Biotechnology Progress

Phase transitions in several plant materials were studied using differential scanning calorimetry (DSC) and small oscillatory amplitude rheology. The glass transition temperature of the unfrozen phase, recrystallization peak of water, and melting point of ice, in fresh plant materials as well as in samples osmotically dehydrated in 15, 30, and 45% sucrose solutions, were identified using both thermal and mechanical analysis. Mechanical analysis showed that the glass transition temperature of the unfrozen phase was approximately ‐45°C for fresh and osmotically dehydrated plant materials at all concentrations. Thermal analysis showed that the glass transition of the unfrozen phase in osmotically dehydrated potato samples varied depending on the amount of freeze‐concentration achieved.

The glass transition temperature (T_g) and structure collapse (volumetric shrinkage) of a freeze‐dried fish protein hydrolyzate (HFP) were studied. An increase in the water activity from 0 to 0.64 reduced the T_g of HFP from 79.1 to −42.8 °C. The Gordon—Taylor equation was a good predictor for the plasticizing effect of water on T_g. At room temperature (19 °C), collapse was initiated at a_w = 0.44 corresponding to a T — T_g value of 35.8 °C, and browning was evident above a_w = 0.55. Above these critical values several structural changes occurred: shrinkage, collapse, browning, and setting into a sticky, high‐viscosity brown liquid. The viscosity of the matrix at the onset of collapse was 10⁵–10⁷ Pa·s, as estimated using the Williams—Landel—Ferry equation.

Soybean peroxidase (SBP) has an extremely high melting temperature of 90.5 °C at pH 8.0 in the presence of 1 mM CaCl₂. The enzyme is substantially more thermostable than the peroxidases from horseradish (HRP) and Coprinus cinereus (CIP). SBP denaturation does not precisely fit the two‐state denaturation model due to the formation of the apoenzyme upon initial melting. A pseudo‐two‐state denaturation can be assumed, however, and this gives rise to apparent kinetics for irreversible inactivation. The apparent kinetics indicate that irreversible deactivation is comprised primarily of enthalpic contributions, with ΔH‡_deact = 22.4 kcal/mol and TΔS‡_deact = 0.2 kcal/mol at 95 °C. Heme transfer studies from the peroxidases to apomyoglobin indicate that SBP holds onto its heme much more tightly than does HRP, and this is consistent with a thermodynamically more stable enzyme.

This article presents two hybrid strategies for the modeling and optimization of the glucose to gluconic acid batch bioprocess. In the hybrid approaches, first a novel artificial intelligence formalism, namely, genetic programming (GP), is used to develop a process model solely from the historic process input‐output data. In the next step, the input space of the GP‐based model, representing process operating conditions, is optimized using two stochastic optimization (SO) formalisms, viz., genetic algorithms (GAs) and simultaneous perturbation stochastic approximation (SPSA). These SO formalisms possess certain unique advantages over the commonly used gradient‐based optimization techniques. The principal advantage of the GP‐GA and GP‐SPSA hybrid techniques is that process modeling and optimization can be performed exclusively from the process input‐output data without invoking the detailed knowledge of the process phenomenology. The GP‐GA and GP‐SPSA techniques have been employed for modeling and optimization of the glucose to gluconic acid bioprocess, and the optimized process operating conditions obtained thereby have been compared with those obtained using two other hybrid modeling‐optimization paradigms integrating artificial neural networks (ANNs) and GA/SPSA formalisms. Finally, the overall optimized operating conditions given by the GP‐GA method, when verified experimentally resulted in a significant improvement in the gluconic acid yield. The hybrid strategies presented here are generic in nature and can be employed for modeling and optimization of a wide variety of batch and continuous bioprocesses.

Commercialization of protein‐based therapeutics is a challenging task in part due to the difficulties in maintaining protein solutions safe and efficacious throughout the drug product development process, storage, transportation and patient administration. Bulk drug substance goes through a series of formulation, fill and finish operations to provide the final dosage form in the desired formulation and container or delivery device. Different process parameters during each of these operations can affect the purity, activity and efficacy of the final product. Common protein degradation pathways and the various physical and chemical factors that can induce such reactions have been extensively studied for years. This review presents an overview of the various formulation‐fill‐finish operations with a focus on processing steps and conditions that can impact product quality. Various manufacturing operations including bulk freeze‐thaw, formulation, filtration, filling, lyophilization, inspection, labeling, packaging, storage, transport and delivery have been reviewed. The article highlights our present day understanding of protein instability issues during biopharmaceutical manufacturing and provides guidance on process considerations that can help alleviate these concerns.

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL‐45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL‐45 on octyl‐Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl‐Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate‐polyacrylamide electrophoresis analysis gel was that corresponding to the SWL‐45, which could be easily desorbed from the support by incubation with triton X‐100, producing a purified enzyme. SWL‐45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL‐45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X‐100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011