# 开放全文证据摘要包 D2（升级「⚠仅摘要」条目用）

> 数字与条件均为原文直引，不得改写；引用时标注 DOI。

## biot.202000572
- 标题：Lyophilization of premixed COVID-19 diagnostic RT-qPCR reactions enables stable long-term storage at elevated temperature.
- 期刊/年：Biotechnology journal 2021｜DOI: 10.1002/biot.202000572｜PMCID: PMC8237061｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1002_biot.202000572.txt

**摘要（直引节选）**：Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) diagnostic tests for SARS‐CoV‐2 are the cornerstone of the global testing infrastructure. However, these tests require cold‐chain shipping to distribute, and the labor of skilled technicians to assemble reactions and interpret the results. Strategies to reduce shipping and labor costs at the point‐of‐care could aid in diagnostic testing scale‐up and response to the COVID‐19 outbreak, as well as in future outbreaks. In this study we test both lab‐developed and commercial SARS‐CoV‐2 diagnostic RT‐qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization. Fully assembled reactions were lyophilized and stored for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS‐CoV‐2 RNA. Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization. We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze‐dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days. We anticipate that the incorporation of these methods into SARS‐CoV‐2 diagnostic testing will improve testing pipelines by reducing labor at the testing facility and eliminating the need for cold‐chain shipping.

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC8237061 PMID: 33964860 Abstract Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) diagnostic tests for SARS‐CoV‐2 are the cornerstone of the global testing infrastructure.
- We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze‐dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days.
- We find that a single RT‐qPCR kit validated for COVID‐19 diagnostics is highly robust to lyophilization, and can be formulated for storage for at least 30 days at up to 37°C while retaining the ability to detect down to 50 copies of SARS‐CoV‐2 RNA.
- The RTX mix fails to detect SARS‐CoV‐2 below 5,000 copies of the target RNA.
- (C) Standard curve of Cq values measured across a 10‐fold serial dilution of SARS‐CoV‐2 synthetic genomes from 5 through 50,000 copies for N1 (black) and N2 (blue) probe mixes.
- These formulations included a concentration gradient of 0–50 mg mL ‐1 of trehalose, sucrose, or dextran alone, each lyoprotectant at 50 mg mL ‐1 in combination with 100 mM of the osmolyte betaine, pairs of lyoprotectants mixed together at 20 mg mL ‐1 each to test synergistic interactions, and a no lyoprotectant control.
- Lyophilized reactions were then incubated for 14 days at room temperature (∼23°C).
- All formulations were tested after 1, 7, and 14 days with 10,000 copies of synthetic SARS‐CoV‐2 RNA to ensure ample template for assessing activity of the reactions.
- However, the effectiveness of the reactions rapidly degraded over the course of the incubation, with only 36% and 32% of reactions successfully detecting SARS‐CoV‐2 RNA after 7 and 14 days at room temperature, respectively (Figure S 2B‐C ).
- PrimeScript responded slightly more robustly to lyophilization, with 64% of reactions retaining activity after 14 days (Figure 3C ).
- GoTaq provided the most promising results in our reaction conditions, with all but one (98.6%) of the reactions detecting SARS‐CoV‐2 RNA across all time points tested (Figure 2A , Figure S 4A‐C ).
- (A) Results for fully assembled, lyophilized diagnostic reactions using the N1 probe mix and the GoTaq commercial reaction mixes after 14 days incubation at ambient temperature (∼23°C) and inoculation with 10,000 copies of SARS‐CoV‐2 RNA.
- (B) Cq values over time for GoTaq reactions tested with 10,000 copies of synthetic SARS‐CoV‐2 RNA.
- Reactions were stable over 14 days under all conditions tested, with only one aberrant reaction at 7 days in the presence of 10 mg mL ‐1 trehalose.
- Reactions tested with 10,000 copies of SARS‐CoV‐2 RNA display increased final fluorescent signal in the presence of higher concentration of lyoprotectant.
- Since final RFU of GoTaq reactions incubated for 14 days in the prior experiment continued to increase up to 50 mg mL ‐1 of lyoprotectant (Figure 2C ), higher concentrations were tested in this experiment.
- GoTaq reaction mix was assembled with N1 primer/probe and lyophilized with no lyoprotectant, sucrose concentrations of 50, 75, or 100 mg mL ‐1 , or with sucrose/trehalose mixes of 20, 30, and 40 mg mL ‐1 each.
- These reactions were incubated at 23°C, 37°C, or 50°C for 30 days.
- After 30 days, reactions were tested with a dilution series of SARS‐CoV‐2 RNA at concentrations ranging from 5 to 50,000 copies.
- After 30 days, reactions incubated at 23°C and 37°C were still capable of detecting SARS‐CoV‐2 RNA at concentrations as low as five total copies (Figure 3A , 3B ).
- However, if the limit of detection (LOD) is defined as the concentration of template at which > 95% of samples containing SARS‐CoV‐2 RNA are identified as positive, [ 30 ] then the optimal LOD is five total copies (1,000 copies mL ‐1 ) for non‐lyoprotected reactions incubated at 23°C and 50 total copies (10,000 copies mL ‐1 ) for lyoprotected reactions incubated at 37°C (Figure 3A‐B , lower table).
- While this value for samples incubated at 37°C is higher than the LOD of the non‐lyophilized CDC assay at 1,000 copies mL ‐1 , [ 31 ] the sensitivity of the lyophilized assay could theoretically be brought up to this value by increasing the scale of the reaction from 5 to 50 μL.
- In addition, reactions incubated at 23°C or 37°C displayed no significant difference in Cq value between the unprotected reactions and the lyoprotected reactions.
- GoTaq diagnostic reactions were premixed with no lyoprotectant (None), 50, 75, or 100 mg mL ‐1 sucrose (Suc50, Suc75, Suc100) or a mixture of 20, 30, or 40 mg mL ‐1 each of sucrose and trehalose (ST20, ST30, ST40) and lyophilized.
- Reactions were incubated for 30 days at (A) 23°C, (B) 37°C, or (C) 50°C to assess stability of reactions exposed to elevated temperatures for long periods.
- Diagnostic reactions were performed on a dilution series from 5 to 50,000 copies of synthetic SARS‐CoV‐2 RNA.

