Protection Mechanisms of Excipients on Lactate Dehydrogenase during Freeze-Thawing and Lyophilization
Problem - The primary objective of this dissertation was to elucidate the protective effects and mechanisms of excipients on a labile model protein, lactate dehydrogenase (LDH), during freeze-thawing and lyophilization. Cryoprotectants are excipients that protect labile molecules during freezing. Some investigators believe that preferential exclusion is the predominant mechanism to explain the cryoprotection of labile proteins by the polyethylene glycols (PEG's). They assume that the hydration shell around a labile protein remains in tact during freezing, as in solution. But freezing is a separation process, that extracts much of the water into a crystalline ice matrix that is distinctly different from pockets containing the solid amorphous protein. Additionally, proponents of the preferential exclusion theory have not presented direct evidence to support integrity of the hydration shell at temperatures beneath the glass transition temperature. Another possible stabilizing mechanism could be that some excipients preferentially bind to a labile protein to stabilize the macromolecule. A preferential binding mechanism to induce protein stabilization in the frozen state would in direct contrast to the proposed Timasheff preferential exclusion mechanism of protein stabilization. Different hyotheses have been proposed to explain the mechanism/s of protection during the drying portion of the lyophilization cycle. Currently, there are two main hypotheses to explain the mechanisms of labile protein lyoprotection (i.e., stabilization during drying) with excipients. The first is the "single amorphous immobilization hypothesis". This theory proposes that amorphous protein stabilization can only be achieved if another amorphous compound provides immobilization and spatial separation in a glassy, solid matrix during dehydration. The second hypothesis is the "water replacement hypothesis". According to this theory, sugars stabilize proteins by hydrogen bonding and other weak forces of attraction to the polar and charged groups of proteins, to replace the water loss, thus, preventing drying-induced denaturation of proteins. Further experimental evidence is needed to support or disprove the water replacement and single amorphous immobilization hypotheses. Methods - For the freeze-thawing experiments, different molecular weights and concentrations of polyethylene glycols (PEG's) were selected. Lyoprotectants are additives that protect labile molecules during drying. Glucose, sucrose, PEG 8000, dextran 37000 (D 37K) and dextran 160000 (D 160K) were selected as model lyoprotectants for the lyophilization experiments. A variety of methods (e.g., enzymatic activity assays, CD, FTIR, UV, SEC-HPLC, SDS-PAGE, binding studies, DSC, cryostage microscopy) were used to correlate the enzymatic activity recoveries with secondary structure recoveries or tetrameric structure recoveries of LDH upon freeze-thawing and lyophilization. Results and Discussion - In this dissertation, the cryoprotection mechanism was tested by using different molecular weights and concentrations of PEG's during freeze-thawing of LDH solutions. PEG's with molecular weights greater than 4000 showed over 90% activity recovery at concentrations as low as 0.1%. PEG 400 and PEG 8000 at different concentrations showed 60-100% conformational recovery during freeze-thawing. Both activity and CD studies showed that the higher the molecular weights and the higher the concentrations of PEG's, the better activity and secondary structure recoveries of LDH after freeze-thawing. 14C-PEG 4000 binding studies showed an extensive non-specific binding between the LDH and PEG molecules. Moreover, the bound-PEG 4000 increased in a concentration-dependent manner when LDH or PEG 4000 concentrations were increased. The conclusion was that extensive non-specific binding instead of preferential exclusion of PEG's from LDH cryoprotected LDH during freeze-thawing. A model was proposed to illustrate how high molecular weight PEG's cryoprotected LDH monomers and tetramers. In the second part of this dissertation, different molecular weights of saccharides, e.g., glucose, sucrose, dextran 37000 (D 37K) and dextran 160000 (D 160K) with or without PEG 8000, were formulated with LDH at various molar ratios. LDH tetrameric structures were destabilized and activity was lost more when dextrans were formulated and lyophilized with LDH. In the enzymatic activity and the SEC-HPLC studies, lower molecular weights saccharides (glucose and sucrose) prevented over 80% activity and tetrameric structure loss, upon lyophilization and reconstitution. In contrast, LDH lyophilized