The conversion of glycolaldehyde and β-hydroxypyruvate to L-erythrulose and carbon dioxide was used as a representative condensation to determine the key data required for rapid process evaluation and the scaleup of useful transketolase-catalyzed biotransformations. The substrates and product exhibited alkaline lability requiring control of reaction pH. The holotransketolase was rapidly deactivated via oxidation and was unstable at pH values less than 6.5 and greater than 10.0. The strength of binding of the cofactors to the enzyme was low at all operational pH values with implications for enzyme immobilization. Glycolaldehyde was found to be toxic to the enzyme and consequently the role of substrate feeding strategies was examined.