Protection of the enzyme l-asparaginase during lyophilisation—a molecular modelling approach to predict required level of lyoprotectant

Ward, Kevin; Adams, G.D.; Alpar, H. Oya; Irwin, W. J.

doi:10.1016/s0378-5173(99)00163-5

Cited by 29 publications

(22 citation statements)

References 30 publications

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“…FTIR spectra of asparaginase IBs revealed the presence of significantly large proportion of β-sheet content in IBs (Fig. 11b) in comparison to native protein [42]. The crystal structure of asparaginase also entails that the protein is rich in β-pleated secondary structures (Swissprot, P00805, PDBe:3eca).…”

Section: Resultsmentioning

confidence: 98%

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Upadhyay

Murmu²,

Singh³

et al. 2012

PLoS ONE

100

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The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

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Section: Resultsmentioning

confidence: 98%

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Upadhyay

Murmu²,

Singh³

et al. 2012

PLoS ONE

100

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“…For this reason, solutes with high T g are often added to increase T g ′ so that the drying can be carried out at a higher temperature, and a high T g of final dried products is obtained to provide a high storage stability. Disaccharide sugars and oligomeric sugars are preferred as additives for freeze‐drying not only because they exhibit a higher T g (Adams and Ramsay 1996), but also because they can be easily vitrified (Franks 1998; Ward and others 1999). For sugar alcohols such as mannitol, caution must be exercised.…”

Section: Role Of Glassy State During Dryingmentioning

confidence: 99%

Role of Glassy State on Stabilities of Freeze‐Dried Probiotics

Santivarangkna¹,

Aschenbrenner²,

Kulozik³

et al. 2011

Journal of Food Science

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High viability of dried probiotics is of great importance for immediate recovery of activity in fermented foods and for health-promoting effects of nutraceuticals. The conventional process for the production of dried probiotics is freeze-drying. However, loss of viability occurs during the drying and storage of the dried powder. It is believed that achieving the "glassy state" is necessary for survival, and the glassy state should be retained during freezing, drying, and storage of cells. Insight into the role of glassy state has been largely adopted from studies conducted with proteins and foods. However, studies on the role of glassy state particularly with probiotic cells are on the increase, and both common and explicit findings have been reported. Current understanding of the role of the glassy state on viability of probiotics is not only valuable for the production of fermented foods and nutraceuticals but also for the development of nonfermented functional foods that use the dried powder as an adjunct. Therefore, the aim of this review is to bring together recent findings on the role of glassy state on survival of probiotics during each step of production and storage. The prevailing state of knowledge and recent finding are discussed. The major gaps of knowledge have been identified and the perspective of ongoing and future research is addressed.

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“…By removing water from the frozen state, rather than the liquid state, morphology (11,12), solubility, and chemical integrity of biological materials are for the most part maintained, provided that the ice crystals formed are not so large as to disrupt the material (13,14). However, the process generates a variety of freezing and drying stresses such as extra-or intravirus ice crystal formation, solution effects, and pH changes (15)(16)(17). These stresses usually lead to the loss of viral infectivity that can be higher than 90% (18,19) and make the production of the product commercially unviable.…”

Section: Introductionmentioning

confidence: 98%

Untitled

et al. 2004

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Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.

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Protection of the enzyme l-asparaginase during lyophilisation—a molecular modelling approach to predict required level of lyoprotectant

Cited by 29 publications

References 30 publications

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Role of Glassy State on Stabilities of Freeze‐Dried Probiotics

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