Surface Plasmon Resonance (SPR): Molecular Interactions ...

LIFE SCIENCE I TECHNICAL BULLETIN FEBRUARY 2014

SURFACE PLASMON RESONANCE: MOLECULAR
INTERACTIONS AND LIGAND BINDING ANALYSIS

AUTHOR: MELANIE VERNERET, PHD, MOLECULAR BIOANALYTICAL SCIENTIST, SGS VITROLOGY, GLASGOW, UK

Characterisation of therapeutic monoclonal antibodies, drugs and other biological products is a necessary step
required during the regulatory approval process. The manner in which biopharmaceuticals and therapeutic
antibodies interact and bind with the target molecule is one important aspect of this characterisation requiring
analysis.

When ligand binding is part of the performance so that the upper and lower x 107 M-1s-1 for low molecular weight
functional activity of a biosimilar or limits of kinetic ranges can be assessed. molecules and a dissociation rate (kd)
therapeutic product, FDA and EMA Moreover, the system enables analysis from 10-5 to 1 s-1.
guidelines require this property be of interactions between biomolecules
quantified1,2. Binding assays are essential ranging from low molecular weight Typically, this data can be obtained with
to defining immunological properties ions to complex viruses. This powerful the antibody of interest immobilized onto
and biological activity of monoclonal system has a wide variety of applications the surface of the sensor chip. Using this
antibodies 3.4,5,6,7. from biological research, vaccine configuration, the Biacore T200 was used
development, bio-therapeutics and small to determine association, dissociation
Efficient selection, optimisation, and molecule drug development, through rate and affinity (Figure 1).
characterisation of bio-therapeutics are to immunogenicity studies and quality
essential throughout the development control8. The Biacore T200 can also be used to
process, as well as for understanding obtain precise information regarding
molecular mechanisms and structure- The Biacore T200 system can be used the specificity of an interaction. Using
function relationships. An efficient to address a multitude of scientific a capture strategy, antibody specificity
development process is crucial to questions. For example, do two can be assessed using a range of
identifying important aspects of product biomolecules interact and, if so, how antigen concentrations from two species
performance and to provide confidence specific is the interaction? What are the (Figure 2). In this experiment, the data
in selection. kinetic parameters for this interaction? demonstrates the antibody has a higher
How strong is this interaction (ie, affinity for antigen from one species
Furthermore, determining concentration affinity)? How fast is the duration of over another, demonstrating that, in this
and monitoring kinetic data during drug- the interaction (ie, rate constants for situation, the antibody/antigen interaction
substance stability studies and batch association and dissociation)? What is is very specific.
production are key elements for quality the active concentration of a specific
control of manufacturing biotherapeutics. biomolecule? The Biacore T200 system can be used
for custom development services and
The GMP compliant Biacore™ T200 Answers to these important questions method validation in compliance with
system is an established and state- can be achieved by determining the GLP or GMP, thus ensuring accurate and
of-the-art instrument for real-time and binding affinity constant (KD), association consistent analysis of all samples. The
label-free biomolecular interaction rate (ka), and dissociation rate (kd). applications of this system are many and
analysis and binding assays. The system Such studies are often limited by low have been performed in our laboratories
is used to deliver high-quality kinetic, sample concentrations, but Biacore include:
binding affinity, concentration, specificity, T200 achieves accurate results where
selectivity, and thermodynamic other systems cannot. The Biacore T200 ƒƒ Screening applications:
interaction data, all with high sensitivity. yields data on association kinetics with a • Clone and candidate selection
Based on Surface Plasmon Resonance sample concentration ≥10 pM and with with ranking of binding affinity
(SPR) technology, the Biacore T200 an association rate (ka) ranking from 103 constant, as well as association
system significantly enhances to 3 x 109 M-1 s-1 for protein or 103 to 5 and dissociation rates

™Biacore T200 is a trademark of General Electric Company

LIFE SCIENCE I TECHNICAL BULLETIN 2

• Screening of putative binding FIGURE 1: CHARACTERIZATION OF A MONOCLONAL ANTIBODY
partners BINDING AFFINITY FOR ITS ANTIGEN

