Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze-and-thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L-lactic dehydrogenase (LDH) in a 700-mL pilot-scale freeze container equipped with internal temperature and pH probes. In 24-hour experiments, target temperature between -10 and -38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of -10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris-HCl and the non-ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE-based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.