Circular dichroism (CD), is a commonly used quantitative technique for HOS which is most sensitive to alpha-helix content, providing information about both secondary and some tertiary structure. Fourier transform infrared spectroscopy (FT-IR) is an orthogonal quantitative method for secondary structure determination that is most sensitive to sheet content. Both techniques can be utilized to give an orthogonal assessment of structure, but should not be expected to provide the same result.
Mass Spectrometry is a sensitive and resolving technique that can be used in various forms, including quantitative MS, to interrogate different aspects of the molecule (e.g. protein sequence and post-translational modifications, including glycosylation). Other characterisation techniques used in BioPharmaSpec laboratories include:
This is the question everyone asks! It is clear from regulatory guidelines that the primary protein structure (i.e. the amino-acid sequence) must be the same. However, it is anticipated that minor differences in post-translational forms or product-related impurities will exist. These differences should be investigated thoroughly during biosimilar testing with regard to their potential impact on safety and efficacy. The US FDA states that analytical characterization should show that the biosimilar is “highly similar to the reference product notwithstanding minor differences in clinically inactive components”.
The biosimilar testing guidelines state that at least 6-10 batches of your biosimilar should be compared to at least 6-10 batches of innovator product. This may be done over multiple comparability studies. Any fewer batches than this and it is difficult to make any statistical claims of biosimiliarity. Further information on designing your comparability study plan can be found here.
We work closely with several companies who are authorized to source reference medicinal product (RMP) in the marketplace and can provide you with their details. It is critical from a regulatory perspective that the comparator molecule can be fully traced and authenticated.
Biosimilar development and approval rely heavily on data obtained from structural characterization and comparability studies performed firstly on batches of the reference medicine and subsequently in head-to-head comparisons against batches of the biosimilar candidates. Biosimilar testing will assess the structural, physicochemical, biophysical and functional properties of the molecules.
Small changes to the primary, secondary, tertiary or higher order structure of a biopharmaceutical can have significant effects on the method of action. For example, glycan structures can control the half-life of a biopharmaceutical in the blood stream, receptor binding or other biological effects. Function can also be affected by higher order structure because if the overall 3D shape of the protein is wrong, receptor binding can be inhibited, immunogenic epitopes can be exposed and aggregation can occur.
Using quantitative mass spectrometry, we can quantitate the amount of something present in a sample, such as the Active Pharmaceutical Ingredient (API) itself or a contaminant (e.g. a process related impurity or Host Cell Protein) through the mass spectrometric measurement of its mass relative to that of a known amount of standard.
Regulatory agencies evaluate acceptable limits of HCPs in a biopharmaceutical product on a case-by-case basis, however most biotechnology companies work towards total HCP levels of less than 100 ng per mg (100 ppm).
The data from protein characterization studies are used to make crucial development decisions based on a thorough understanding of the structural attributes of a biopharmaceutical. For this reason, characterization or comparability studies are not performed to GMP, since they are not testing the product against a set specification. Characterization reports are supportive to a regulatory submission and therefore developers want to be confident that the data has been generated using scientifically sound protein characterization methods. Methods and instruments should be qualified and a meaningful and well-designed quality system should be implemented and followed. Standard Operating Procedures (SOP) should be in place and should be regularly monitored and updated.