**工艺/辅料句（直引）**：
- Lyophilization of premixed COVID-19 diagnostic RT-qPCR reactions enables stable long-term storage at elevated temperature.
- In this study we test both lab‐developed and commercial SARS‐CoV‐2 diagnostic RT‐qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization.
- Fully assembled reactions were lyophilized and stored for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS‐CoV‐2 RNA.
- Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization.
- Keywords: COVID‐19 diagnostics, cell‐free, synthetic biology, lyophilization, RT‐qPCR RT‐qPCR diagnostics are at the foundation of the global fight against the COVID‐19 pandemic, but the scale‐up, distribution, and administration of these tests in great numbers has proven a great logistical challenge.
- In this work, we demonstrate that fully premixed RT‐qPCR reactions for the detection of the SARS‐CoV‐2 virus may be lyophilized and stored for extended periods at ambient or elevated temperatures without loss of activity.
- We anticipate that lyophilized RT‐qPCR diagnostic tests will allow shipping outside the cold chain, reduce labor at the point of testing, and aid in the development of automated testing pipelines.
- One strategy to enable distribution of preassembled RT‐qPCR diagnostic reactions without the need for the cold‐chain storage is lyophilization (i.e., freeze drying), which would reduce distribution and storage costs and labor in the diagnostic lab.
- Lyophilization is a common strategy to confer stability to biological samples and biochemical reactions, enabling the storage of samples as a dry powder at ambient temperature for later rehydration.
- [ 6 ] In recent years, lyophilization has been used by synthetic biologists to enable cell‐free systems for on‐demand biomanufacturing, biosensing, and educational kits.
- [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ] Further, lyophilized in vitro transcription and PCR‐based detection mixtures have demonstrated superior qualities for providing diagnostics in resource‐limited settings.
- [ 13 , 15 , 16 ] To prevent the loss of activity during lyophilization and storage, additives referred to here as lyoprotectants can be implemented and optimized to stabilize biological molecules in freeze‐dried mixes.

## s11095-023-03607-9
- 标题：Practical Advice on Scientific Design of Freeze-Drying Process: 2023 Update.
- 期刊/年：Pharmaceutical research 2023｜DOI: 10.1007/s11095-023-03607-9｜PMCID: PMC10661802｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1007_s11095-023-03607-9.txt

**摘要（直引节选）**：Objective The purpose of this paper is to re-visit the design of three steps in the freeze-drying process, namely freezing, primary drying, and secondary drying steps. Specifically, up-to-date recommendations for selecting freeze-drying conditions are provided based on the physical–chemical properties of formulations and engineering considerations. Methods and Results This paper discusses the fundamental factors to consider when selecting freezing, primary drying, and secondary drying conditions, and offers mathematical models for predicting the duration of each segment and product temperature during primary drying. Three simple heat/mass transfer primary drying (PD) models were tested, and their ability to predict product temperature and sublimation time showed good agreement. The PD models were validated based on the experimental data and utilized to tabulate the primary drying conditions for common pharmaceutical formulations, including amorphous and partially crystalline products. Examples of calculated drying cycles, including all steps, for typical amorphous and crystalline formulations are provided. Conclusions The authors revisited advice from a seminal paper by Tang and Pikal (Pharm Res. 21(2):191-200, 2004) on selecting freeze-drying process conditions and found that the majority of recommendations are still applicable today. There have been a number of advancements, including methods to promote ice nucleation and computer modeling for all steps of freeze-drying process. The authors created a database for primary drying and provided examples of complete freeze-drying cycles design. The paper may supplement the knowledge of scientists and formulators and serve as

