Regeneration of Capto Core 700 resin through high throughput and laboratory scale studies and impact on production of a SARS-CoV-2 vaccine candidate.

During the development of a SARS-CoV-2 vaccine candidate, at the height of the COVID-19 pandemic, raw materials shortages, including chromatography resins, necessitated the determination of a cleaning in place (CIP) strategy for a multimodal core-shell resin both rapidly and efficiently. Here, the deployment of high throughput (HT) techniques to screen CIP conditions for cleaning Capto Core 700 resin exposed to clarified cell culture harvest (CCCH) of a SARS-CoV-2 vaccine candidate produced in Vero adherent cell culture are described. The best performing conditions, comprised of 30% n-propanol and ≥0.75 N NaOH, were deployed in cycling experiments, completed with miniature chromatography columns, to demonstrate their effectiveness. The success of the CIP strategy was ultimately verified at the laboratory scale. Here, its impact was assessed across the entire purification process which also included an ultrafiltration/diafiltration step. It is shown that the implementation of the CIP strategy enabled the re-use of the Capto Core 700 resin for up to 10 cycles without any negative impact on the purified product. Hence, the strategic combination of HT and laboratory-scale experiments can lead rapidly to robust CIP procedures, even for a challenging to clean resin, and thus help to overcome supply shortages.

A simple and efficient derivatization strategy combined with switchable solvent liquid-liquid microextraction hydroxychloroquine methyl acetate-d3 -based quadruple isotope dilution gas chromatography mass spectrometry for the determination of hydroxychloroquine sulfate in biological fluids.

RATIONALE: A derivatization switchable solvent liquid-liquid microextraction quadruple isotope dilution gas chromatography mass spectrometry (D-SS-LLME-ID4 -GC/MS) method is presented for the determination of hydroxychloroquine sulfate in human biofluids. METHODS: While mixing type/period and concentration of NaOH were optimized via a univariate optimization approach, a multivariate optimization approach was used to determine optimum values for relatively more important parameters such as volumes of derivatization agent (acetic anhydride), NaOH and switchable solvent. RESULTS: Under the optimum experimental conditions, limit of detection and limit of quantification were calculated as 0.03 and 0.09 mg/kg (mass based), respectively. An isotopically labelled material (hydroxychloroquine methyl acetate-d3 ) was firstly synthesized to be used in ID4 experiments which give highly accurate and precise recovery results. After the application of D-SS-LLME-ID4 , superior percent recovery results were recorded as 99.9 ± 1.6-101.3 ± 1.2 for human serum, 99.9 ± 1.7-99.8 ± 1.8 for urine and 99.6 ± 1.5-101.0 ± 1.1 for saliva samples. CONCLUSIONS: The developed D-SS-LLME-ID4 -GC/MS method compensates the complicated matrix effects of human biofluids and provides highly accurate quantification of an analyte with precise results.

Fast, simple and cheap: method modified from conventional cultivation for tuberculosis diagnosis allows seeding on Löwenstein-Jensen of any swab-embedded pulmonary samples decontaminated with sodium hydroxide.

BACKGROUND: Few tuberculosis (TB) control programmes in low-income countries have access to culture facilities in their primary care diagnostic centres and this scenario may have worsened with the coronavirus disease 2019 pandemic. Thus the aim was to develop and evaluate a simpler TB test that allows seeding on Löwenstein-Jensen (LJ) medium of several swab-embedded samples decontaminated with sodium hydroxide (NaOH). METHODS: A cotton swab containing each sample was decontaminated in NaOH before being dipped into a slightly acidic solution to neutralize the pH in order to allow the culture to develop on LJ medium. Samples (n=543) from suspected or confirmed pulmonary TB were analysed in two phases: standardization (n=167) and evaluation of the study method (n=376). RESULTS: The study method showed sensitivity >95% and specificity >93% using Ogawa-Kudoh (OK) and modified Petroff (MP) as standards and was comparable to MP-LJ (p>0.05) and slightly superior to OK (p=0.03) for sputum culture and more comprehensive than the latter for other pulmonary specimens. CONCLUSIONS: This article reports a more comprehensive, simpler and less costly method for diagnosing TB in the laboratory with fewer economic resources and biosafety equipment. Thus a patent application was filed (BR1020190103841).