MELANIE ZERULLA-WERNITZ: Depending on the type of substance, different analysis methods are used. Excipients, such as salts, acids or alkalis, are subjected to chemical identity testing by means of the methods described in pharmacopoeias. For proteins, complex bioanalytical methods, e.g. capillary electrophoresis, are used.
Oligonucleotides are a completely different substance class; for this reason, the commonly used and established methods are not perfectly suited for ID determination. A specific determination of the mass and sequence of the oligonucleotide is decisive.
Therefore, one possibility for the ID determination of oligonucleotides is the sequence analysis (e.g. mass spectrometry), which is already routinely used by API manufacturers.
During the incoming-goods inspection at Vetter, the ID determination of the API manufacturer must be confirmed, preferably with a sequence-specific method. A repetition of the very complex mass-spectroscopic analysis is not required for this purpose.
Which solution did Vetter develop for the ID determination of oligonucleotides?
ALEXANDRA HEUSSNER: We have researched oligonucleotides extensively to understand their specific requirements and challenges.
Our idea was to develop a platform method based on the determination of the melting point. The melting point is defined as the temperature at which half of the oligonucleotides are present as a single strand and the other half as a double strand.
The determination of the melting point is sequence-specific. We wanted to show that the method is robust and sensitive enough to meet our own requirements as well as those of our customers and the regulatory authorities. And we have succeeded in doing this.
How exactly does the procedure work?
ALEXANDRA HEUSSNER: A UV spectrophotometer suitable for use in the pharmaceutical industry is required for the melting point determination. This means that in addition to the necessary measurement accuracy, the data security and data integrity required by the authorities must also be guaranteed. After an extensive search, we found an instrument that meets all requirements.
The measurement itself only takes approx. 15 minutes. In addition, parallel measurements are possible which further increases the speed and efficiency.
The analysis takes place as follows: The absorbance of a sample under UV radiation is measured continuously, while the sample temperature is increased at the same time (figure 1). The absorption increases continuously until it reaches a maximum level. The inflection point of the curve is the melting point (Tm) of the substance, i. e. 50 % of the double strands are then present as single strands. This melting point is sequence-specific for oligonucleotides and, as we now know, a robust parameter. Thus, the ID can be confirmed as required.