Protein-based Formulations Research Articles

BSA/HSA ratio modulates the properties of Ca2+-induced cold gelation scaffolds

An effective tissue engineering approach requires adjustment according to the target tissue to be engineered. The possibility of obtaining a protein-based formulation for the development of multivalent tunable scaffolds that can be adapted for several types of cells and tissues is explored in this work. The incremental substitution of bovine serum albumin (BSA) by human serum albumin (HSA), changing the scaffolds’ hydrophilic/hydrophobic ratio, on a previously optimized scaffold formulation resulted in a set of uniform porous scaffolds with different physical properties and associated cell proliferation profile along time. There was a general trend towards an increase in hydrophilicity, swelling degree and in vitro degradation of the scaffolds with increasing replacement of BSA by HAS. The set of BSA/HSA scaffolds presented distinct values for the storage (elastic) modulus and loss factor which were similar to those described for different native tissues such as bone, cartilage, muscle, skin and neural tissue. The preferential adhesion and proliferation of skin fibroblasts on the BSA25%HSA75% and HSA100% scaffolds, as predicted by their viscoelastic properties, demonstrate that the BSA/HSA scaffold formulation is promising for the development of scaffolds that can be tuned according to the tissue to be repaired and restored.

Simulation and Experimental Validation of the Denaturation of a Whey Protein-Based Coating During Convection and/or Infrared Drying on a Plastic Film and Influence on its Oxygen Barrier Properties

ABSTRACTThe in-process denaturation of native whey protein-based coatings, film temperature, residual humidity, and protein degree of denaturation was simulated with combined existing models. The simulation was validated experimentally through determining the degree of denaturation of the protein in the coating. Whey protein-based formulations were applied, and denaturation was induced via infrared heating during the drying process. The oxygen transmission rate was measured and correlated with the degree of denaturation. The observed influence of the denaturation degree allowed the identification of a critical denaturation degree improving oxygen barrier. The developed simulation appears as an efficient tool for optimizing the processing and properties of protein-based materials.

Gel-like pea protein Pickering emulsions at pH 3.0 as a potential intestine-targeted and sustained-release delivery system for β-carotene

In recent years, there is increasing interest in the development of food-grade Pickering emulsions as promising delivery systems for bioactive compounds. Our previous work reported that most of the proteins in pea protein isolate (PPI) at pH3.0, present in the nanoparticle form, can effectively perform as a kind of food-grade Pickering stabilizers for oil-in-water emulsions (LWT, 2014). The purpose of this study was to further report that PPI-stabilized emulsions at pH3.0 exhibited great potential to act as intestine-targeted and sustained-release delivery systems for β-carotene. The emulsions were produced by microfluidization at a specific protein concentration of 6.0% (w/v) and varying oil fractions (ϕ) of 0.2–0.6. The results indicated that increasing ϕ was favorable for the gel-like network strengthening of these emulsions. The gel formation was largely related to the droplet flocculation as well as inter-floc attractive interactions. The in vitro simulated digestion results showed that the release of β-carotene during the intestinal digestion of these emulsions could be well modulated by altering ϕ. The gel-like emulsion at ϕ=0.6 exhibited much lower release of β-carotene, but higher stability towards degradation during the digestion, than that at ϕ=0.3. The findings provide important information not only for the design of novel delivery systems for lipophilic bioactive components, but also for the development of plant protein-based formulations.

Standardization of formulations for the acute amino acid depletion and loading tests.

The acute tryptophan depletion and loading and the acute tyrosine plus phenylalanine depletion tests are powerful tools for studying the roles of cerebral monoamines in behaviour and symptoms related to various disorders. The tests use either amino acid mixtures or proteins. Current amino acid mixtures lack specificity in humans, but not in rodents, because of the faster disposal of branched-chain amino acids (BCAAs) by the latter. The high content of BCAA (30-60%) is responsible for the poor specificity in humans and we recommend, in a 50g dose, a control formulation with a lowered BCAA content (18%) as a common control for the above tests. With protein-based formulations, α-lactalbumin is specific for acute tryptophan loading, whereas gelatine is only partially effective for acute tryptophan depletion. We recommend the use of the whey protein fraction glycomacropeptide as an alternative protein. Its BCAA content is ideal for specificity and the absence of tryptophan, tyrosine and phenylalanine render it suitable as a template for seven formulations (separate and combined depletion or loading and a truly balanced control). We invite the research community to participate in standardization of the depletion and loading methodologies by using our recommended amino acid formulation and developing those based on glycomacropeptide.