ƒƒ Quantitative kinetic evaluation ka (M-1 s-1) kd (s-1) KD (nM)
(quality control release & stability): Association rate Dissociation rate Affinity
• Binding affinity constant, and
association and dissociation 1.0 ± 0.1 x 106 2.67 ± 0.02 x 10-3 2.6 ± 0.3
rates
• Relative binding assessments This sensorgram depicting the response signal obtained during injection of various concentrations of antigen
and consideration of biosomilar (consider as the analyte) onto the antibody of interest, immobilised on the surface of the sensor chip
molecules (consider as the ligand), using the Biacore T200. 0-120 sec = association period; 120-250 sec = dissociation.
Antigen/Analyte = human beta-2-microglobulin; Ligand = mouse monoclonal antibody anti- human beta-
ƒƒ Antibody characterisation: 2-microglobulin.
• Antibody isotype determination

ƒƒ Immunogenicity assessments,
such as Anti-Drug Antibody
measurements (ADA)

The SGS Vitrology team is highly
experienced in the capabilities of the
Biacore T200. This system has been
qualified and validated in our laboratories
and is a routinely used tool for rapid
timelines and cost-effective solutions for
many types of studies and a broad range
of applications.

FIGURE 2: SPECIFIC BINDING OF A MONOCLONAL ANTIBODY
BETWEEN TWO ANTIGENS

This sensorgram depicting 0-250 sec = association period; 250-600 sec = dissociation period. In this experi-
ment the antibody anti-GST (glutathione S-transferase) is captured onto the surface of the sensor chip and
the antigen (analyte) either Schistosoma japonicum GST (green) or Human GST (red) is injected separately
using the same concentration (500 nM). Ligand = mouse monoclonal antibody anti- Schistosoma japonicum
GST.

LIFE SCIENCE I TECHNICAL BULLETIN 4

REFERENCES

1. ICH Topic Q 6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (1999).
2. FDA Draft Guidance for Industry, Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product (2012).
3. FDA Point to consider in the manufacturing and testing of monoclonal antibody products for human use (1997).
4. FDA Guidance for Industry Monoclonal Antibodies Used as Reagents in Drug Manufacturing (2001).
5. The European Pharmacopoeia - Monoclonal antibodies for human use (current edition).
6. Guideline on development, production, characterisation and specification for monoclonal antibodies and related products, EMEA/CHMP/BWP/157653/2007.
7. Guideline on similar biological medicinal products containing monoclonal antibodies – non-clinical and clinical issues, EMA/CHMP/BMWP/403543/2010.
8. GE Healthcare, Biacore T200 product description. Available at: https://www.biacore.com/lifesciences/products/systems_overview/Biacore_T200/System-

Information/index.html. Retrieved Jan 28, 2014.

To receive future articles on current trends and regulatory updates, subscribe to SGS’ Life Science News at www.sgs.com/lss_subscribe
Read more about SGS’s biosafety testing services.

CONTACT INFORMATION

EUROPE ASIA NORTH AMERICA
BELGIUM INDIA (CHENNAI) CANADA
+32 10 42 11 11 +91 44 2254 2601 + 1 905 364 3757
[email protected] [email protected] [email protected]

FRANCE (PARIS) INDIA (MUMBAI) USA (FAIRFIELD, NJ)
+33 1 41 06 95 93 +91 22 6457 2540 45 + 1 888 747 8782
[email protected] [email protected] [email protected]

FRANCE (POITIERS) SINGAPORE USA (LINCOLNSHIRE, IL)
+33 (0) 5 49 57 04 04 +65 677 53 034 +1 847 821 8900
[email protected] [email protected] [email protected]

GERMANY (BERLIN) CHINA USA (WEST CHESTER, PA)
+49 30 3460 7500 +86 21 6115 2197 + 1 610 696 8210
[email protected] [email protected] [email protected]

GERMANY (FREIBURG) TAIWAN WWW.SGS.COM/LIFESCIENCE
+49 761 6116 7760 +886 2 2299 3279 ext 2500
[email protected] [email protected]

GERMANY (TAUNUSSTEIN)
+49 6128 744 245
[email protected]

ITALY (LIVORNO)
+39 0586 852591
[email protected]

SWITZERLAND (GENEVA)
+41 22 794 8374
[email protected]

UK (GLASGOW)
+44 141 952 0022
[email protected]

UK (WOKINGHAM)
+44 (0) 1189 896940
[email protected]