Mechanistic Modeling of Size Exclusion Chromatography-Assisted In Vitro Refolding of the Recombinant Biosimilar Teriparatide (PTH-34).

In vitro protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using Escherichia coli as host cells. This study is focused on designing size exclusion chromatography-assisted in vitro refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and in vitro refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on in vitro refolding of recombinant teriparatide was studied using the Box-Behnken design. Size exclusion chromatography (SEC)-assisted in vitro refolding was performed at 8 °C at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post in vitro refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.

High frequency of ofloxacin resistance patterns of Mycobacterium leprae from India: An indication to revisit second line anti-leprosy treatment regimen.

OBJECTIVES: Drug resistance in leprosy is an emerging concern, leading to treatment failures, recurrences, and potential spread of resistant Mycobacterium leprae in the community. In this study, we aimed to assess drug resistance prevalence and patterns amongst leprosy patients at a tertiary care referral hospital in India. METHODS: Mutations in drug resistance determining regions for dapsone, rifampicin, and ofloxacin of the M. leprae genome in DNA extracted from skin biopsies of 136 leprosy patients (treatment-naive = 67, with persistent skin lesions = 35, with recurrence = 34) were analysed by polymerase chain reaction followed by Sanger sequencing. Wild-type strain (Thai-53) was used as a reference strain. RESULTS: Resistance mutations were identified in a total of 23 patients, constituting 16.9% of the cohort. Within this subset of 23 cases, resistance to ofloxacin was observed in 17 individuals (12.5%), while resistance to both dapsone and rifampicin was detected in three patients each (2.2% for both). The occurrence of ofloxacin resistance showed minimal disparity between recurrent and treatment-naive cases, at 17.6% and 16.4%, respectively. Dapsone resistance emerged in two treatment-naive cases and one case with persistent skin lesions. Notably, none of the treatment-naive cases or those with recurrence/relapse exhibited rifampicin resistance. Subsequently, no statistically significant correlation was identified between other clinical variables and the presence of antimicrobial resistance. CONCLUSIONS: The occurrence of resistance to the current multidrug therapy regimen (specifically dapsone and rifampicin) and to ofloxacin, a secondary antileprosy medication in M. leprae, represents a concerning scenario. This calls for an expansion towards bactericidal drug options and the establishment of robust surveillance for drug resistance in countries burdened with high leprosy rates. Moreover, the introduction of stringent antimicrobial stewardship initiatives is imperative. As a single centre study, it represents a limited, cross-sectional view of the real situation in the field.

An atypical presentation of multiple myeloma in a young patient with pathological fracture.

We report a case of a 34-year-old male with a history of pulmonary tuberculosis and pathological fracture of shaft of long bone presented with symptoms of lower respiratory tract infection. The patient did not have any typical symptoms of multiple myeloma or hypercalcemia on presentation. Throughout his hospitalization, his serum globulin level was very high along with mild normocytic normochromic anemia and mild renal function derangement without apparent cause. Acute phase markers of inflammation, for example, erythrocyte sedimentation rate (ESR) were not elevated in this patient and there was no lytic lesion in bone radiographs. He was eventually diagnosed as a case of stage 3 multiple myeloma by immuno-fixation electrophoresis and bone marrow study. Multiple myeloma represents a pathology of diverse distribution and has varied unusual presenting symptoms. We consider it an underdiagnosed disease often missed especially in young because it is not considered by clinicians.

Survival in human rabies but left against medical advice and death followed - Community education is the need of the hour.

Human survival after developing rabies is very scary to humanity. We report a case of a 58-year-old woman from Uttar Pradesh (north India), who presented with 5-days of fever and 1-day of altered sensorium associated with agitation, hydrophobia, and bedwetting after 20 days of WHO category 3 bite in the face by a rabid dog. She had taken three doses of anti-rabies vaccinations but not immunoglobulin of postexposure prophylaxis. Laboratory investigation showed a rising titer of virus-neutralizing antibodies in both serum and cerebrospinal fluid (CSF). We treated the patient according to the modified Milwaukee protocol. The patient remained to survive and had a recovery trend during hospital stays of 15 days before relatives took her left against medical advice (LAMA). As we know rabies has approximately 100% mortality rate but by using the aggressive treatment approach (like Milwaukee protocol), the patient may survive. Rabies can be effectively prevented by using adequate postexposure vaccine prophylaxis and rabies immunoglobulin (in category-3) after bite of a rabid animal. Our report along with other published reports should give more motivation to clinicians and education to the public to have an intensive treatment approach and patience, respectively to make rabies survival.

Efficient method for Agrobacterium mediated transformation of Artemisia annua L.

Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.