Appropriate amino acid mixtures for tryptophan depletion and tyrosine/phenylalanine depletion and the safety of long-term amino acid depletion in humans: reply

Dear Editor, Thank you for giving me the opportunity to respond to the comments raised by Professor Young (2013) in response to my recent review entitled ‘Novel nutritional treatment for manic and psychotic disorders: review of tryptophan and tyrosine depletion studies and the potential of protein-based formulations using glycomacropeptide’ (Badawy 2013). In my article, I proposed the use of the whey protein glycomacropeptide (GMP), which lacks phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) as a platform for a number of formulations for use in the tests of separate and combined depletion of, or loading with, the above amino acids and also for a nutritional treatment of manic and psychotic disorders. Professor Young's comments were under four headings, which will now be addressed under the following suitable titles. Amino acid profile and safety of long-term therapy with GMP

Novel nutritional treatment for manic and psychotic disorders: a review of tryptophan and tyrosine depletion studies and the potential of protein-based formulations using glycomacropeptide

Current amino acid (AA) mixtures used in acute tryptophan (Trp) and tyrosine (Tyr) plus phenylalanine (Phe) depletion and loading tests are unpalatable and lack specificity. Specificity is improved by reducing content of branched-chain amino acids (BCAA) and palatability to a certain extent by dose reduction. This study aims to identify a palatable naturally occurring alternative(s) to amino acids with the desired BCAA content for use in the above tests. A palatable alternative lacking in Trp, Tyr and Phe has been identified in the whey protein fraction caseino-glycomacropeptide (c-GMP). The absence of these three aromatic amino acids renders GMP suitable as a template for seven formulations for separate and combined depletion or loading and a placebo control. The absence of Phe and Tyr enables GMP to provide a unique nutritional therapy of manic and psychotic disorders by inhibition of cerebral dopamine synthesis and release and possibly also by enhancing glutamatergic function, in general, and in patients resistant to anti-psychotic medication, in particular. Seven GMP-based formulations for the above tests are proposed, two of which can be used in the above nutritional therapy and a third formulation as a placebo control in clinical trials. Development of these formulations should advance the above research and diagnostic tests, open new avenues for neuroscience research on monoamine function, and improve the therapy of bipolar and psychotic disorders and enhance the quality of life of sufferers.

Cold, gel-like soy protein emulsions by microfluidization: Emulsion characteristics, rheological and microstructural properties, and gelling mechanism

In this paper we reported the rheological and microstructural properties of a kind of novel cold, gel-like soy protein isolate (SPI) emulsions obtained by means of microfluidization. These gel-like emulsions were formed from untreated and preheated (95 °C, 15 min) SPI at a protein concentration of 6% (w/v), and various oil volume fractions (Φ; 0.2–0.6) and NaCl concentrations (0–500 mM). The rheological properties and microstructure were characterized using steady viscosity and dynamic oscillatory measurements, as well as confocal laser scanning microscopy (CLSM). The characteristics (e.g. droplet size distribution and creaming stability) of the emulsions, formed at lower protein concentrations (e.g. 0.5–4.0%), were also characterized, aiming to reveal the mechanism of the gel-like network formation. The dynamic oscillatory data indicated that both untreated and preheated SPI emulsions exhibited gel-like rheological properties, but the specific apparent viscosity (η) and storage modulus (G′) of the latter ones were much higher at a comparable Φ. Both η and G′ progressively increased upon Φ increasing, indicating enhanced inter-droplet interactions. At a given Φ value (0.3), increasing NaCl concentration progressively increased η and G′ of the preheated SPI emulsions, indicating the importance of electrostatic screening for the gel-like network formation. The CLSM analyses confirmed formation of the gel-like network, mainly composed of aggregated oil droplets, which was closely dependent on the Φ and NaCl concentration. The gel-like network was formed by bridging flocculation of oil droplets, mainly through inter-droplet hydrophobic interactions between the proteins adsorbed at the interface. These results suggested that soy proteins exhibit excellent potential to produce cold, gel-like emulsions, especially through a heat pretreatment followed by microfluidization, which might be of vital importance for the development of soy protein-based formulations, especially as carriers for heat-labile ingredients with health effects.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Co-administration of Tacrolimus Suppresses Pharmacokinetic Modulation of Multiple Subcutaneously Administered Human Interferon-alpha in Beagle Dogs