**含数值句（直引）**：
- Freeze-dryers are usually loaded at either room temperature (usually 20 to 25°C) or 5°C when liquid stability is a potential concern.
- On rare occasions, a product is loaded onto shelves cooled below 0°C (e.g., to -50°C);
- After loading a freeze-dryer, vials are typically equilibrated for at least 30 min before cooling to minimize vial-to-vial temperature variations.
- As noted in [ 1 ], “ it is not practical to manipulate the supercooling by changing the cooling rate in a freeze dryer because the cooling rates are usually limited to less than 2°C/min, and the degree of supercooling is unlikely to change within such a small range .” It remains a true statement based on almost two decades since the publication of that paper.
- Gitter et al.[ 19 ] conducted ice nucleation was achieved at -5°C using ice fog or depressurization methods in systems containing excipients (sucrose, trehalose) or their mixtures (mannitol and sucrose at a 4:1 ratio) and in monoclonal antibodies containing formulation (in sucrose, or trehalose, or mannitol-sucrose and 10 mM histidine buffer and 0.02% w/v polysorbate 80).
- Ice nucleation was conducted at either -3°C or -10°C in monoclonal antibody formulations (10 mg/mL) in either sucrose or trehalose (disaccharide at 7.5% w/v).
- Luoma and coworkers investigated the effect of depressurization, partial vacuum, and ice fog techniques on product attributes of a monoclonal antibody formulation (10 and 100 mg/mL mAb in sucrose and histidine buffer-based formulations) and also an enzyme formulation (2.5 mg/mL in 500 mM arginine phosphate and polysorbate 80) post drying and during storage in vials of different sizes.
- In the author's experience, an increase in cooling rate from 0.3 to 1°C/min resulted in a notable improvement of cake appearance for one of the small molecule drugs due to a reduction in phase separation.
- As for commercial cycles, cooling rates of 0.5°C/min or lower are recommended to ensure product temperature homogeneity across freeze-dryer shelves [ 26 ].
- Annealing temperatures between -15°C and -10°C for 3–5 h is recommended as a temperature range above Tg’ for most of the products that use lyophilization.
- If product Tg’ is relatively high (e.g., -7 to -15°C), annealing may not be beneficial.
- In the glycine-sucrose-water system, for example, the critical cooling rate decreased from > 20°C/min for a solution with glycine/sucrose ratio of 3.0 to < 0.5°C/min at glycine/sucrose ratio of 0.9 [ 34 ].
- For 5% mannitol solution in glass vials, the critical cooling rate was estimated to be between 0.2°C/min and 0.5°C/min [ 35 ] .
- Final Freezing Temperature and Hold Time Regarding final freezing temperature and hold time, Tang and Pikal[ 1 ] recommended targeting a final freezing product temperature of -40°C in most cases.
- Due to the radiation effect, shelf temperature should be lower (-45°C or lower) to ensure that product temperature in edge vials remains below Tg’.
- 2 Freezing time to reach -44.8 °C is calculated using the freezing model [ 38 ] .
- The top panels ( a , b ) represent a total freezing time for the product (including the ramp) to reach -44.8°C at a cooling rate of 1°C/min (left panel) and 0.5°C/min (right panel) as a function of fill volume for different vial sizes.
- Figure 2 demonstrates that 1 h hold time would be sufficient for fill volumes of less than 1.5 mL (2R vial) and 5.5 mL (20R vial) at 1C/min ramp rate (fill depth 1 cm for both 2R and 20 R- table A1 ) which is in alignment with Tang and Pikal [ 1 ].
- Additional soak time (about 30 min) can be added to calculated values shown in Fig.
- The critical first step in establishing primary drying conditions is selecting the target product temperature, commonly chosen as 2°C below the critical product temperature.
- Nevertheless, the recommendation by Tang and Pikal [ 1 ] represents a very reasonable approach for the majority of situations nowadays: “ a small safety margin (2°C) be used if freeze-drying time is long (e.g., more than two days), a large safety margin (5°C) be used if freeze-drying time is short ( < 10 h), and safety margin of 3°C be used if primary drying time is somewhere between 2 days and 10h.
- The Tsc (temperature of secondary crystallization/melt) is commonly above -5°C for both mannitol- and glycine-based formulations (exact value depends on the concentration and nature of other solutes present);
- therefore, the target product temperature could be conservatively selected as -10°C.
- However, performing primary drying at such high target product temperatures could overload an old freeze-dryer, as described in [ 1 ], who therefore recommended target product temperature not to exceed -15°C.
- Recent advances in freeze-dryer design could allow higher sublimation rates, so the target temperature could increase to at least -10°C.
- The transition from freezing to the primary drying step is usually straightforward, with a vacuum applied at the end of the freezing segment, followed by heating the shelf to the pre-determined set-point, usually at the ramp rate of 0.5 or 1 °C/min.

