scholarly journals Applying Microfluidics for the Production of the Cationic Liposome-Based Vaccine Adjuvant CAF09b

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1237
Author(s):  
Signe Tandrup Schmidt ◽  
Dennis Christensen ◽  
Yvonne Perrie
Keyword(s):  
Large Scale ◽  
Colloidal Stability ◽  
Scale Up ◽  
Cationic Liposome ◽  
Poly I:C ◽  
Particle Sizes ◽  
Adjuvant Activity ◽  
Lipid Film ◽  
Gmp Production

Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20–40% v/v), [Lipid] (DDA and MMG, 6–12 mg/mL) and dimethyl sulfoxide [DMSO] (0–10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.

scholarly journals Essential Role of Host Double-Stranded DNA Released from Dying Cells by Cationic Liposomes for Mucosal Adjuvanticity

Vaccines ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 8
Author(s):  
Rui Tada ◽  
Akihiro Ohshima ◽  
Yuya Tanazawa ◽  
Akari Ohmi ◽  
Saeko Takahashi ◽  
...  
Keyword(s):  
Essential Role ◽  
Cationic Liposome ◽  
Cationic Liposomes ◽  
Mucosal Vaccine ◽  
Nasal Administration ◽  
Adjuvant Activity ◽  
Mucosal Adjuvant ◽  
Double Stranded Dna ◽  
Dying Cells

Infectious disease remains a substantial cause of death. To overcome this issue, mucosal vaccine systems are considered to be a promising strategy. Yet, none are approved for clinical use, except for live-attenuated mucosal vaccines, mainly owing to the lack of effective and safe systems to induce antigen-specific immune responses in the mucosal compartment. We have reported that intranasal vaccination of an antigenic protein, with cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl], induced antigen-specific mucosal and systemic antibody responses in mice. However, precise molecular mechanism(s) underlying the mucosal adjuvant effects of cationic liposomes remain to be uncovered. Here, we show that a host double-stranded DNA (dsDNA), released at the site of cationic liposome injection, plays an essential role for the mucosal adjuvanticity of the cationic liposome. Namely, we found that nasal administration of the cationic liposomes induced localized cell death, at the site of injection, resulting in extracellular leakage of host dsDNA. Additionally, in vivo DNase I treatment markedly impaired OVA-specific mucosal and systemic antibody production exerted by cationic liposomes. Our report reveals that host dsDNA, released from local dying cells, acts as a damage-associated molecular pattern that mediates the mucosal adjuvant activity of cationic liposomes.

Practical Considerations for Large-Scale Cryopreservation of a Tissue Engineered Human Dermal Replacement

1999 ◽  
Author(s):  
Dawn R. Applegate ◽  
Kang Liu ◽  
Jonathan Mansbridge
Keyword(s):  
Large Scale ◽  
Stress Proteins ◽  
Scale Up ◽  
Tissue Growth ◽  
Volume Distribution ◽  
Isolated Cells ◽  
Successful Design ◽  
Tissue Systems ◽  
End Use

Abstract Tissue engineering is redefining the field of transplantation by providing a readily available, off-the-shelf supply of consistent, easy to use, safe and effective products. Successful design and scale-up of both tissue growth and preservation processes have enabled maintenance of tissue integrity, functionality and viability from product manufacture to end-use and afforded clinical efficacy, feasibility of large-volume distribution and cost-effectiveness. Advances in tissue preservation are being realized through extension of mathematical models and biological principles for isolated cells to bench-scale tissue systems. Hence, implementation problems inherent to large-scale systems are not often considered. Furthermore, the effects of preservation on tissues are not always tested in vivo. This paper addresses the practical obstacles to the design and implementation of a large-scale cryopreservation process. The effects of cryopreservation on in vivo tissue functionality including alteration of cell signal transduction pathways and expression of stress proteins in response to cryopreservation are also reviewed.

scholarly journals Coordinating the in vivo processes for minicircle production - a novel approach to large scale manufacturing

2018 ◽  
Author(s):  
Peter Mayrhofer ◽  
Hana Jug ◽  
Aleš Štrancar ◽  
Alexandre Di Paolo ◽  
Laurent Jost ◽  
...  
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Process Development ◽  
Scale Up ◽  
Hydrophobic Interaction Chromatography ◽  
Viral Gene ◽  
Novel Approach ◽  
Dna Production ◽  
Minicircle Dna

ABSTRACTSafety as well as efficiency issues in connection with bacterial backbone sequences should be carefully considered when designing new DNA vaccines or non-viral gene therapy approaches. Bacterial backbone sequences like antibiotic resistance markers or regulatory bacterial elements constitute biological safety risks and reduce the overall efficiency of the DNA agent. To overcome these problems the minicircle technology has been developed. But, despite all the obvious advantages, minicircles have so far not replaced their problem laden conventional counterpart in gene transfer applications what can be contributed to efficiency issues in large scale manufacturing. In this article we describe the combined efforts of experts in the field of minicircle development, large scale biomanufacturing and downstream process development to provide a new approach. The Recombination Based Plasmid Separation (RBPS) Technology, which has already solved crucial problems associated with minicircle-DNA production, has been developed further for this purpose. A novel parental plasmid exploiting advanced in vivo process coordination for restriction and subsequent degradation of miniplasmid-DNA will be introduced. Furthermore we describe the scale-up of minicircle-DNA production by fermentation in combination with high performance downstream processes including purification by ion exchange and hydrophobic interaction chromatography on monolithic material.

