The enzymatic synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) starting from D-mannose and pyruvic acid using Neu5Ac-aldolase has been scaled up. A repetitive batch ultrafiltration bioreactor was used for the KDN synthesis on 100 g scale with a conversion of up to 85%. Furthermore, a 440 mt pilot-scale enzyme membrane reactor (EMR) was performed for the continuous production of KDN. Conversion of mannose was 75% at a space-time yield of 375 g/(L d). KDN was advanteageously isolated by crystallization with an overall yield of 75%.