**工艺/辅料句（直引）**：
- Abstract Objective The purpose of this paper is to re-visit the design of three steps in the freeze-drying process, namely freezing, primary drying, and secondary drying steps.
- Specifically, up-to-date recommendations for selecting freeze-drying conditions are provided based on the physical–chemical properties of formulations and engineering considerations.
- Methods and Results This paper discusses the fundamental factors to consider when selecting freezing, primary drying, and secondary drying conditions, and offers mathematical models for predicting the duration of each segment and product temperature during primary drying.
- Three simple heat/mass transfer primary drying (PD) models were tested, and their ability to predict product temperature and sublimation time showed good agreement.
- The PD models were validated based on the experimental data and utilized to tabulate the primary drying conditions for common pharmaceutical formulations, including amorphous and partially crystalline products.
- 21(2):191-200, 2004) on selecting freeze-drying process conditions and found that the majority of recommendations are still applicable today.
- There have been a number of advancements, including methods to promote ice nucleation and computer modeling for all steps of freeze-drying process.
- The authors created a database for primary drying and provided examples of complete freeze-drying cycles design.
- Keywords: freeze-drying, lyophilization, mathematical modeling, process design status released display-pdf yes is-in-collection-domain yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2023 Mar 20;
- Introduction Freeze-drying (lyophilization) is a common pharmaceutical manufacturing process used to produce various drug products.
- Freeze-drying consists of 3 segments, i.e., freezing, primary drying (ice sublimation), and secondary drying (desorption of unfrozen water), with primary drying (ice sublimation) being the longest.
- Freezing is a critical stage of a freeze-drying cycle, as the structure and morphology of a frozen cake can influence product behavior during primary drying.

## j.talanta.2021.122797
- 标题：Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics.
- 期刊/年：Talanta 2021｜DOI: 10.1016/j.talanta.2021.122797｜PMCID: PMC8353973｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1016_j.talanta.2021.122797.txt

**摘要（直引节选）**：As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

**含数值句（直引）**：
- In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing.
- The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C.
- Using this method, the quantification cycle (Cq) values of degraded reagents would be larger than those with 100 % activity ( Fig.
- The extracted nucleic acids were stored in a 1.5-mL sample tube and maintained at −80 °C before PCR.
- Freezing step All PCR components were added to a 15 mL centrifuge tube, supplemented with trehalose [10 % final concentration (w/v), Sigma-Aldrich], mannitol [2.5 % final concentration (w/v), Sigma-Aldrich] and polyethylene glycol 20,000 [ PEG20000 , 1.5 % final concentration (w/v), Sigma-Aldrich].
- After solidification, the beads were transferred to a metallic tray and stored at −20 °C for 1 h to anneal.
- The tray containing above beads was then transferred to a freeze dryer (Advantage 2.0, VITRIS) whose shelf was pre-cooled to −40 °C.
- (C) The freeze-dried reagents are convenient to use because they only require reconstitution in water, and the reconstitution process can be done in less than 5 s.
- Freeze-drying process After loading the samples, the shelf temperature of the freeze dryer was maintained at −40 °C for 720 min, followed by 25 °C for 180 min.
- The entire freeze-drying process was performed in an environment with a humidity of less than 2 %.
- Real-time and accelerated stability testing The packed freeze-dried PCR mixes were stored at RT (18–25 °C), 37 °C, 56 °C, and 80 °C for a long time.
- And the reconstitution process could be done within 5 s ( Fig.
- 19 PCR mixes for different target detection purposes were able to be stored at RT (18–25 °C) for 1–2 years, thus far ( Fig.
- 3 In above 19 PCR mixes, except for the sequence specific primers and probes ( Table S2 ), the other components, including 10 × PCR Buffer, dNTPs, DNA polymerase, and reverse transcriptase (for RNA target detection only) were all the same.
- Because the packaging consumables could not withstand temperatures above 200 °C, temperatures of 60, 80, 100, 120, 140, 160, 180, and 200 °C were chosen to determine the highest temperature that could be used for testing.
- 4 Ai) was stored at 100 °C or higher, it shrank rapidly ( Fig.
- By contrast, no obvious changes in appearance or activity were observed when the bead was stored at 60 °C or 80 °C for more than 12 h.
- Thus, 80 °C was chosen as the highest temperature.
- (A) Appearance changes over time in freeze-dried beads when stored at 100 °C or higher.
- 4 To obtain comprehensive evaluation results, 37 °C and 56 °C were also examined, in addition to 80 °C and RT.
- Therefore, the temperature gradient selected for accelerated stability testing was RT, 37 °C, 56 °C, and 80 °C.
- Shortened evaluation period for the freeze-dried PCR mix Accelerated stability testing was further performed following storage at the selected temperatures (37 °C, 56 °C, and 80 °C) to verify the feasibility of shortening the evaluation period.
- The freeze-dried PCR mixes for RNA target detection could be stored at 37 °C, 56 °C, and 80 °C for 99 days, 6 days, and 1 day, respectively ( Fig.
- The mixes for DNA target detection could be stored at 37 °C, 56 °C, and 80 °C for 617 days, 26 days, and 21 days, respectively ( Fig.
- These results indicated that the evaluation process for freeze-dried PCR mixes could be accelerated using higher temperatures: for the reagents used for RNA detection, 99 days at 37 °C, 6 days at 56 °C or 1 day at 80 °C would be equivalent to 567 days at RT.
- Similarly, 26 days at 56 °C or 21 days at 80 °C of reagents for DNA detection would be equivalent to 617 days at RT or even 37 °C.

