Purified protein derivatives (PPDs) previously prepared by two different methods from the cultured filtrate of Mycobacterium avium subsp. paratuberculosis (MAP) ATCC strain 19698 were further characterized and compared. The traditional production method, which incorporates autoclaving prior to filtration of the culture media, was compared to a modified method that removed autoclaving during the process. PPD preparations were characterized utilizing mass spectrometry and compared for skin test responses using the guinea pig potency test. Mass spectrometry identified 131 MAP proteins among the three PPD preparations, ten of which present in all preparations. Guinea pig potency testing between PPD preparations resulted in significant difference between production lots. The overall potency of each tested lot was acceptable based upon the measured reactivity. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon gamma production from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.