Superparamagnetic Iron Oxide Nanoarticles for Biomedical Applications: a focus on PVA as a coating

2003 ◽  
Vol 789 ◽  
Author(s):  
M. Chastellain ◽  
A. Petri ◽  
H. Hofmann
Keyword(s):  
Iron Oxide ◽  
Biomedical Applications ◽  
Large Scale ◽  
Colloidal Stability ◽  
Superparamagnetic Iron Oxide ◽  
Narrow Size Distribution ◽  
Water Based ◽  
Co Precipitation

ABSTRACTNanoscaled particles showing a superparamagnetic behavior have been intensively studied these past years for biomedical applications and water-based ferrofluids turned out to be promising candidates for various in vivo as well as in vitro applications. Nevertheless, the lack of well-defined particles remains an important problem. One of the major challenges is still the large-scale synthesis of particles with a narrow size distribution. In this work iron oxide nanoparticles are obtained by classical co-precipitation in a water-based medium and are subsequently coated with polyvinyl alcohol. The thus obtained ferrofluids are studied and a focus is made on their colloidal stability.

scholarly journals Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation

2021 ◽  
Vol 22 (8) ◽  
pp. 3828
Author(s):  
Laura Nicolle ◽  
Jens Casper ◽  
Melanie Willimann ◽  
Céline M. A. Journot ◽  
Pascal Detampel ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Colloidal Stability ◽  
Transfection Efficiency ◽  
Scale Up ◽  
Water Soluble ◽  
Viral Gene ◽  
In Vivo Evaluation ◽  
Hepatic Expression

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

scholarly journals Enhanced ability of plant-derived PGT121 glycovariants to eliminate HIV-1-infected cells

2021 ◽  
Author(s):  
Sai Priya Anand ◽  
Shilei Ding ◽  
William D. Tolbert ◽  
Jérémie Prévost ◽  
Jonathan Richard ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Scale Up ◽  
Scale Production ◽  
Viral Neutralization ◽  
Antiviral Activities ◽  
Infected Cells ◽  
Hiv 1

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. Additionally, different strategies are currently being tested to scale-up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo . However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4+ T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 mAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

A Hybrid Bioprinting Approach for Scale-Up Tissue Fabrication

2014 ◽  
Vol 136 (6) ◽  
Author(s):  
Yin Yu ◽  
Yahui Zhang ◽  
Ibrahim T. Ozbolat
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Large Scale ◽  
Vascular System ◽  
Scale Up ◽  
Organ Printing ◽  
Promising Application ◽  
Organ Models

Tissue engineering has been focused on the fabrication of vascularized 3D tissue for decades. Most recently, bioprinting, especially tissue and organ printing, has shown great potential to enable automated robotic-based fabrication of 3D vascularized tissues and organs that are readily available for in vitro studies or in vivo transplantation. Studies have demonstrated the feasibility of the tissue printing process through bioprinting of scaffold-free cellular constructs that are able to undergo self-assembly for tissue formation; however, they are still limited in size and thickness due to the lack of a vascular network. In this paper, we present a framework concept for bioprinting 3D large-scale tissues with a perfusable vascular system in vitro to preserve cell viability and tissue maturation. With the help of a customized Multi-Arm Bioprinter (MABP), we lay out a hybrid bioprinting system to fabricate scale-up tissues and organ models and demonstrated envision its promising application for in vitro tissue engineering and its potential for therapeutic purposes with our proof of concept study.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Solid Lipid Nanoparticles: A Promising Drug Delivery Technology

2009 ◽  
Vol 2 (2) ◽  
pp. 509-516
Author(s):  
S. Pragati ◽  
S. Kuldeep ◽  
S. Ashok ◽  
M. Satheesh
Keyword(s):  
Drug Delivery ◽  
Solid Lipid Nanoparticles ◽  
Large Scale ◽  
Colloidal Particles ◽  
Scale Up ◽  
Lipid Nanoparticles ◽  
Scale Production ◽  
Research Activity ◽  
New Approach ◽  
Solid Lipid

One of the situations in the treatment of disease is the delivery of efficacious medication of appropriate concentration to the site of action in a controlled and continual manner. Nanoparticle represents an important particulate carrier system, developed accordingly. Nanoparticles are solid colloidal particles ranging in size from 1 to 1000 nm and composed of macromolecular material. Nanoparticles could be polymeric or lipidic (SLNs). Industry estimates suggest that approximately 40% of lipophilic drug candidates fail due to solubility and formulation stability issues, prompting significant research activity in advanced lipophile delivery technologies. Solid lipid nanoparticle technology represents a promising new approach to lipophile drug delivery. Solid lipid nanoparticles (SLNs) are important advancement in this area. The bioacceptable and biodegradable nature of SLNs makes them less toxic as compared to polymeric nanoparticles. Supplemented with small size which prolongs the circulation time in blood, feasible scale up for large scale production and absence of burst effect makes them interesting candidates for study. In this present review this new approach is discussed in terms of their preparation, advantages, characterization and special features.

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