**工艺/辅料句（直引）**：
- In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis.
- Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily.
- As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced.
- When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees.
- Keywords: Microfluidic molecular diagnostics, Room-temperature-storable, Freeze-dried PCR mixes, Accelerated stability testing Graphical abstract Image 1 status released display-pdf yes is-in-collection-domain yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2021 Jun 12;
- However, traditional PCR reagents typically made for central laboratories are not applicable for microfluidic molecular diagnosis, unless a freeze-drying method is introduced.
- Freeze-drying allows for the preservation of activity in qPCR reagents over the long-term storage at RT because this process removes most of the water molecules [ 7 ].
- Thus, microfluidic chip contained the freeze-dried reagents can be stored everywhere irrespective of local preservation conditions.
- Secondly, liquid-form PCR reagents are cumbersome and complicated to prepare, whereas freeze-dried reagents are convenient to use because they only require reconstitution in water.
- Thirdly, instead of loading each component of PCR reagent to microfluidic chip separately, one can transfer all these components into the chip easily if they are freeze-dried as beads ( Fig.
- Freeze-dried PCR mixes can make up for it if they are reconstituted with sample instead of water ( Fig.
- 1 Schematic diagrams of the freeze-dried PCR mixes and related methods.

## pnas.86.2.520
- 标题：Membrane phase transitions are responsible for imbibitional damage in dry pollen.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 1989｜DOI: 10.1073/pnas.86.2.520｜PMCID: PMC286503｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.86.2.520.txt

**摘要（直引节选）**：Membrane phase transitions are responsible for imbibitional damage in dry pollen. PMCID: PMC286503 DOI: 10.1073/pnas.86.2.520 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 1989 Jan;86(2):520–523. doi: 10.1073/pnas.86.2.520 Membrane phase transitions are responsible for imbibitional damage in dry pollen John H Crowe John H Crowe * Department of Zoology, University of California, Davis, CA 95616 Find articles by John H Crowe *, † , Folkert A Hoekstra Folkert A Hoekstra * Department of Zoology, University of California, Davis, CA 95616 ‡ Plant Physiology Department, Agricultural Uni

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC286503 PMID: 16594011 Abstract We have found that the most probable cause of the leakage seen when dry cells or organisms such as seeds, pollen, or yeast cells are plunged into water is a gel to liquid crystalline phase transition in membrane phospholipids accompanying rehydration.

**工艺/辅料句（直引）**：
- Preservation of membranes in anhydrobiotic organisms: the role of trehalose.
- Preservation of freeze-dried liposomes by trehalose.
- The influence of temperature on the reconstitution of active dry yeast.

## fchem.2018.00004
- 标题：Detection of Collapse and Crystallization of Saccharide, Protein, and Mannitol Formulations by Optical Fibers in Lyophilization.
- 期刊/年：Frontiers in chemistry 2018｜DOI: 10.3389/fchem.2018.00004｜PMCID: PMC5790775｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.3389_fchem.2018.00004.txt

