scholarly journals Taking Advantage of Nature’s Benefits: Soluble and Stable Antigen Straight Out of the Pathogen

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 137
Author(s):  
Didier Clénet ◽  
Léna Clavier ◽  
Benoit Strobbe ◽  
Christel Le Bon ◽  
Manuela Zoonens ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Process Development ◽  
Storage Conditions ◽  
Full Length ◽  
Soluble Form ◽  
Drying Process ◽  
Liquid Form ◽  
Freeze Dried

Integral membrane proteins (MP) exhibit specific tridimensional conformation and topology that define their various functions. Pathogen surface antigens, encompassing many MP, are at the forefront of the viral strategy which is broadly targeted by the host immune response. These antigens are present in equilibrium under different oligomeric forms with distinctive epitopes, and to obtain them in a soluble form and/or stable constitutes a real risk. The solubilization of a full-length MP directly from a pathogen to rapidly obtain a native antigen mimicking the original conformation of the MP at the pathogen surface is the process development reported in this work. Rabies virus (RABV) was used as a model for this demonstration and its full-length glycoprotein (G) was stabilized in amphiphatic polymers (A8-35 amphipols). The stability of the soluble RABV-G was evaluated under various stress conditions (temperatures, agitation and light exposures) and a long-term stable RABV-G formulation, suitable for the freeze-drying process, was defined using a design of experiment approach. RABV-G/A8-35 in liquid form was shown to be antigenically stable at 5 °C and 25 °C for one month, and a dedicated kinetic model predicted its stability up to 1 year at 5 °C. To mitigate the RABV-G/A8-35 sensitivity to mechanical stress, a solid form of RABV-G/A8-35 and a freeze-drying process were considered, resulting in a 2-year thermally stable product at 5 °C, 25 °C and 37 °C. To the best of our knowledge, this is the first time that a natural full-length MP, extracted from a virus and trapped in amphipols, was kept antigenically stable in the long term, in a defined freeze-dried form out of any refrigerated storage conditions. These results described an easy process to obtain a pure, well conformed native-like antigen of interest, from a circulating pathogen which is of concern for diagnostic (quantification/characterization assays), therapeutic and vaccine strategies. After the physical characterization of the protein, the identification of RABV G/A8-35 neutralizing epitopes has been underway before in vivo testing.

The Effect of Storage Temperature and Physical State on the Performance of Antisera in Radioimmunoassay after Long-Term Storage

1988 ◽  
Vol 25 (1) ◽  
pp. 89-95
Author(s):  
B A Middleton ◽  
L M Morgan ◽  
G W Aherne ◽  
V Marks
Keyword(s):  
Room Temperature ◽  
Storage Temperature ◽  
Storage Conditions ◽  
Liquid Form ◽  
Long Term Storage ◽  
Freeze Dried ◽  
Control Serum ◽  
Term Storage ◽  
Temperature Storage

The performance in radioimmunoassay of four antisera after storage at temperatures ranging from −40°C to room temperature, in three physical states (frozen, liquid or freeze dried) was investigated over a 3-year period. No deterioration in antiserum performance in terms of precision and accuracy of quality control serum measurement or recovery of ligand was apparent under any of the storage conditions studied. Some lowering of titre became apparent in two of the antisera over the study period. Deterioration was most marked when antiserum was stored lyophilised at room temperature. Storage of antiserum frozen confers no advantage over storage at 4°C provided precautions are taken to minimise bacterial contamination when storing antiserum in liquid form.

scholarly journals Freeze drying as a method of long-term conservation of mammalian semen – a review

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Iwona Rajska
Keyword(s):  
Freeze Drying ◽  
Genetic Material ◽  
Simultaneous Application ◽  
Preservation Method ◽  
Freeze Dried ◽  
Unlimited Time ◽  
Specialized Equipment

Abstract With the development of biotechnological methods that allow for the manipulation and free exchange of genetic material, there is a need to improve the methods of collecting and storing such material. Until now, freezing in liquid nitrogen has allowed the storage of cells and entire plant and animal tissues for practically unlimited time. Despite this, alternatives are still being sought that will eliminate the constant need to keep samples at low temperature. Lyophilization or freeze drying can be an alternative to standard freezing procedures. The storage of samples (lyophilisates) does not require specialized equipment but only the refinement of the preservation method itself. In the case of cells capable of movement, e.g. sperm, as a result of the lyophilization process, they lose the ability to reach the oocyte in vivo and IVF. However, it is possible to use freeze-dried sperm for in vitro fertilization by ICSI, which is observed on the basis of the results obtained in cleavage, embryo development and production of live born offspring after embryo transfer. Studies on lyophilization of sperm are carried out on many animal species, both laboratory and livestock. This conservation method is seen as the possibility of creating biobanking for genetically valuable and endangered species with the simultaneous application of ICSI. This review article was intended to present the issues of the freeze-drying process of mammalian semen and help in finding solutions that will improve this technique of long-term preservation of biological material.

