Making good’s buffers good for freezing: The acidity changes and their elimination via mixing with sodium phosphate

Veselý, Lukáš; Susrisweta, Behera; Heger, Dominik

doi:10.1016/j.ijpharm.2020.120128

Cited by 7 publications

(3 citation statements)

References 67 publications

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“…1d, e, left panel) which suggests a modest pH decrease at the cryogenic temperature. It has been reported that the pH of CAPS can decrease about 1 unit upon freezing, opposite to other Good's buffers, which tend to increase their pH [34,37]. Our findings suggest that the decrease in pH for CHES is even more pronounced than for CAPS; however, to our knowledge no information is reported about this particular buffer system.…”

Section: Discussioncontrasting

confidence: 56%

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

2021

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Due to fast relaxation processes of transition metal ions, electron paramagnetic resonance (EPR) spectroscopy of metalloproteins needs to be performed at cryogenic temperatures. To avoid damaging the biological system upon freezing, a cryoprotectant is generally added to the sample as a glassing agent. Even though cryoprotectants are expected to be inert substances, evidences in literature show their non-innocent role in altering the shape of EPR spectra of proteins and biological objects in general. In this work we conduct a systematic study on the impact of several experimental factors—such as buffer composition, choice of cryoprotectant, pH and temperature—on the EPR spectrum of myoglobin, taken as a reference system for being a well-characterized heme-containing protein. We focus on high-pH buffers to induce and investigate the alkaline transition of ferric myoglobin (pKa ~ 8.9). A combined approach of continuous-wave EPR and UV–visible absorption spectroscopy shows that using particular pairs of buffers and cryoprotectants determines a considerable pH variation in the sample and that this effect is enhanced at cryogenic temperature. In addition, phase memory times were measured to evaluate the efficiency of different cryoprotectants and compared with spectral linewidths in continuous-wave EPR. Our findings suggest that among the selected cryoprotectants ethylene glycol is rather effective, even more than the widely used glycerol, without having unwanted effects.

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Section: Discussioncontrasting

confidence: 56%

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

2021

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“…17−19 Additionally, a peculiar property of buffers upon freezing is the induction of pH gradients. 20,21 For example, commonly used phosphate-buffered saline (PBS) can experience pH changes of as much as 4 units upon freezing. 22 These effects may be suppressed, although not fully eliminated by common sugar cryoprotectants such as mannose, sucrose, or trehalose.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Although LNPs are normally perceived as condensed hydrophobic matter, they can entrap a significant amount of water depending on the composition − and nucleic acid cargo. , LNPs containing messenger RNA can consist of up to 30% water, , which suggests that a seemingly benign variable in LNP formulationchoice of neutralization or storage buffercould have dramatic consequences on LNP formation, properties, and stability, especially since mRNA-LNPs solutions require subzero temperatures for long-term storage . Crystallization of buffering solutions upon freezing can severely affect the properties of biologics and can induce LNP rupture and aggregation as captured in recent reports. − Additionally, a peculiar property of buffers upon freezing is the induction of pH gradients. , For example, commonly used phosphate-buffered saline (PBS) can experience pH changes of as much as 4 units upon freezing . These effects may be suppressed, although not fully eliminated by common sugar cryoprotectants such as mannose, sucrose, or trehalose. ,, Phospholipid headgroups also reportedly interact with buffering agents, causing lipid membrane softening .…”

Section: Introductionmentioning

confidence: 99%

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

et al. 2022

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Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers�HEPES, Tris, and phosphate-buffered saline�with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze−thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.

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Beyond pH: Acid/Base Relationships in Frozen and Freeze-Dried Pharmaceuticals

Heger

Govindarajan

et al. 2023

Principles and Practices of Lyophilization in Product Development and Manufacturing

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Making good’s buffers good for freezing: The acidity changes and their elimination via mixing with sodium phosphate

Cited by 7 publications

References 67 publications

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

Beyond pH: Acid/Base Relationships in Frozen and Freeze-Dried Pharmaceuticals

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