**摘要（直引节选）**：The collapse temperature (Tc) and the glass transition temperature of freeze-concentrated solutions (Tg') as well as the crystallization behavior of excipients are important physicochemical characteristics which guide the cycle development in freeze-drying. The most frequently used methods to determine these values are differential scanning calorimetry (DSC) and freeze-drying microscopy (FDM). The objective of this study was to evaluate the optical fiber system (OFS) unit as alternative tool for the analysis of Tc, Tg' and crystallization events. The OFS unit was also tested as a potential online monitoring tool during freeze-drying. Freeze/thawing and freeze-drying experiments of sucrose, trehalose, stachyose, mannitol, and highly concentrated IgG1 and lysozyme solutions were carried out and monitored by the OFS. Comparative analyses were performed by DSC and FDM. OFS and FDM results correlated well. The crystallization behavior of mannitol could be monitored by the OFS during freeze/thawing as it can be done by DSC. Online monitoring of freeze-drying runs detected collapse of amorphous saccharide matrices. The OFS unit enabled the analysis of both Tc and crystallization processes, which is usually carried out by FDM and DSC. The OFS can hence be used as novel measuring device. Additionally, detection of these events during lyophilization facilitates online-monitoring. Thus the OFS is a new beneficial tool for the development and monitoring of freeze-drying processes.

**含数值句（直引）**：
- Adel Mhamdi, RWTH Aachen University, Germany ✉ *Correspondence: Wolfgang Friess wolfgang.friess@lrz.uni-muenchen.de This article was submitted to Chemical Engineering, a section of the journal Frontiers in Chemistry 26 1 2018 6 4 4 12 2 2018 Copyright © 2018 Horn and Friess.
- Tc values are usually 1–3°C higher than Tg' values and drying above Tc may result in macrocollapse of the lyophilizate.
- Nevertheless, since it is known that an 1°C increase in Tp can shorten primary drying times by about 13%, the interest is to dry at the highest possible Tp (Pikal, 1990 ).
- Furthermore, the high heating rates of 5–20°C/min in DSC analysis do not correspond to the typical 0.5–1°C/min during lyophilization but facilitates the Tg' analysis due to a more distinct baseline shift (Her and Nail, 1994 ).
- Materials and methods Materials Sucrose (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), trehalose (Hayashibara, Okayama, Japan), stachyose (A & Z Food Additives, Hangzhou, China), and D(-)-mannitol (VWR International, Ismaning, Germany) were used in different concentrations (5, 10, or 20% [m/V]) as excipient formulations.
- 150 mg/mL mAb with 10% (m/V) sucrose in 10 mM sodium phosphate buffer pH 7.0 and 100 mg/mL lysozyme with 10% (m/V) trehalose in purified water served as protein formulations.
- All formulations were filtered with 0.2 μm polyethersulfone membrane syringe filters (VWR International GmbH, Ismaning, Germany) prior to use.
- T range: −40 to 120°C, short term accuracy ± 0.2°C, response time 0.3 s).
- OFS data were recorded every 10 s for the complete run time (ENLIGHT 1.0 Sensing Analysis Software by Micron Optics).
- 6R or 10R glass vials (Fiolax™ clear, MGlas AG, Münnerstadt, Germany) filled with either 4 or 6 mL were stoppered for freeze/thawing experiments or semi-stoppered for lyophilization runs with corresponding lyophilization stoppers (type: Westar®, West Pharmaceutical Services, Eschweiler, Germany).
- OFS–determination of the Tc and Tcry during freeze/thawing Freeze/thaw cycles between +20 and −50°C at 2.5, 1.0, or 0.5°C/min with 90 min hold times were carried out using a FTS LyoStar™ 3 (SP Scientific, Stone Ridge, NY, USA) freeze-dryer.
- Table 1 Collapse and non-collapse freeze-drying cycles of sucrose (20%/10%) and stachyose (20%) solutions.
- 30 μL sample in 40 μL aluminum crucibles were cooled down to −60°C and heated to + 20°C at 10, 2.5, 1.0, or 0.5°C/min.
- Freeze-drying microscopy (FDM) Collapse temperatures Tc were determined by FDM (Linkam FDCS 196 freeze-drying stage with LinkSys32 software, Linkam Scientific Instruments, Tadworth, United Kingdom) equipped with a Duo 2.5 vacuum pump (Pfeiffer Vakuum GmbH, Asslar, Germany) and an AxioImager A1 microscope used with a 200-fold magnification (Carl Zeiss AG, Oberkochen, Germany).
- Samples were cooled down to −50°C with 1°C/min.
- A pressure of 0.1 mbar was applied and the samples were heated to −40 or −30°C with 5°C/min.
- Tc was detected as onset of collapse in the following drying step at 1°C/min to 0°C.
- The combination of ice crystal growth, solidification of the product and increasing viscosity due to cryoconcentration led to a shift to lower wavelengths with decreasing the Tp to −50°C.
- During thawing the unshielded OFS sensors showed a pronounced peak close to −30°C, which could be explained by the rapid change in viscosity of the amorphous freeze-concentrate and thus release of mechanical strain (Akyurt et al., 2002 ;
- Figure 2 OFS thermogram of sucrose 20% (m/V).
- Detailed investigation of the T OFS peak First, it was to be proven whether the T OFS peak at around −30°C resulted from the glass transition and was not an artifact.
- The two commonly used disaccharides sucrose and trehalose both showed Tg' and T OFS values of around −32 to −28°C (Figure 3 ).
- Stachyose, a rarely used tetrasaccharid, showed a Tg' value of −23.8 ± 0.2°C and a T OFS peak at −23.8 ± 0.4°C.
- black line: 20%, dark gray line: 10%, gray line: 5%;
- Typically, in literature heating at 5 to 20°C/min is performed as the Tg can be detected with higher sensitivity and sharpness of the transition as compared to analyzing at lower rates (Coleman and Craig, 1996 ;
- Nevertheless it would be beneficial to measure a more realistic Tg' value at slower heating rates e.g., at 1°C/min.

