Table 2.

Examples of tests to establish equivalence between plant proteins and microbially produced protein surrogates

Parameter compared	Method	Contribution to equivalence assessment	Interpretation of results
Intactness	Western blot analysis	Detection of potential amino acid sequence differences owing to insertions, truncations or degradation; detection of modifications such as glycosylation	Insertions, truncations or modifications indicate potential differences in physicochemical properties including functional activity. Differences may be acceptable depending on results of other parameters identified and the purpose of the safety study
Immuno-reactivity	Western blot analysis	Detection of potential differences in immuno-reactivity	Differences in binding to specific antibodies indicate differences in protein structure; further tests should be conducted to judge impact on equivalence
Intact mass	Mass spectrometry	Detection of insertions, truncations, substitutions and other modifications with higher accuracy and precision than western blotting	As for intactness
Protein sequence	N-terminal sequencing; mass spectrometry	Detection of potential differences in amino acid sequence	Amino acid sequence contributes to the structure and function of a protein. Differences in sequence may be acceptable depending on results of activity assays and the purpose of the safety study
Glycosylation	Immuno-blot analysis	Detection of potential differences in glycosylation status	Glycosylation affects many properties of proteins including stability and function. It has been claimed that glycosylation affects the allergenicity of proteins, although recent work casts doubt on this suggestion. Nevertheless, differences in glycosylation status might be regarded seriously owing to potential variation in physicochemical properties of the proteins
Functional activity	Enzymatic activity assay, insecticidal bioassay	Detection of potential differences in specific catalytic activity (enzymes) or insecticidal bioactivity (toxins)	Confirmation of equivalent activities confirms equivalent protein folding (tertiary and quaternary structure). Depending on the results of other equivalence tests, differences in activity may be acceptable. Differences in activity may be allowed for in safety studies. For example, margins of exposure could be based on comparisons of activity, not concentration

Equivalence is judged separately for each test substance based on a weight of evidence