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.

Scientists from Porton Biopharma recently investigated the production of ErA, during which they detected an acidic species of the enzyme using an analytical technique called ion-exchange HPLC (IEX).
 
Efforts were undertaken by scientists from Porton Biopharma, University College London, the University of Sheffield, and Diamond to characterise this acidic variant of ErA. To begin with, samples of the ErA variant were isolated from IEX peaks and a series of analytical techniques were initially applied that included isoelectric focussing and liquid chromatography-mass spectrometry (LC-MS).
 
After discovering that primary-structure modifications were not likely to be the cause of the charged species, SAXS was performed at B21 to closely analyse the structure of the enzyme in high-resolution. SAXS data were collected at 15°C as a series of 30 frames of 10 s duration using 25 µL aliquots of ErA isolates. The data were processed using scÅtter software, which was developed by B21’s Principal Beamline Scientist Rob Rambo.
 
 

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.

Dr David Gervais of Porton Biopharma concluded: “This is a powerful technique that should be used if you have an indication that there is a conformational change in your product”. Porton Biopharma will continue to explore the manufacturing process of ErA to pinpoint the source of the variants and hope to further characterise them in the future.
 
 
To find out more about using the B21 beamline, or to discuss potential applications, please contact Principal Beamline Scientist Dr Robert Rambo: robert.rambo@diamond.ac.uk

 

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).

Related publication

Gervais et al. Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry. Pharmaceutical Research. 32(11) pp. 3636-3648 (2015). DOI: 10.​1007/​s11095-015-1722-2.