**工艺/辅料句（直引）**：
- Abstract The collapse temperature (Tc) and the glass transition temperature of freeze-concentrated solutions (Tg') as well as the crystallization behavior of excipients are important physicochemical characteristics which guide the cycle development in freeze-drying.
- The most frequently used methods to determine these values are differential scanning calorimetry (DSC) and freeze-drying microscopy (FDM).
- The objective of this study was to evaluate the optical fiber system (OFS) unit as alternative tool for the analysis of Tc, Tg' and crystallization events.
- The OFS unit was also tested as a potential online monitoring tool during freeze-drying.
- Freeze/thawing and freeze-drying experiments of sucrose, trehalose, stachyose, mannitol, and highly concentrated IgG1 and lysozyme solutions were carried out and monitored by the OFS.
- The crystallization behavior of mannitol could be monitored by the OFS during freeze/thawing as it can be done by DSC.
- Online monitoring of freeze-drying runs detected collapse of amorphous saccharide matrices.
- Additionally, detection of these events during lyophilization facilitates online-monitoring.
- Thus the OFS is a new beneficial tool for the development and monitoring of freeze-drying processes.
- Keywords: freeze-drying, lyophilization, optical fiber system, glass transition, collapse, crystallization, monitoring status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2017 Nov 6;
- Introduction Freeze-drying is commonly used for the long-term stabilization of biopharmaceuticals which cannot be stabilized adequately in the liquid state.
- Efficient development of freeze-drying cycles is of utmost importance as the process is time and cost consuming.

## pharmaceutics16101346
- 标题：Effectiveness of Lyoprotectants in Protein Stabilization During Lyophilization.
- 期刊/年：Pharmaceutics 2024｜DOI: 10.3390/pharmaceutics16101346｜PMCID: PMC11510631｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.3390_pharmaceutics16101346.txt

**摘要（直引节选）**：Background: Proteins are commonly used in the healthcare industry to treat various health conditions, and most proteins are sensitive to physical and chemical changes. Lyophilization, also known as freeze-drying, involves sublimating water in the form of ice from a substance at low pressure, forming a freeze-dried powder that increases its shelf life. Extreme pressure and varying temperatures in the freeze-drying process may damage the protein’s structural integrity. Lyoprotectants are commonly used to protect protein conformations. It is important to choose a suitable lyoprotectant to ensure optimal effectiveness. Method: Twenty articles screened from Scopus, Web of Science, and PubMed were included in this review that discussed potential lyoprotectants and their effectiveness with different protein models. Results: Lyoprotectants were categorized into sugars, polyols, surfactants, and amino acids. Lyoprotectants can exhibit significant protective effects towards proteins, either singularly or in combination with another lyoprotectant. They exert various interactions with the protein to stabilize it, such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, and osmoprotection. Conclusions: This review concludes that disaccharides are the most effective lyoprotectants, while other groups of lyoprotectants are best used in combination with other lyoprotectants.