with higher molecular weight saccharides (dextrans), even at high molar ratios prevented less than 70% tetrameric structure loss. After PEG 8000 was formulated with LDH and various saccharides, activity and tetrameric structure recoveries of LDH were significantly increased (most reached over 90% recovery). SEC-HPLC studies confirmed the results from enzymatic activity assay studies, indicating that enzymatic activity loss and recovery were correlated with tetrameric structure loss and recovery of LDH. LDH formulations with lower molecular weight saccharides (i.e., glucose and sucrose) were able to better protect the thermal stability of LDH than those with higher molecular weight saccharides (dextrans). This was demonstrated by the higher melting temperature (Tm) of LDH when LDH was formulated and lyophilized with lower molecular weight saccharides. PEG 8000 in the LDH-saccharide formulations shifted the Tm higher than the Tm without PEG 8000 in the formulations. Only when LDH was combined with both a saccharide and PEG 8000, was the LDH able to attain maximum enzymatic activity, thermal stability and tetrameric structure recovery. In FTIR studies, LDH lyophilized with saccharides was incapable of fully protecting the LDH secondary structure from degradation. After PEG 8000 was added into the formulations, most of LDH-PEG 8000-saccharide formulations at 1:100:1000 molar ratios showed better preservation of the LDH secondary structures. LDH-PEG 8000-glucose or sucrose formulations upon lyophilization preserved the LDH secondary structures better than the LDH-glucose or sucrose or LDH-PEG 8000 formulations. One LDH-PEG 8000-sucrose formulation showed full preservation of the LDH secondary structure. Since PEG 8000 has been confirmed an effective cryoprotectant that specifically protects proteins during freezing only, saccharides protected the proteins from destabilization during drying. Therefore, PEG 8000 and the saccharides that protected the LDH secondary structure loss worked on different freezing and drying stresses. FTIR studies confirmed the destabilizing effects of the polysaccharide dextrans from SEC-HPLC studies, even though LDH-PEG 8000-D 37K and LDH-PEG 8000-D 160K formulations upon lyophilization preserved the LDH secondary structure better than LDH-D 37K and LDH-D 160K formulations. LDH-PEG 8000-D 37K and LDH-PEG 8000-D 160K formulations upon lyophilization showed worse preservation of LDH secondary structures than the LDH-PEG 8000 formulation. This indicated that dextrans not only did not stabilize LDH during drying, but they disrupted the stabilization effect of PEG 8000 on LDH during freezing. Compared with the LDH secondary structure changes in lyophilized cakes from FTIR, SEC-HPLC and CD, results showed less significant tetrameric and secondary structure changes of LDH-saccharide or LDH-saccharide-PEG 8000 formulations upon lyophilization. This indicated that after reconstitution, some of the unfolded or denatured structures of LDH were refolded. Rehydration often can favor a refolding of protein structures, provided that the unfolding of the protein is reversible. The results of these studies supported the water replacement theory as the predominant mechanism for lyoprotection during drying of LDH. The single amorphous immobilization hypothesis was not supported by these results. Based on the steric hindrance of the bulky dextrans and the "water replacement mechanism", a matrix theory was proposed to explain why sucrose with PEG 8000 had the synergistic protective effect and dextrans with PEG 8000 had antagonistic effects on stabilization of LDH during lyophilization. DSC and freeze-dry cryostage microscopy were used to further explore the thermal properties of the formulations. It was clear that for similar types of saccharide molecules, the larger the molecules, the higher were the phase transition temperatures. D 37K did not show better activity and secondary structure recovery of LDH even though it had a higher collapse temperature and is a large polysaccharide that is amorphous upon solidification. This finding disproved the "amorphous immobilization hypothesis" and showed that amorphous immobilization was not necessary to stabilize labile proteins during lyophilization. As a "proof of principle", the experimental findings were used to produce an acceptable formulation of lyophilized LDH.
LDH, PEG, cryoprotection, lyoprotection, stabilization, freeze-thawing, lyophilization, non-specific binding, CD, SEC-HPLC, FTIR, Freeze-dry cryostage microscope, Medicinal and Pharmaceutical Chemistry, Medicine and Health Sciences, Pharmacy and Pharmaceutical Sciences
University of Tennessee Health Science Center
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