Performance of a fast Surface Plasmon Resonance based MAb quantification method

T. Björkman, E. Monié and T. Eriksson GE Healthcare Bio-Sciences AB, R&D, Björkgatan 30, SE-751 84 Uppsala, Sweden

First presented at the 241st ACS meeting, Spring 2011 (27-31 March)

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Introduction •  Monoclonal antibodies (MAb) are large complex molecules

with many possible modifications that may affect safety and efficacy. Hence, many different analytical methods and techniques are needed to characterize and analyze the product.

•  Target molecule concentration is a key attribute that needs to be followed throughout the process, all the way from cell culture to final formulation and from development to final manufacturing. With the introduction of high throughput process development technologies, the number of samples for analysis has increased, and automated, fast, robust and precise analytical methods are essential.

•  In this work we show how Surface Plasmon Resonance (SPR) can be used for MAb quantification.

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Surface Plasmon Resonance (SPR) •  SPR technology allows label-free monitoring of biomolecular-interactions

as they occur.

•  In SPR, an electric field is generated when polarized light strikes a metal surface on a special sensor. This generates electron charge density waves called plasmons that reduce the intensity of the reflected light.

•  The angle at which the intensity minimum occurs is a function of the refractive index of the solution close to the metal layer on the opposing face of the sensor surface and can be used to track molecular interactions.

•  In Biacore™ systems, SPR is used to monitor interactions occurring on a biospecific surface attached to the metal layer.

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SPR measurement principle

Figure 1. An increased sample concentration at the surface coating of the sensor chip causes a corresponding increase in refractive index which alters the SPR angle. By monitoring the SPR-angle as a function of time the kinetic events in the surface are displayed in a sensorgram.

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Figure 2. After receiving a stable baseline the activation reagents, EDC/NHS are applied to the surface. After a short wash the ligand is subjected to the surface and finally the surface is inactivated with ethanolamine. EDC = (1-Ethyl-3-[3dimethylaminopropyl]carbodiimidehydrochloride; NHS = N-hydroxysulfosuccinimide

Immobilization of ligand

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Principle of concentration determination using SPR

Figure 3. To generate a sensorgram a sample is injected over an immobilized surface. By calculating the difference between the response at the report-point stable and baseline, a value that is directly correlated to the concentration is obtained.

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Sensograms from a standard curve for Mab quantification

Figure 4. Examples of sensorgrams obtained with Biacore T-200 for different known concentrations of MAb in a standard curve where the MabSelect SuRe™ ligand (SuRe ligand, a Staphylococcus protein A derivative) has been immobilized on the surface. The difference between the maximum signal and the baseline signal are plotted for each sensorgram. The corresponding difference for any unknown sample can be compared to the standard curve to obtain the concentration.

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Correlation with other methods

Figure 5. The left panel shows correlation between immobilized SuRe ligand and a murine anti human IgG Fc (work from masters thesis “New SPR based assays for plasma protein titer determination of albumin, IgG and IgG subclasses” Feb. 2011, by Johan Kärnhall). The right panel shows correlation between the SPR method with immobilized SuRe ligand and a standard concentration method using a 1 ml HiTrap™ MabSelect SuRe column for the concentration determination. The samples used in the comparisons are cell culture supernatants taken at different times during cell culture from two different fed batch cultures.

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SPR stability over repeated injections

Figure 6. Determinations of two control samples 30 and 50 µg/ml after repeated injections with MAb containing cell culture supernatant in between. The surface shows stability for at least 578 cycles.

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Testing for matrix effects

Figure 7. After purification of a MAb on MabSelect SuRe the flow through (FT) was collected and used for studying “matrix effects” due to interference from sample components. The FT spiked standard curve shows excellent correlation to the normal standard curve in hepes buffered saline containing 0.05 % polysorbate and 30 mM EDTA (HBS-EP).

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Determination of samples against a master standard curve

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Conclusions

•  SPR is an analytical technique that provides fast (5 min/sample) and reliable analyses with high precision

•  The method is characterized by high sensitivity and robustness

•  Due to its high sensitivity, sample dilution can often be used to reduce background interference (matrix effects)

•  The technique can be adapted to almost any biomolecular interaction e.g. for monoclonal antibody concentration measurement

•  A single master standard curve can be used for analyses over a one week period

•  The immobilized surface is durable for at least 578 cycles

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Acknowledgments

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