antigen binding steps were performed in the respective
media for all analyte samples. When the binding rate wasdeemed low enough to suggest potential signal quenching
due to matrix effects, sample dilution in 1X Kinetics Buffer
was performed. Figure 9 shows the binding rate data for theminimum dilution required for each media type for therespective recombinant proteins.

Factor VIII

0.04

0.03
0.03
0.02
0.02

0.01

0.01

0

8x

x

O

1/
O

SF

X-

C

H

C
X

H

10
1/

21

BL

D

SF

21

BP

ER

3

BL

O
H

29

4x
1/

C
D

C

EM
M

EM

44

S0

M
D

N

KB

G
D

1/
O

H
C

XSF

Factor IX

0.04

1x

x
16

x

O
H

C

20

1/

X
SF

x

21
BL

BL

21

3

16

C

D

D

C

H

29

O

3

1/

x
16
1/

29

x

O
H

16
1/

C
D

C

x

M

EM

16

EM

1/

M

N

D

x

S0

N

S0

44

16

44

1/

G
D

G

D

1x

KB

0

GCSF

0.05

0.04

0.03

0.03
0.02
0.02
0.01

0.01

0

0.04

1/
SF 16x
X
SF
C
H
XO
C
H
O
1/
2x

BP

ER

BP

ER

x

21

1/

16

1
BL
2

BL

29
3

KB
G
44
D
G
44
1/
8x
N
S0
D
M
EM
D
M
EM
1/
8
C
D x
C
H
O

1x

D

8x

X-

C

H

O

1/

H
O

x

C
X

SF

SF

BL
2

11
/4

ER

BP

21
BL

3
29

H
O

x

C

1/
4

D
C

D

EM

S0
M
EM

D
M

N

D
D G4
4
G
44
1/
4x

1x

KB

0

EPO

0.03

0.02

0.01

Figure 9: Binding rate analysis at constant protein concentration incommon cell culture media. Low binding rates in undiluted samples
suggest potential matrix effect hence the need for sample dilution.

C

D

itr
a

te

Bu
ﬀ
D er pG
H
G 44 4.
44 p 0
1/ H 48x.0
N pH
N S0 4.0S0p1/ H 4
4x
.
p 0
D
M D H
EM M 4.0
EMC 1/8
4C D x p .0
D
C
C HO H 4
H.
O
p 0
1/ H 48x.0p
29 H
29 3 p 4.0
3H1/
4x 4.0
BL pH
BL 2 421 1 p .0
1/ H
8x 4.
p 0
BP BPE H 4
.0
ER R
1/ pH
2 2
SF
SF x p .5
X
H
C XC 2.
HO HO 51/
8x 4.0
pH
4.
0

0

11

Octet® System Comparison to HPLCThe advantages of using HPLC for the quantitation of biomolecules include its applicability in diverse sample types
including small organic molecules, large biomolecules andpolymers amongst others. HPLC however has the disadvantage of lack of a “universal” detector. The fact that for proteins, the detection in HPLC is achieved at a wavelength of
280 nm (not specific to any given protein) means that thedetection has to be accompanied with good column separation resolution otherwise data accuracy is compromised.
HPLC also lacks the through-put most critical in bioprocessing laboratories hence can be time consuming, especially for regulated analysis under good manufacturing
practices (GMP). The BLI assays on Octet® systems on theother hand can be optimized for specificity towards target
biological molecules. The simple dip and read plate design
method also allows for the rapid analysis of samples during
protein expression and early on in the drug development
process and can therefore facilitate necessary adjustmentsin the development of optimal conditions
To compare the performance of the Octet® systems with
HPLC, recombinant insulin was used as an example. Samples were analyzed side by side at Aragen Biosciences using
the Octet® R8 instrument and a Zorbax 300SB-C18
RP-HPLC column.

