A polypeptide having proline-specific endoprotease activity, wherein the polypeptide is a variant polypeptide having less than 70% residual activity on acetyl-AlaAlaPro-paranitroaniline (Ac-AAP-pNA) as a substrate after the polypeptide has been kept at a temperature of 65°C for 15 min as compared to the polypeptide of SEQ ID NO: 1 and is selected from the group consisting of i. a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one combination of amino acid substitutions selected from the group consisting of the combinations (K238E, I204V, V460A), (F279S, A242V, N507I), (T145A, K424M), (T359A, F379S), (M170I, A421T), (N441S, G484S), (L470H, Q288R), (L470H, E387G), (T281S, L373I), (P304A, P469A), and (P466T, P469Q), and optionally one further amino acid substitution I204F, Y205N or P477A wherein the polypeptide does not comprise the amino acid substitution I204F when the polypeptide has the amino acid substitutions K238E, I204V, and V460A, wherein the positions are defined with reference to SEQ ID NO: 1; ii. a polypeptide according to i), but lacking a signal sequence and/or a proprotein sequence; iii. a polypeptide according to i) to ii), wherein the polypeptide has at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence according to SEQ ID NO: 1; and iv. a polypeptide encoded by a nucleic acid which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, or to a mature coding sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises at least one mutation encoding at least one combination of amino acid substitutions selected from the group consisting of (K238E, I204V, V460A), (F279S, A242V, N507I), (T145A, K424M), (T359A, F379S), (M170I, A421T), (N441S, G484S), (L470H, Q288R), (L470H, E387G), (T281S, L373I), (P304A, P469A), and (P466T, P469Q), and optionally one further amino acid substitution I204F, Y205N or P477A wherein the amino acid substitution I204F is not present, when the polypeptide has the amino acid substitutions K238E, I204V, and V460A, wherein the amino acid substitutions are defined with reference to SEQ ID NO: 1. i. a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one combination of amino acid substitutions selected from the group consisting of the combinations (K238E, I204V, V460A), (F279S, A242V, N507I), (T145A, K424M), (T359A, F379S), (M170I, A421T), (N441S, G484S), (L470H, Q288R), (L470H, E387G), (T281S, L373I), (P304A, P469A), and (P466T, P469Q), and optionally one further amino acid substitution I204F, Y205N or P477A wherein the polypeptide does not comprise the amino acid substitution I204F when the polypeptide has the amino acid substitutions K238E, I204V, and V460A, wherein the positions are defined with reference to SEQ ID NO: 1; ii. a polypeptide according to i), but lacking a signal sequence and/or a proprotein sequence; iii. a polypeptide according to i) to ii), wherein the polypeptide has at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence according to SEQ ID NO: 1; and iv. a polypeptide encoded by a nucleic acid which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, or to a mature coding sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises at least one mutation encoding at least one combination of amino acid substitutions selected from the group consisting of (K238E, I204V, V460A), (F279S, A242V, N507I), (T145A, K424M), (T359A, F379S), (M170I, A421T), (N441S, G484S), (L470H, Q288R), (L470H, E387G), (T281S, L373I), (P304A, P469A), and (P466T, P469Q), and optionally one further amino acid substitution I204F, Y205N or P477A wherein the amino acid substitution I204F is not present, when the polypeptide has the amino acid substitutions K238E, I204V, and V460A, wherein the amino acid substitutions are defined with reference to SEQ ID NO: 1.