Manufacturing arthropod and mammalian allergen extracts
Richard F. Lockey, Dennis K. Ledford in Allergens and Allergen Immunotherapy, 2020
A general guide for the preparation of allergen extracts of mites and insects follows: Weigh the desired amount of source material in a 1:20 weight/volume ratio of a specific buffer, i.e., phosphate buffer saline (PBS), at 4°C for 4–8 hours under continuous stirring conditions. Other weight/volume ratios can also be used. Afterward, adjust the pH between 7.5 and 8.5 by the addition of NaOH or HCl. The careful selection of the extraction buffer is a key issue, as has been previously demonstrated [40]. In some cases, a double extraction of the source material can be performed. Once the extraction is completed, centrifuge the recovered extract volume in a centrifuge at 10,000 rpm for 30 minutes at 4°C (the centrifuge must be cooled down in advance). Afterward, carefully separate the supernatant and discard the pellet. Filtrate the obtained extract in a filter cascade until going through a 0.2 μm pore size filter. A diafiltration (dialysis) step is now performed in a Pellicon tangential flow filtration system (Pellicon, Merck Millipore), using a cassette of 5 kDa cutoff. The dialysis process should be performed with five times the same volume of ultrapure. The conductivity in the last step must be lower than 500 ppm (approximately 800 μS/cm). Once the extract is dialyzed, aliquot the extract in 50 mL freeze-drying vials (previously cleaned and labeled) and freeze-dry it according to the program selected. After the freeze-drying cycle is completed, the extract is ready to be diluted to the desired concentration, and after several manufacturing steps, it is ready to be used.
Bioprocess Parameters of Production of Cyanobacterial Exopolysaccharide
Gokare A. Ravishankar, Ranga Rao Ambati in Handbook of Algal Technologies and Phytochemicals, 2019
Ramus (Ramus 1972) reported segregation of the “encapsulating polysaccharide” from Porphyridium biomass. Red microalgae produces sulfated polysaccharides dissolvable within the nutrient media. The removal of color from biomass was achieved by applying the acetone along with ethanol, further solubilization of exopolysaccharide glue from porphyridium cells was achieved using warm water. Another study also reports the use of absolute alcohol or isopropanol (Liu et al. 2015; Patel et al. 2013). The yield of polysaccharide is influenced by temperature of precipitation and polarity of alcohol. This technique robustly yields EPS. The method possesses specific merits like further recycling the alcohol via distillation to modify extremely viscid solution. Patel et al. (Patel et al. 2013) delineated the extraction and salting out of EPS from Porphyridium cruentum applying the alcoholic precipitation, separation via membrane method. Furthermore, they have inferred the application of diafiltration employing 300 kDa membrane as a highly effective technique. Occasionally, an additional refinement phase is necessary to remove the undesired molecules like proteins, pigments, salts and another compounds via trichloroacetic acid modification, peripheral ultrafiltration, or precipitation by using alcohol (Li et al. 2011; Patel et al. 2013).
Biologic Drug Substance and Drug Product Manufacture
Anthony J. Hickey, Sandro R.P. da Rocha in Pharmaceutical Inhalation Aerosol Technology, 2019
Once proteins are purified through downstream chromatography steps, the next steps involve transitioning the protein into the formulation buffer (called buffer exchange or diafiltration, or DF) and increasing the protein concentration (called ultrafiltration, or UF). These steps are usually carried out together in a single unit operation, utilizing a process called tangential flow filtration (TFF). This unit operation involves continuous flow of the protein solution under high pressure in a direction tangential to the UFDF membrane. Buffer exchange happens by introduction of the target buffer into the protein solution. The semi-permeable membrane causes retention of the protein molecules in the circulation path (retentate), while certain amount of the buffer mixture is allowed to pass through the membrane (permeate). For buffer exchange, the retentate volume is held constant through pressure control mechanisms. Thus, over time, as more of the target buffer is introduced, the overall percentage of the target buffer increases and the levels of the initial buffer species remaining become minimal. To increase protein concentration, more permeate is pushed through the membrane than fresh buffer is introduced, so that the overall retentate volume is reduced and the concentration is increased.