**含数值句（直引）**：
- It is crucial to preserve materials that require a sterile and delicate preservation technique and minimal moisture (less than 1%) to maintain stability [ 4 , 5 ].
- Disaccharides Lactose AH (Anhydrous): ~−28 °C, Lactose MH (Monohydrate): ~−31 °C, Sucrose: ~−32 °C, Trehalose: ~−27 °C, Maltose: ~−34 °C Disaccharides are commonly used as lyoprotectants due to their relatively high Tg′ values compared with monosaccharides, which help maintain stability in the glassy state.
- Trisaccharide Raffinose: ~−35 °C Trisaccharides like raffinose have Tg′ values that are somewhat intermediate between mono- and disaccharides, providing different stabilization characteristics.
- Polysaccaride Poly-vinylpyrimidine: 100 °C to 175 °C, Dextran-40: −15 °C to −20 °C, Dextran 6 kDa: −25 °C to −30 °C, Dextran: −10 °C to −20 °C (Tg′ increases with molecular weight due to reduced molecular mobility.) PVP has a high Tg′ and is excellent for forming stable glassy matrices, providing good protection for proteins during freeze-drying.
- Amino acids Glycine: ~−35 °C (when used in combination with other excipients), Alanine: ~−45 °C, Proline: ~−52 °C Amino acids generally have lower Tg′ values and are often used in combination with other stabilizers to achieve optimal protective effects.
- Solubilizers b-cyclodextrin, PEG 6000: −55 °C to −65 °C.
- also, with sucrose and glucose at 5% to 10% concentrations, no adverse reactions such as shrinkage, melting, puffing, or collapsing were observed [ 19 ].
- The addition of Tween-80 (0.04%) to the formula improved porosity, reduced bacterial aggregation, achieved minimal moisture (0.4%), and enhanced water reabsorption [ 19 ].
- The mRNA-lipid nanoparticles (LNPs) lyophilized with sucrose/trehalose/mannitol mixture were stable at 2–8 °C, and they did not reduce immunogenicity in vivo or in vitro with a rigid structure with large porosity, which tolerated rapid temperature increases and efficiently removed water [ 14 ].
- This mixture maintained the enzymatic activity of lactate dehydrogenase (LDH) by 50% compared with the control [ 29 ].
- Studies from the 1960s and 1970s recognized the stabilizing effects of these sugars on biological materials.
- The use of synthetic polyols and polymers like polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) emerged in the late 1980s and 1990s.
- From the 1990s to the 2000s, the discovery of amino acids highlighted their role in both cryoprotection and lyoprotection.
- The discovery of naturally occurring antifreeze proteins (AFPs) and glycopeptides in the 1990s opened new avenues for protein stabilization.
- Polysaccharides like dextran started to be used in the late 2000s more frequently due to their excellent film-forming abilities and compatibility with various proteins.
- The results showed that an addition of 0.50 mmol/L Ca 2+ to the culture medium significantly improved the freeze-drying survival rates of the strain ( p < 0.05).
- After non-enzymatic glycation, the glass transition temperature of GAPP rose to 21.4 °C, up from 27.6 °C.
- This study confirmed that FMD (ficoll/maltitol/DMSO) and DMD (dextran/maltitol/DMSO) systems maintained ~90% activity compared with fresh extract at 4 °C for 3 months.
- At 25 °C, lyoprotectants were more effective than at 4 °C;
- DMD and FMD samples retained over 36% activity.
- FMD-protected samples had >30% full extract activity at 37 °C after 3 months, around 40% better than the extract-only control.
- FMD demonstrated the best protection at 50 °C for the first month [ 20 ].
- Presence of sucrose or trehalose improved the enzyme activity and achieved an 80% release of loaded activity of lyophilized Vegetal diamine oxidase (vDAO) [ 15 ].
- To support this, the Bacillus Calmette Guerin (BCG) strain that was lyophilized with trehalose showed a longer shelf-life with 12% heat stability (28 days for 37 °C), while sucrose exhibited declining activity of the strain.
- Sucrose incorporation into freeze-dried trivalent antivenom (FDTAV) has maintained the protein concentration with 80% immunoglobulin activity, with no visual alteration in turbidity and no aggregates over the time [ 22 ].

**工艺/辅料句（直引）**：
- Lyophilization, also known as freeze-drying, involves sublimating water in the form of ice from a substance at low pressure, forming a freeze-dried powder that increases its shelf life.
- Extreme pressure and varying temperatures in the freeze-drying process may damage the protein’s structural integrity.
- Lyoprotectants are commonly used to protect protein conformations.
- It is important to choose a suitable lyoprotectant to ensure optimal effectiveness.
- Method: Twenty articles screened from Scopus, Web of Science, and PubMed were included in this review that discussed potential lyoprotectants and their effectiveness with different protein models.
- Results: Lyoprotectants were categorized into sugars, polyols, surfactants, and amino acids.
- Lyoprotectants can exhibit significant protective effects towards proteins, either singularly or in combination with another lyoprotectant.
- Conclusions: This review concludes that disaccharides are the most effective lyoprotectants, while other groups of lyoprotectants are best used in combination with other lyoprotectants.
- Keywords: lyoprotectant, protein stabilization, freeze-drying, cryodesiccation status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2024 Sep 11;
- Introduction Freeze-drying, also known as lyophilization, is widely used to preserve proteins in a dry state for long-term storage and transportation.
- Lyophilized protein powder can be stocked for future outbreaks, and this strategy can even be used in experiments with more extended time scales [ 1 , 2 ].
- Lyophilization is an ideal process to convert protein solution into a solid form.