Insulin samples were serially diluted 2-fold in either PBS or
DMEM media at 800, 400, 200, 100, 50, 25, 12.5, 6.25,
3.125, 1.56, 0.78 and 0 µg/mL. Half of the prepared samples
were used for the RP-HPLC studies while the other half wasused to run concurrent Octet® assay quantitation studies.
For the Octet® system, the assay used was as per the assay
protocol section while for RP-HPLC the following conditions were used:
Sample flow rate: 1 mL/min
Eluents:
A: 0.1% Trifluoroacetic acid (TFA) in water
B: 0.08% TFA in Acetonitrile
Elution Gradient: 30–50 % B in 10 minutes
Detection: 280 nm
Run time per sample: 10 minutes
The results suggest that HPLC exhibits excellent sample
recovery at high insulin concentrations but significantly
over estimates concentrations at the low end of the
dynamic range. The Octet® systems on the other hand havea more limited linear dynamic range of quantitation forinsulin. However, the systems exhibit excellent recovery of
low concentration insulin samples implying better sensitivity (Table 3). Moreover, it takes significantly less time toassay the same number of samples on an Octet® system as
it does via HPLC, providing significant time savings that canlower overall analysis costs.

Table 3: A comparison of data accuracy for insulin quantification between Octet® systems and HPLC.
% Recovery

Insulin measured using
RP-HPLC, µg/mL

Insulin measured using
Octet®, µg/mL

Insulin measured using
RP-HPLC (PBS), µg/mL

0.78

not determined

0.77

98.2

1.56

not determined

1.56

100.0

3.13

not determined

3.06

97.8

12

Insulin measured using Octet(1X Kinetic Buffer), µg/mL% Recovery
Starting insulin,
µg/mL

®

6.25

20.0

6.24

319.7

99.8

12.50

26.7

12.50

213.8

100.0

25.00

39.2

25.00

156.7

100.0

50.00

62.6

50.00

125.1

100.0

100.00

113.5

113.5

200.00

212.8

106.4

400.00

414.1

103.5

800.00

813.6

101.7

GMP Operations
The Octet® platform is equipped with the necessary tools
for use in GMP environments. The platform includes both
IQOQ tools and 21 CFR Part 11 features to help meet GMP
requirements including electronic security, audit trails, user
access management and other compliance options.
In quantitation assays, precision, linearity and accuracy are
key parameters that must be demonstrated to allow theassay to be validated for use in manufacturing. In addition,
the limit of quantitation (LOQ) should be determined toincrease confidence in the assay’s performance. As an
example, insulin was used to determine precision and accuracy, while the LOQ was determined for all samples asshown in Table 2.
Three samples (25, 6.25 and 1.56 µg/mL) of insulin werespiked in 1X Kinetics Buffer and treated as unknown samples. A standard curve, also in 1X Kinetics Buffer, was generated for the 0–50 µg/mL range. All samples were run in
triplicate. Data was analyzed using the initial slope binding
rate. The standard samples exhibited concentration %CVs
of < 3% (Table 4) while the unknown samples exhibited
concentration %CVs well below 5% indicating excellent
precision. Dose recovery for the unknown samples wasfound to be within 90–110 %, indicating excellent method
accuracy (Table 5).
Table 4: Insulin Quantification: Standard samples exhibit excellent
precision, < 3% CV.
Conc.
µg/mL

Binding rate,
nm/s

Conc. avg,
µg/mL

Conc.
% CV

50.00

1.020050.0

2.390025.00

0.500025.0

1.880012.50

0.187212.5

1.24006.25

0.06116.2

1.48003.13

0.01893.1

0.34761.56

0.00581.6

2.83000.78

0.00120.8

0.28310.00

0.00010.0

0.0000

Table 5: Insulin Quantification: Unknown samples, n=3, exhibit
excellent precision and accuracy.
Conc.
µg/mL

Binding rate,
nm/s

Conc. avg,
µg/mL

Conc.
% CV

% Recovery

25.00

0.518225.9

1.4100104

6.25

0.06406.4

0.1638102

1.56

0.00661.7

1.4000106Best Practices for Custom RecombinantProtein Quantitation Assays
1.

Always hydrate the biosensors for at least 10 minutes inthe expected assay matrix. Note that in some cases, it
may be necessary to hydrate the biosensors for longer
than 10 minutes. A good rule of thumb is to hydrate thebiosensors for approximately 20 minutes in cell culture
spent media if the analyte is to be analyzed in its crude
matrix during cell line expression monitoring.

