Work undertaken in Germany is demonstrating how a new particle size measurement technique is opening up many new applications areas in the laboratory. Heike Krischollek reports on the specific problems posed when dealing with biological materials and how one new measurement system has been designed to overcome these difficulties.
The life sciences, and biotechnology in particular, are rapidly-expanding, innovative fields that attract much attention. The focus is on research and the development and commercialisation of technologies and services for a wide variety of applications.
Work in these areas demands people with specialist skills. It also requires a whole range of laboratory techniques and instrumentation that can meet the analytical challenges that this environment brings. Suppliers and scientists alike are regularly faced with the need to carry out novel measurements on novel and perhaps difficult materials.
Measuring particle size in biological materials poses some specific problems. Photon correlation spectroscopy (PCS) is a suitable method for materials in the size range generally demanded for biotechnology applications, but the properties of the sample can make measurement very difficult. Material tends to be available only in small amounts. Scattering properties may be very weak and samples are often not robust. These factors mean that only highly-sensitive instruments are suitable.
Malvern's Zetasizer HS has been designed to meet all the criteria for measurement of this type of sample (Fig.1).
Proof of process
At Boehringer Ingelheim Pharma KG, particle size measurement of antibodies is a major challenge. Below is a description of work that was undertaken using the Zetasizer HS to demonstrate the relationship between antibody size and the production process temperature, to optimise the process steps and to set the physico-chemical conditions.
Heat inactivation of viruses is an efficient non-invasive physical treatment for high-value pharmaceutical proteins. A disadvantage is the limited stability of the proteins at high temperature and longer retention times. In addition, it is difficult to efficiently control heating of the product solution in large volumes. The necessary validation of an inactivation process is only possible when the process solution is homogeneous and there is no temperature gradient.
A new technology has been developed by Charm Bioengineering Inc of Malden, USA, in co-operation with Boehringer Ingelheim Pharma KG of Biberach, Germany, for a high-temperature, short heating time (HTST) using microwave energy. This makes possible the heating of product solutions in a continuous flow with flow rates up to 80 litres per hour. Inactivation of viruses can be achieved while still maintaining the integrity of the pharmaceutical product.
In the examples shown, a buffered solution of recombinant antibody was exposed to the HTST process with the temperature increased in steps from 20oC to 95oC and PCS used to measure particle size. It can be seen that with increasing temperature there is a shift of the product peak, and a polymer or aggregate peak becomes visible.
In order to use the highest possible temperature, it was first necessary to determine the optimum physico-chemical buffer conditions that would allow the protein solution to be exposed to the maximum temperature without reducing the efficiency of virus inactivation. To do this, the product in a variety of buffer solutions was exposed to microwave treatment and the resulting solutions were screened for aggregates using the Zetasizer HS.
Optimising the process steps
Depending on the physico-chemical conditions, proteins in solution have a tendency to aggregate. Aggregation is often irreversible and leads to an undesirable loss of product.
Using PCS to check the behaviour of proteins under different buffer conditions during the development of the chromatographic and filtration cleaning process allows optimisation of the process steps. This way, consideration is given very early to the buffer solutions to ensure that the proteins retain the desired monomeric shape. Ultimately this delivers improvements in product security and a more robust production process.
PCS measurement is also used in formulation for the development of biotechnologically-produced proteins. Here the challenge is to maintain the active monomeric form of the protein by adjusting the physico-chemical conditions of the solution.
With its new avalanche photo diode (ADP) technology, the Zetasizer HS opens up many new application areas. The signal is 10-20 times greater than with the standard APD detector, providing higher resolution, enhanced reproducibility, and significantly shorter measurement times, even for demanding applications.
Heike Krischollek is with Boehringer Ingelheim, Biberach, Germany.