scholarly journals Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 841-846 ◽  
Author(s):  
Yosuke Kawase ◽  
Toshio Hani ◽  
Nobuo Kamada ◽  
Kou-ichi Jishage ◽  
Hiroshi Suzuki
Keyword(s):  
Freeze Drying ◽  
Developmental Rate ◽  
Storage Conditions ◽  
Blastocyst Stage ◽  
Influential Factor ◽  
Effect Of Pressure ◽  
Freeze Dried ◽  
Preservation Potential ◽  
Long Term Preservation

Freeze-dried spermatozoa are capable of participating in normal embryonic development after injection into oocytes and thus useful for the maintenance of genetic materials. We recently reported that long-term preservation of freeze-dried mouse spermatozoa by conventional methods requires temperatures lower than −80 °C. Successful permanent preservation of mouse spermatozoa at much higher temperatures requires thorough investigation of the freeze-drying procedure. Thus, we examined the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse spermatozoa. Three different primary drying pressures were applied to evaluate the effect of pressure on freeze-dried spermatozoa under varying storage conditions and the rate of development measured. The developmental rate of embryos to the blastocyst stage from intracytoplasmic sperm injection by freeze-dried spermatozoa at pressures of 0.04, 0.37, and 1.03 mbar without storage were 59% (337/576), 71% (132/187), and 33% (99/302) respectively. When stored at 4 °C for 6 months, the rate was 13% (48/367), 50% (73/145), and 36% (66/182) respectively. These results show that primary drying pressure is an influential factor in the long-term preservation of freeze-dried mouse spermatozoa.

scholarly journals Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 839
Author(s):  
Karina Jasińska ◽  
Bartłomiej Zieniuk ◽  
Dorota Nowak ◽  
Agata Fabiszewska
Keyword(s):  
Freeze Drying ◽  
Antioxidant Properties ◽  
Antibacterial Properties ◽  
Synthetic Activity ◽  
Drying Process ◽  
Enzyme Preparations ◽  
Ester Derivative ◽  
Freeze Dried ◽  
The Impact ◽  
Geranyl Ester

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2–6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 15-24 ◽  
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Freeze Drying ◽  
Chelating Agents ◽  
Sperm Head ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Freeze Dried ◽  
Boar Sperm

SUMMARYSuccessful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

scholarly journals 314 FULL-TERM DEVELOPMENT OF RAT OOCYTES MICROINSEMINATED WITH FREEZE-DRIED SPERMATOZOA

2005 ◽  
Vol 17 (2) ◽  
pp. 307
Author(s):  
M. Hirabayashi ◽  
M. Kato ◽  
S. Hochi
Keyword(s):  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Pure Water ◽  
Full Term ◽  
Female Rats ◽  
Membrane Disruption ◽  
Developmental Potential ◽  
Fresh Freeze ◽  
Freeze Dried

Since freeze-dried spermatozoa can be stored at ambient or refrigerated temperature, the costs required for maintenance and shipping of spermatozoa can be reduced. To date, viable offspring in mice (Wakayama and Yanagimachi 1998 Nat. Biotech. 16, 639) and rabbits (Liu et al. 2004 Biol. Reprod. 70, 1776) have been produced by intracytoplasmic sperm injection (ICSI) using freeze-dried samples. The objectives of the present study were to examine whether freeze-dried rat spermatozoa can participate in full-term development by ICSI, and whether sonication prior to freeze-drying of the spermatozoa influences the offspring rate. Spermatozoa from cauda epididymides of Sprague-Dawley (SD) rats were collected in 10 mM TRIS/HCl buffer supplemented with 50 mM NaCl and 50 mM EGTA. A 2 × 3 factorial-designed experiment was conducted. The sperm suspensions were either sonicated for 10 s using a 10% power output from an ultrasonic cell disruptor or not sonicated. The sperm suspensions were then processed for freeze-thawing (100-μL sample in 1.0-mL cryotube was cooled in liquid nitrogen vapor, stored at -196°C for 48 h, and thawed in a 25°C water bath) and freeze-drying (100-μL sample in 1.5-mL polypropylene tube was frozen in liquid nitrogen for 20 s, lyophilized for 6 h by a freeze-drying apparatus, stored at 4°C for 48 h, and rehydrated with 100 μL ultra pure water), or were subjected to immediate use for ICSI. The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4 μm in diameter, as described previously (Hirabayashi et al. 2002 Transgenic Res. 11, 221). The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. The in vivo developmental potential of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa is shown in the table below. Viable rat offspring were produced in all six experimental groups, with the offspring rates at 2.5–35.0%. Sonication treatment of rat spermatozoa to induce membrane disruption and tail/midpiece dissociation from the heads was effective in increasing the offspring rate after ICSI. The positive effect of sperm sonication may be explained as facilitating decondensation of sperm heads by membrane disruption in the spontaneously activating rat oocytes. Thus, successful participation of freeze-dried rat spermatozoa into full-term development was demonstrated by applying the ICSI. Table 1. In vivo development of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa

scholarly journals Emerging Freeze-Drying Process Development and Scale-up Issues