Specific antibody production is an important issue in crossover pharmacokinetic (PK) studies of protein-based formulations. We recently reported that intravenous co-administration of tacrolimus with multiple human interferon-alpha (h-IFN) administrations successfully suppressed the production of anti-h-IFN antibodies in rats. Since crossover PK studies are preferentially carried out using larger animals such as dogs or monkeys that are capable of accepting the same dosage formulations as those for clinical use, we extended our study of co-administration of tacrolimus with multiple h-IFN administrations to beagle dogs in the present study. Beagle dogs were subcutaneously administered 0.5 million IU/kg of h-IFN once a week for 4 weeks. In some experiments, tacrolimus at 0.01 or 0.1 mg/kg was intravenously co-administered at the same time as the h-IFN administration. Co-administration of the lower dose of tacrolimus (0.01 mg/kg) failed to suppress the anti-h-IFN IgG responses, while co-administration of the higher dose (0.1 mg/kg) successfully suppressed these responses. Moreover, co-administration of tacrolimus had little effect on the serum creatinine concentrations, suggesting that multiple administrations of tacrolimus at the concentrations examined did not cause severe renal disorders. Taken together, the present data confirm that co-administration of tacrolimus is a promising way to assess crossover PK studies of human or humanized proteinic formulations in beagle dogs.

Antibody mediated protection of immunized macaques against pathogenic SHIV challenge

The primary objective of our current HIV vaccine research program is to evaluate novel alphavirus-based and Envelope (Env) protein-based vaccine formulations either alone or in prime boost regimen for improved immunogenicity and efficacy over other HIV vaccine approaches. The outcome of this work will be the production of candidate vaccine components that can be further advanced to clinical trials in human subjects. For these studies we employ viral genes derived from SF162 a primary subtype B HIV-1 isolate in alphavirus replicon particle-based vaccines. A chimeric alphavirus vector system (VEE/SIN) is used that is composed of the Venezuelan Equine Encephalitis (VEE) replicon combined with the Sindbis virus structural proteins. We have performed a proof of concept study where macaques were primed with VEE/SIN, boosted with o-gp140DV2SF162, and challenged intravenously by pathogenic SHIVSF162P4. In the next study we evaluated these recombinant VEE/SIN vaccines alone, and also in combination with DV2-trimer derived from SF162 for their ability to protect against mucosal SHIVSF162P4 challenge. Both parenteral and mucosal routes of immunization have been evaluated to identify the optimal regimen(s) for the induction of potent and protective humoral and cellular responses in rhesus macaques. In the third study, we evaluated the ability of DV2SF162 trimer to protect against against mucosal SHIVSF162P4 challenge. Both parenteral and mucosal routes of immunization have been evaluated. Results so far indicate that the recombinant VEE/SIN particles induced antigen-specific CD4+ and CD8+ T cell responses as well as neutralizing antibodies in small animals (mice and rabbits) and non-human primates. Moreover, intramuscular immunization with VEE/SIN particles encoding SIV gag or gagpol and HIV env is quite effective in priming for potent anti-Env antibody responses. However, it seems intramuscular boosting of alpha primed animals with o-gp140DV2SF162 in MF59 is critical for blocking and/or reducing acute phase viremia following either intravenous or intrarectal SHIVSF162P4 challenges of rhesus macaques. Furthermore, macaques that were immunized intranasally with o-gp140DV2SF162 protein in the LTK63 mucosal adjuvant followed by intramuscular immunization with o-gp140DV2SF162 in MF59 were also protected against SHIVSF162P4 challenge. Protection in these studies appears to occur after the induction of high titer serum neutralizing antibodies that recognize the challenge virus.

Liposomes for Protein Delivery: Selecting Manufacture and Development Processes

The use of liposomes as protein carriers has been investigated for a number of possible potential applications in pharmaceutical formulations. These are for sustained release to improve adjuvancy for vaccine development, protein absorption enhancement, and drug targeting carriers and as vehicles for administration of hydrophobic compounds. The majority of current liposomal protein formulations are still in various preclinical research stages, with relatively little known or reported human clinical findings to date. There are still pending challenges to creating commercially stable and bioactive protein formulations with lipids. In addition to lipid developmental issues, because the stability of proteins may be affected by many physical or chemical manipulations, the choice of manufacturing designs for liposomal incorporation becomes somewhat more complex. For this reason, solvent-free vesicle procedures should be the primary approach to the preparation of protein-based formulations. In this regard a variety of recently reported methods are discussed. The advantages or disadvantages of these procedures are compared to those of procedures involving solvents. Consideration to large-scale manufacturing and protein recovery issues is also given.