Virus-like particle-based nanocarriers as an emerging platform for drug delivery
Published in Journal of Drug Targeting, 2023
Bingchuan Yuan, Yang Liu, Meilin Lv, Yilei Sui, Shenghua Hou, Tinghui Yang, Zakia Belhadj, Yulong Zhou, Naidan Chang, Yachao Ren, Changhao Sun
To produce VLPs for clinical use, purification is required. The main purpose of purification is to remove host cell proteins and process-derived impurities from the VLP concentrate [129]. Super-centrifugal purification methods in sucrose or CsCl gradients are usually sufficient to obtain suitable VLPs for subsequent application, especially on a laboratory-scale or using small-scale processes. Hillebrandt et al. reported a new purification method known as crossflow filtration [143]. The application of a super-centrifugal method is limited in industrial production because of the risk of VLP aggregation, high labour intensity and the lack of scalability [138]. Therefore, it is necessary to purify VLPs using special chromatography techniques rather than super-centrifugation. Depending on the VLP properties, different ion exchange-, affinity- and size-exclusion columns can be used for purification. Diafiltration and tangential flow filtration are also used to scale up VLP production.
Systematic determination of the relationship between nanoparticle core diameter and toxicity for a series of structurally analogous gold nanoparticles in zebrafish
Published in Nanotoxicology, 2019
Lisa Truong, Tatiana Zaikova, Brandi L. Baldock, Michele Balik-Meisner, Kimberly To, David M. Reif, Zachary C. Kennedy, James E. Hutchison, Robert L. Tanguay
In the second step, TMAT-AuNPs were synthesized through a biphasic ligand exchange reaction of TOAB-stabilized particles in toluene with TMAT (Kearns 2007). TMAT (100 mg) was dissolved in 30 mL of water and neutralized by passing through a column of poly (4-vinylpyridine) before adding it to the toluene solution obtained from the first step. The biphasic reaction mixture was stirred rapidly at room temperature for 23 h. The reaction was deemed complete when the dark-colored NPs transferred from the organic to aqueous phase. The aqueous layer was isolated and extracted with 30 mL × 5 of DCM to remove remaining toluene. Traces of organic solvents remaining after the extractions were removed by rotary evaporation at room temperature. The crude material was purified by diafiltration using a 100 kDa membrane with 2000 mL (100 volumes) of nanopure water. After lyophilization, the powder was collected and characterized by 1H NMR, UV-vis spectroscopy, SAXS, and TEM.
Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process
Published in mAbs, 2018
Cheng Du, Yunping Huang, Ameya Borwankar, Zhijun Tan, Anthony Cura, Joon Chong Yee, Nripen Singh, Richard Ludwig, Michael Borys, Sanchayita Ghose, Nesredin Mussa, Zheng Jian Li
Two IgG4 molecules (1 and 2) and two IgG1 molecules (3 and 4) were used in this study. The IgG4 molecules had a single point mutation of serine to proline in the hinge region motif CPSC of original IgG4 to resemble the IgG1 inter-chain disulfide bond structure CPPC.11,20 All the mAbs were produced in CHO cell culture with cell viabilities ranging from 50 to 90%. The CB was usually generated with depth filtration unless otherwise specified, in which case it was generated by centrifugation. The mAb 1 cell culture in some cases was treated with low pH plus dextran sulfate before depth filtration. Downstream purification was achieved by Protein A chromatography, additional polishing chromatographic steps and a final ultrafiltration/diafiltration step into a histidine-sugar-based buffer. Protein concentrations were determined by absorption at 280 nm (A280) by a Dropsense-96 spectrometer (Trinean NV, 9050 Gent, Belgium).