2011 ◽  
Vol 12 (1) ◽  
pp. 372-378 ◽  
Author(s):  
Sajal Manubhai Patel ◽  
Michael J. Pikal
Keyword(s):  
Freeze Drying ◽  
Process Development ◽  
Scale Up ◽  
Drying Process

scholarly journals Effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa

2020 ◽  
Vol 45 (3) ◽  
pp. 189-196
Author(s):  
S. Said ◽  
T. Maulana ◽  
S. Setiorini ◽  
G.E. Ibrahim ◽  
M.N. Ramadhan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Freeze Drying ◽  
Sperm Head ◽  
Test Method ◽  
Dna Integrity ◽  
Drying Process ◽  
Freeze Dried ◽  
Bovine Spermatozoa ◽  
Amino Acid Glycine

The objective of the current study was to investigate the effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa. Spermatozoa were freeze-dried in medium with the addition of an amino acid glycine, cysteine, glutamine, or its combination with EDTA. Sperm head morphometry was identified at 400X magnification using Axio Vision(Zeiss Company, Germany), while for membrane plasma integrity (MPI) determined by calculation of the percentage of spermatozoa having intact plasma membrane by osmotic resistance test method and DNA integrity analysis using acridine orange staining. Sperm head had declined in size after the freeze-drying process, MPI of FD spermatozoa gradually increased significantly when FD solution was added with an amino acid solution (glycine, cysteine) and its combination with EDTA. DNA integrity of all freeze-dried spermatozoa treatments was remaining intact, no significantly different (P>0.01) among treatments. The present study concluded that the addition of an amino acid (glycine, cysteine) or its combination with EDTA could be reduced morphometric sperm heads and plasma membrane damage of freeze-dried bovine spermatozoa, however, DNA integrity of bovine sperm nucleus remaining intact after the freeze-drying process without addition both amino acids and EDTA. 

scholarly journals Solubilization of ibuprofen for freeze dried parenteral dosage forms

2019 ◽  
Vol 69 (1) ◽  
pp. 17-32
Author(s):  
Maja Preskar ◽  
Tomislav Vrbanec ◽  
Franc Vrečer ◽  
Primož Šket ◽  
Janez Plavec ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Aqueous Solubility ◽  
Pharmaceutical Formulations ◽  
Dosage Forms ◽  
Drying Process ◽  
Solid Dosage Forms ◽  
Solid Dosage ◽  
Combined Use ◽  
Freeze Dried

Abstract Ibuprofen, a weakly acidic non-steroidal anti-inflammatory drug having poor aqueous solubility, is a challenging drug for the development of pharmaceutical formulations, resulting in numerous research attempts focusing on improvement of its solubility and consequently bioavailability. Most studies have been done for solid dosage forms, with very little attention paid to parenterals. Hence, the main purpose of the present study was to enhance ibuprofen solubility as a result of formulation composition and the freeze drying process. Moreover, the purpose was to prepare a freeze dried dosage form with improved ibuprofen solubility that could, after simple reconstitution with water for injection, result in an isotonic parenteral solution. Solubility of ibuprofen was modified by various excipients suitable for parenteral application. Drug interactions with selected excipients in the final product/lyophilisate were studied by a combined use of XRPD, DSC, Raman and ss-NMR. Analyses of lyophilized samples showed solubility enhancement of ibuprofen and in situ formation of an ibuprofen salt with the alkaline excipients used.

Repeatability of in vitro digestibility assays of forages when using fresh, frozen or freeze-dried cow faeces instead of sheep rumen liquor as sources of micro-organisms

1995 ◽  
Vol 1995 ◽  
pp. 110-110 ◽  
Author(s):  
S Akhter ◽  
E Owen ◽  
M K Theodorou ◽  
S L Tembo ◽  
E R Deaville
Keyword(s):  
Freeze Drying ◽  
Rumen Fluid ◽  
In Vitro Digestibility ◽  
Two Stage ◽  
Freeze Dried ◽  
Fresh Frozen ◽  
Sheep Rumen ◽  
Micro Organisms

Previous studies (El Shaer, Omed and Axford, 1987; Akhter, Owen, Fall, O'Donovan and Theodorou, 1994) with the two-stage in vitro procedure of Tilley and Terry (1963) have shown a high correlation between digestibilities of forages as determined using either sheep rumen liquor, sheep faeces or cow faeces as the microbial inoculum. In the first study of the of the present investigation one objective was to examine the repeatability of these digestibility measurements when made on different occasions. A second objective was to assess whether the correlations between faecal and rumen fluid based inocula could be improved if microorganisms were obtained from pairs rather than individual animals. The objective in the second study using forages of known in vivo digestibility, was to investigate the effect of freezing or freeze-drying of faeces on the repeatability of digestibilities of forages determined in vitro using micro-organisms from cow faeces.

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