The document describes methods for using surface plasmon resonance (SPR) to compare the binding properties of proposed biosimilar products to reference products. SPR was used to assess potency and kinetics of trastuzumab binding to FcRI and bevacizumab binding to VEGF. Assays showed good linearity, accuracy, precision and ability to distinguish differences between biosimilars and references, making SPR suitable for biosimilar comparability studies.

1. FIGURE 1: POTENCY BINDING CURVES OF
TRASTUZUMAB (HERCEPTIN) WITH FCRI/CD64
The standard deviations of the triplicate measurements were smaller
than the plotted symbols.
COMPARABILITY METHODS FOR BIOSIMILAR TESTING USING THE BIACORE T200
INTRODUCTION
Regulatory organizations have issued detailed guidelines for biosimilar products which includes a demonstration of biosimilarity with the licensed originator
product. Because monoclonal antibodies represent a major class of biosimilar products, target ligand binding assays and Fc Receptor binding assays
(FcRI/CD64, FcRII/CD32, FcRIII/CD16 and FcRn) are essential to support characterization, development, lot-to-lot variability and product release. Our
laboratory has developed and validated methods using Surface Plasmon Resonance (SPR) technology that conform to ICH, FDA, and EMA guidelines to
assess the comparability of functional activity of the proposed biosimilar product to its reference product.
METHODS
For the presented studies, the Biacore™ T200 (GE Healthcare) was used to perform side-by-side comparability binding assays.
Ligands (R&D systems) or Fc Receptors (R&D systems or SinoBiologicals) were immobilized or captured respectively onto a CM5 sensor chip. Originator
(Bionical Ltd) and biosimilar product were flowed over the ligand/FcR of interest and binding assay endpoints information reported appropriately. All
experiments were carried out in HBS-EP+ buffer (GE Healthcare) at a constant temperature of 25 °C.
For potency comparability (relative binding), triplicate independent serial dilutions (8 concentrations) of the originator and biosimilar product were prepared
and injections performed at a flow rate of 5 μL/min over two flow cells; one reference surface and one with captured Fc Receptor (FcR) or immobilized
ligand. After each sample injection, the sensor chip was regenerated and FcR/ligand re-captured appropriately. BiacoreTM T200 Control Software 2.0 was
used for data collection and export of data to PLA 2.1 Software (Stegmann systems) for potency evaluation.
For kinetics association constant (ka or k-on), dissociation constant (kd or k-off) and affinity constant (KD) comparability, independent dilutions of the
originator and biosimilar product were prepared and injections performed in quadruplicate at a flow rate of 30 μL/min. After each sample, the sensor chip
was regenerated. BiacoreTM T200 Control Software 2.0 was used for data collection. BiacoreTM T200 Evaluation Software 2.0 was used with 1:1 binding
model fitting for kinetics/affinity evaluation.
RESULTS
Potency comparability:
In each potency experiment up to three test samples can be compared to a reference sample (with an assigned potency of 1). The following potency results
present the binding of Trastuzumab (Herceptin) to recombinant FcRI/CD64. Trastuzumab (standard/reference) and the test samples were injected in
triplicate and analyzed using a 4PL model with all test sample curves were deemed to be parallel to the reference sample curve (p>0.05). Between each
injection the surface was regenerated with 30 μL of 10 mM glycine pH1.5. The obtained potency binding curves are shown in Figure 1.
MELANIE VERNERET, JOHN BLACK, CHRISTOPHE IOCHEM, ARCHIE LOVATT
SGS LIFE SCIENCE SERVICES, GLASGOW, UK
The assay showed good linearity with an r2 = 0.974 with all measured potencies in the range of 88.9% to 101.6% as show in Table 1. Moreover, the assay
showed good repeatability, with a %CV < 15% for each concentration per potency levels and high precision with a %CV < 5% for replicate potency levels.
TABLE 1: TRASTUZUMAB - FCRI/CD64 ASSAY ACCURACY
Kinetics/Affinity comparability:
The following kinetics results present the binding of Bevacizumab to recombinant Vascular Endothelial Growth Factor A (VEGFA). In the presented
experiment, five test samples were compared to a reference sample. Between each injection the surface was regenerated with 30 μL of 10 mM glycine
pH1.5. The obtained kinetics binding curves are shown in Figure 3.
During qualification, to assess the assay linearity, accuracy and precision evaluation, the test samples and reference samples were all prepared from a
single batch of Bevacizumab and the concentration of the test samples were adjusted to simulate samples of different response.
