Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review

To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing.     

Microbial Dynamics and Diversity of Bacillus thuringiensis in Textile Effluent Polluted and Non-polluted Rice Field Soils of Orissa, India

Microbial diversity was assessed in the soils of non-polluted rice fields of Central Rice Research Institute and Choudwar, and textile effluent contaminated (about 30 years) rice fields of Choudwar about 4 years after cessation of pollution. The soils contained 0.62–1.01 % organic C and 0.07–0.12 % total N, and measured 6.18–8.24 pH and 0.6–2.68 mS/cm Eh which were more in the polluted Choudwar soil. The microbial populations (×10⁶ cfu/g soil) in the soils were: heterotrophs 1.21–10.9, spore formers 0.9–2.43, Gram (−)ve bacteria 4.11–8.0, nitrifiers 0.72–1.5, denitrifiers 0.72–2.43, phosphate solubilizers 0.14–0.9, asymbiotic nitrogen fixers 0.34–0.59, actinomycetes 0.07–0.11, fungi 0–0.5 and Bacillus thuringiensis (Bt) 0.4–0.61 which predominated in the polluted soil of Choudwar. The fungi were scarce in the polluted rice fields. The Bt isolates belonged to three motile and one non-motile group. Two motile Bt isolates were phenotyped as Bt subsp. sotto and israelensis, whereas, the non-motile isolate was Bt subsp. wahuensis. All Bt isolates produced extracellular protease, lipase and amylase enzymes. The microbial guilds had positive correlation among themselves, as well as, with soil physico-chemical characters but the fungi had negative relation and the nitrogen fixers were unrelated with the biotic and abiotic components.     

Isolation and structural identification of the trihydroxamate siderophore vicibactin and its degradative products from Rhizobium leguminosarum ATCC 14479 bv. trifolii

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C₃₃H₅₅N₆O₁₅) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C₂₀H₃₆N₄O₁₀, C₃₁H₅₅N₆O₁₅, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Infradian rhythmicity in lactogenic hormone (prolactin,growth hormone,cortisol and thyroid hormone) secretion throughout the lactation cycle in mithun cows (Bos frontalis): variation among strains

The role of lactogenic hormones (prolactin, growth hormone, cortisol and thyroid hormone) on lactation yield in Mithun cows as well as their rhythmicity throughout the lactation cycle were studied in Mizoram (n = 4) and Nagaland (n = 7) strain of mithun (Bos frontalis). Blood samples were collected from all the animals from the day of calving to the complete dry off at an interval of 15 days. All the hormones were estimated in the serum by commercially available ELISA kits. Plasma level of cortisol (μg/dl), growth hormone (GH, in ng/ml), prolactin (PRL, in μIU/ml), triiodothyronine (T₃, in nmol/μl) and thyroxin (T₄, in ng/ml) were 20.84 ± 0.29, 28.08 ± 0.56, 9.87 ± 0.20, 27.82 ± 0.56 and 51.33 ± 0.48, respectively, in mithun irrespective of strains during the lactation period. Levels of all the hormones varied significantly (p ≤ 0.01) during different days of lactation cycle but, there was no significant difference among strain. Levels of PRL, GH, cortisol and T₃ were significantly (p < 0.01) higher around calving and declined sharply. The hormones remained in almost steady state during mid-lactation and declined during late lactation. All the hormones stated above were positively correlated with lactational yield thus their role on lactogenesis and galactopoiesis was established.