2. An antibody-loading scouting experiment is recommended to ensure the optimal antibody is selected.
Although the selected antibody concentration is
5 µg/mL for the five analytes mentioned in this application note, it is important to note that for some analytes
the optimal concentration may be different. In principle,
antibody loading should saturate the biosensor surface
without a post-loading drift and should yield the highest analyte response signals. In addition, the rate ofantibody loading should be gradual to ensure uniformity in loading. A rapid loading slope may indicate
non-optimal biotinylation process that may affect antigen binding. A key advantage of the Octet® platform isthat the software allows the user to increase assay steptime if necessary. This allows for flexibility in using low
sample concentrations as the assay loading step can beincreased accordingly during the scouting experiment.
3. During assay development, make sure to test for potential non-specific binding interactions (NSB) by runningreference biosensors and samples. A reference analysis
is two-fold: first test for NSB of the analyte to a biosensor blocked with an irrelevant antibody; second test for
NSB between the assay media and the coated antibody.
In the first case, the presence of NSB should suggest
further assay development while in the second case the
NSB can be subtracted using reference biosensors.
Further assay development if necessary, is best done in
the instrument’s Kinetics.
4. For data accuracy, it is important to ensure matrix
matching between the standard sample and the analyte. For example, if the cell culture media is SFX-CHO
media, the standard samples should be spiked into
SFX-CHO spent media to allow for comparable analyte
environments, hence comparable analyte specificity. If
the systems cannot be matrix matched, it is recommended that the analyte be diluted 1:10 in 1X Kinetics
Buffer to allow for matrix consistency between the standard samples and the analyte.

13

5. Determine the useful standard sample concentration
range and work in the linear portion of the standard
curve. Standard samples should always be run in replicates to increase confidence in the assay.
6. Note that for recombinant protein quantitation, the recommended assay shaking speed is 1000 RPM. However, there may be analytes where a different shaking
speed may be required. 1000 RPM is typically recommended in tandem with a longer assay time to improveassay sensitivity. In general, the higher the RPM, thebetter the sensitivity. In cases where the analyte is highin concentration, the assay dynamic range may be
expanded by lowering the shaking speed to the default
400 RPM.
7.

Although 1X KB is used in these studies, sample diluent
buffer (higher % BSA) can also be used and is recommended for most proteins not included in this Application Note.

Determine assay goals
and target analyte
concentrations

Biotinylate analyte
speciﬁc antibody

The simple Dip and Read approach on Octet® platforms in
combination with High Precision Streptavidin Biosensors(SAX) provide an easy to use, time-saving technique thatenables high throughput, accurate quantitation of recombinant proteins. The LOQ for insulin concentration when determined on the Octet® was 0.78 µg/mL in both DMEM and 1X
Kinetics Buffer while it was much higher (6.25 µg/mL) in both
media when determined on RP-HPLC. Moreover, while ittakes more than 10 minutes to assay one sample on
RP-HPLC, the Octet® systems take approximately the sametime to assay as many as 8 samples (Octet® R8), 16 samples(Octet® RH16) and 96 samples (Octet® RH96). The technique
can be a suitable replacement for HPLC and ELISA as it ismore robust to various cell culture media and can be usedboth upstream and downstream in the therapeutic protein
drug development process. In addition, Octet® platforms areequipped with GMP-compliant features that allow for thedevelopment of robust methods that can be quickly validated for assay transfer to QC laboratories.

Perform antibody
loading optimization
assaysFigure 10: A summary of the work-flow for custom quantitation assays

14

Conclusion

Evaluate sample
matrix considerations
and dilution requirements

Generate standard curvefrom puriﬁed analyte andquantitate unknown samples

References1.

Current State and Perspectives on Erythropoietin
Production, Lee, J S, et al., Applied Microbiology andBiotechnology, 95:1405–1416, 2012.
2. Granulocyte-colony stimulating factor (G-CSF)induced mechanical hyperalgesia in mice: Role for
peripheral TNFα, IL-1β and IL-10 Carvalho TT et al;European Journal of Pharmacology,749:62-72, 2015
3. Biopharmaceutical Benchmarks 2014, Walsh, G.,
Nature Biotechnology, 32:992–100, 2014.

15

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