Successful chemo drug monitoring
Variants in enzyme proved to not hinder drug’s activity
Biopharmaceuticals are substances used for medical or diagnostic purposes that are derived from a biological source. One particular biopharmaceutical product, known as L-asparaginase, is used as chemotherapy to treat a form of leukaemia known as acute lymphoblastic leukaemia (ALL). This drug is an enzyme that breaks down a vital amino acid known as asparagine, thereby slowing the proliferation of cancer cells. L-asparaginase can be produced commercially from two different types of bacteria: Erwinia chrysanthemi and Escherichia Coli, and the two resulting enzymes are referred to as ErA and EcA, respectively.
ErA is manufactured and marketed (as Erwinase®) by Porton Biopharma. This company recently carried out an investigation into the manufacturing processes of ErA and noted that small amounts of ErA were subjected to post-translational modifications. In light of this, Porton Biopharma sought to characterise the ErA variant using the capabilities of Diamond Light Source.
Using SAXS (small-angle X-ray scattering) at the Solution State SAXS beamline (B21), it was shown that the aberrant ErA enzyme was a conformational variant that was not chemically different from the parent enzyme. The highly-sensitive technique allowed Porton Biopharma to analyse the ErA variant in solution and provide reassurance to regulatory bodies that the subtle changes did not hinder the activity of the drug.
Monitoring the variants
The procedures involved in the manufacturing of biopharmaceuticals like ErA can introduce product-related variants. These variants, which are often only found in negligible quantities, may be the result of post-translational modifications such as deamidation, methionine oxidation, or even protein-protein aggregation. By monitoring and tracking the origins of variants it is hoped that the quality of a medicinal product can be improved.
SAXS shows conformational change
The application of SAXS showed that the ErA variant had undergone a slight conformational change that led to the enzyme being slightly less compact than the parent protein. This modification was not a chemical change to the enzyme and did not affect the activity of the product.
A further analytical technique known as ion mobility mass-spectrometry (IM-MS) was conducted by Dr Konstantinos Thalassinos at University College London to characterise the variant. This technique corroborated the SAXS results and demonstrated that the acidic ErA was a conformational variant, with very subtle structural differences from the original enzyme. It was postulated that the slight conformational change led to the exposure or shielding of charged residues within the enzyme that prompted the early eluting from the IEX column.
Normalised Kratky Plots from Small-Angle X-ray Scattering Analysis of IEX Peak Isolates. The data are presented for analyses of 1.25 mg/mL enzyme samples in entirety (a) and as a close up to the area of interest (b). The cross hairs indicate the position of an ideal globular protein obeying Guinier’s approximation. The nomenclature of the two axes is as follows: q, scattering vector; I(q), scattered intensity; Rg, radius of gyration; I(0), scattering at zero angle. The parameters Rg and I(0) were obtained from Guinier analyses of the collected SAXS data (not shown).