Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes.

Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product.

Endotoxin contamination of ovalbumin suppresses murine immunologic responses and development of airway hyper-reactivity.

The reversible airway hyper-reactivity (AHR) of asthma is modeled by sensitizing and challenging mice with aerosolized ovalbumin. However, the C57BL/6 murine strain does not display the large increase in circulating IgG and IgE antibodies found in human atopy and asthma. We found that commercial ovalbumin was contaminated with lipopolysaccharide (LPS) in amounts sufficient to fully activate endothelial cells in an in vitro assay of the first step of inflammation. Desensitization of TLR4 by LPS pretreatment suppressed the inflammatory effect of ovalbumin. The presence of LPS was occult, because it does not require serum presentation and, like the LPS of Salmonella minnesota, was not suppressed by polymyxin B. Purified ovalbumin did not activate endothelial cells in vitro; however, endotoxin-free ovalbumin was far more effective than commercial material in stimulating IgE production and respiratory dysfunction in a C57BL/6 murine model of AHR. Moreover, endotoxin-free ovalbumin induced lung inflammation with alveolar enlargement and destruction in a histologic pattern that differed from the changes caused by commercial, endotoxin-contaminated ovalbumin. Reconstitution of purified ovalbumin with S. minnesota LPS decreased lung inflammation, decreased changes in lung function, and suppressed anti-ovalbumin antibody production. We conclude endotoxin contaminates ovalbumin preparations and that endotoxin co-administration with the ovalbumin antigen creates a state of tolerance in a murine model of AHR. Co-exposure to endotoxin and antigen occurs in humans through organic dusts, so murine models of AHR may reflect the clinical situation, but models based on commercial ovalbumin do not accurately reflect the effect of protein antigen alone on animal physiology.

Effect of storage and layer age on quality of eggs from two lines of hens.

Eggs from ISA-White and ISA-Brown hens between 28 and 59 wk of age were stored for up to 10 d to produce a sample of 5,763 eggs differing in the three major determinants of albumen quality. Eggs from ISA-Brown hens were larger and had less yolk, more albumen, and a greater percentage of shell than those from ISA-White hens. Egg size increased with increasing age of the hen, although more for the ISA-White hens than the ISA-Brown hens, and the yolk increased more in size than did the shell and albumen. During storage, albumen weight decreased and yolk weight increased slightly. The height of the inner thick albumen of eggs from ISA-White hens was greater than that of eggs from ISA-Brown hens, and it decreased as the hen age increased and with increasing time in storage. The pH of the albumen was not different between strains, and the effect of hen age was small, but it increased with time in storage. Regression coefficients of the height of the inner thick albumen on the weight of the egg were between -0.058 and 0.102, showing that the fixed regression of 0.05-mm albumen height per gram of egg implied by the Haugh unit is wrong. The statistical association between albumen pH and egg weight was very low. If albumen quality is being used as a measure of freshness, then the albumen height is biased by the strain and age of hen, whereas the albumen pH is not.

The effect of shell egg pasteurization on the protein quality of albumen.

The effect of a low-temperature, extended-time, in-shell pasteurization process on the protein quality of egg albumen was evaluated. Ten dozen fresh chicken eggs were pasteurized in a hot-air oven at 55 C for 180 min. The eggs were refrigerated and broken out for analysis on Days 0, 7, 14, 28, 42, and 56 following pasteurization. There were no significant differences in total or soluble protein over the experimental period for the pasteurized or unpasteurized albumen. Mean protein content was 80.6 +/- 0.5% for the pasteurized albumen and 80.9 +/- 0.5% for the unpasteurized albumen. In vitro digestibility, as measured by the AOAC method, was 82.4 +/- 0.7% for the pasteurized albumen and 81.7 +/- 0.6% for the unpasteurized albumen. There were no significant differences over the experimental period in the digestibility of the samples. Free amino acids and discriminant-computed protein efficiency ratio (DC-PER) also did not differ between the pasteurized and unpasteurized albumens or over the experimental period. The in-shell pasteurization process used had no effect on the protein quality of albumen.

Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.

Ahemeral lighting of turkey breeder hens. 1. Cycle length effects on egg production and egg characteristics.

A study was conducted to determine the effective range of light-dark cycle lengths for reproductive performance in turkey hens. The treatments consisted of seven different light-dark cycle lengths: 21, 23, 24, 26, 27, 28, and 30 h each with a 15-h photophase per cycle. Data were collected for BW, feed intake, livability, onset and rate of egg production, egg weight (EW), shell thickness, and weight of egg components. The results indicate that turkey hens can be induced to lay eggs with light-dark cycle lengths other than 24 h (ahemeral) with practical extremes being about 23 to 28 h. Egg production in the ahemeral treatments never significantly exceeded that occurring in the 24 h group and the percentage of floor eggs increased at cycle lengths greater than 26 h and at 21 h. Cycle lengths of 21 and 30 h provided the most extreme deviations in the quantity and quality of eggs from those obtained on a 24 h cycle length. Shell thickness increased consistently as cycle lengths increased from 23 to 30 h. Egg weight increased in a curvilinear manner as cycle lengths increased or decreased from 24 h. This increase was associated with increases in shell, yolk, and albumen weight; however, the contribution by each was inconsistent, varying by cycle length as well as time on treatment. It may be concluded that an effective practical range of light-dark cycles for turkeys is 23 to 28 h and that 28 h is a reasonable limit to maximize EW and shell thickness while minimizing a reduction in egg production.