The correlation between measured and nominal response for three separate experiments, over different flow cells, is shown in Figure 4. The nominal
response is the concentration at which the test samples were prepared relative to the reference and the measured response is the test sample response
which is calculated from assay data relative to the reference.
The assay showed good linearity with an r2 = 0.959 for the nominal response range of 0.5 - 1.5 and all measured response were within 15% of the nominal
value (range 85.2% - 106.6%) as show in Table 2. Moreover, the assay showed good repeatability, with a %CV < 10% for each concentration and high
precision with a %CV < 15% for replicate nominal response levels (Table 3).
TABLE 2: BEVACIZUMAB - VEGFA ASSAY ACCURACY
TABLE 3: BEVACIZUMAB - VEGFA ASSAY PRECISION
Conclusions
Biacore potency or kinetic assays can provide valuable in vitro data regarding functional interaction properties of the products without the need for animal
studies and the variability due to biological complexity and/or statistical noise of the cell-based assays (e.g. Antibody Dependant cell-mediated Cytotoxicity -
ADCC). Furthermore, when considering costs and speed, Biacore assays provide a more economical and faster alternative to cell-based assays.
More importantly, as shown in the presented examples, Biacore assays can easily distinguish differences in the biosimilars sample in comparison to the
reference product with high levels of accuracy, precision and linearity, making them fit-for purpose for comparability studies in compliance with regulatory
requirements for quality control and biosimilar license approval.
Similar approaches can be used with the full range of FcRs and target binding for any monoclonal antibody.
References
1. ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology (1995)
2. Bioanalytical Method Validation – FDA (2011) – Draft 2013
3. Guideline on bioanalytical method validation – EMA (2012)
4. Guideline on Similar Biological Medicinal Products – EMA (2014)
5. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues – EMA
(2015)
6. Guideline on similar biological medicinal products containing monoclonal antibodies – non-clinical and clinical issues – EMA (2012)
7. Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product – FDA (2015)
8. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product – FDA (2015)
9. ICH Topic Q 6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (1999)
10. The United States Pharmacopeial Convention – Chapter <1032> <1033> <1034>
11. European Pharmacopeia, European Pharmacopoeia. 8.0. Section 5.3. Statistical Analysis of results of Biological Assays and tests.
During qualification, to evaluate the assay, linearity, accuracy and precision,
the test samples and reference samples were all prepared from a single batch
of Trastuzumab and the concentration of the test samples were adjusted to
simulate samples of different nominal potencies.
The correlation between measured and nominal potencies for three separate
experiments is shown in Figure 2. The nominal potency is the concentration at
which the test samples were prepared relative to the reference and the
measured potency is the concentration which is calculated from assay data
relative to the reference.
FIGURE 2: CORRELATION BETWEEN MEASURED AND NOMINAL
POTENCY FOR TRASTUZUMAB - FCRI/CD64
0
0.5
1
1.5
2
0 0.5 1 1.5 2
Measuredpotency
Nominal potency
PARAMETERS
ACCEPTANCE
CRITERIA
VALUE
r² > 0.95 0.974
Intercept -0.2 to 0.2 0.04
Slope 0.8 to 1.2 0.91
POTENCY ACCEPTANCE CRITERIA ACCURACY (%)
0.5 80 to 120 % 97.4
0.7 80 to 120 % 92.0
0.8 80 to 120 % 88.9
1.0 80 to 120 % 100.8
1.2 80 to 120 % 101.6
1.3 80 to 120 % 91.1
1.5 80 to 120 % 93.3
FIGURE 3: KINETIC BINDING CURVES OF BEVACIZUMAB
(AVASTIN) WITH VEGFA
FIGURE 2: CORRELATION BETWEEN MEASURED AND NOMINAL
RESPONSE FOR BEVACIZUMAB - VEGFA
0
0.5
1
1.5
2
0 0.5 1 1.5 2
MeasuredResponse
Nominal Response
PARAMETERS
ACCEPTANCE
CRITERIA
VALUE
r² > 0.95 0.959
Intercept -0.2 to 0.2 0.16
Slope 0.7 to 1.3 0.77
NOMINAL RESPONSE ACCEPTANCE CRITERIA ACCURACY (%)
0.5 80 to 120 % 106.6
0.8 80 to 120 % 103.5
1.0 80 to 120 % 99.9
1.2 80 to 120 % 93.2
1.5 80 to 120 % 85.2
NOMINAL RESPONSE % CV
0.5 10.7
0.8 11.8
1.0 4.9
1.2 14.5
1.5 0.9
GSK PCGSK PC