Analytical Techniques for Biotherapeutics

Analysing Biotherapeutic Formulations

There is growing interest in the use of spray drying to create biotherapeutic dry powder formulations. Spray drying is becoming increasingly viewed as a more cost-effective method for enhancing the stability of thermo-sensitive molecules (such as proteins, peptides, monoclonal antibodies and vaccines).

A key aspect in the successful development of dry powder formulations is identifying suitable analytical techniques to evaluate the biologic in question. These tests will be needed to: aid formulation development, determine the impact of different spray drying parameters on the product and to determine stability profile. There are a wide range of assays available so it is important to choose suitable assays that can provide insight into two important aspects of the final powders produced, namely assays that show:

The biologic in question retains its structure and activity both during the spray drying process and on subsequent storage.
The spray drying process is generating powders with the necessary physical properties required for powder handling, reconstitution and storage stability.

There is a range of potential assays that can be used to evaluate the spray dried formulation, the list is extensive therefore ,some of the key tests that practitioners might want to consider have been highlighted. The assays have been divided into two groups: those used to evaluate the physical properties of the spray dried powders and those assays designed to evaluate the structure, function and chemical properties of the biotherapeutic itself.

Physical properties of the spray dried powder

Test

Rationale

Particle size analysis
(Laser light scattering, impaction by ACI / NGI)

Particle sizing will give an insight into powder handling requirements, storage stability and performance in delivery devices such as dry powder inhalers.

Residual moisture
(loss on drying, Karl Fischer)

Tight control of residual moisture is an important read out in the optimisation of spray drying conditions and tight specification will be needed in order to meet storage stability targets.

Thermal properties
(DSC / DVS)

Thermal properties, such as moisture uptake, melting points or glass transition temperatures of the powders produced will impact on handling and storage stability.

Powder flow properties
(Bulk / tapped density, angle of repose)

One of the major challenges of spray drying is engineering powders that have suitable handling and flow characteristics.

Electron microscopy
(scanning, transmission)

Important for evaluating particle morphology, evidence of any crystallinity and retention of physical structure of biologic (e.g. virosomes).

Structure, function and chemical properties of the biologic

Test

Rationale

HPLC
analysis (size exclusion, reverse phase)

Established technique for evaluating aggregation of biologic molecules (during spray drying and on storage) and for evaluating chemical modification(s) of specific amino acids.

Gel electrophoresis
(denaturing / non-denaturing, Western blotting)

Technique for evaluating covalent and non-covalent molecular weight changes of structural components (including aggregation, dissociation and scission).

Biologic functional assays
(e.g. enzyme activity, antigenicity, ELISA)

Testing the activity of the molecule being formulated is an important test of the molecule’s integrity during spray drying and on subsequent storage.

Mass spectroscopy

Sensitive technique especially useful for detecting oxidation and deamidation of amino acid side chains that can occur during spray drying and on storage.

Spectrophotometric assays
(reconstituted powder)

Assess reconstitution time, presence of insoluble material (aggregation), substrate binding studies.

Circular Dichroism

Used to determine protein folding, looking at secondary and tertiary structures.