Reactor Design for Continuous Monoclonal Antibody Precipitation Based Upon Micro‐mixing

BACKGROUND: Precipitation has been applied for the processing of important therapeutics, including monoclonal antibodies (mAbs). The scale‐up has proven to be a challenging task due to the complexity of the reactions and transport processes involved. This requires a good understanding of the molecular processes underpinning precipitate formation. The aim of this study was to build a micro‐mixing model for the precipitation of a mAb in continuous tubular reactors using ammonium sulphate. The effect of micro‐mixing on precipitate formation (with respect to size, strength, and nature) was evaluated. An ultra scale‐down (USD) centrifugation methodology was applied to determine the ease of precipitate clarification. RESULTS: The results demonstrated that the final mean particle size decreased with increased micro‐mixing, and was obtained with short residence times. Antibody yields in the tubular reactors were consistently above 90% and were shown to be independent of the mixing. Similar particle sizes between a lab and pilot‐scale reactor were correlated with the average energy dissipation rate. The smaller particles obtained from improved micro‐mixing had higher fractal dimensions that correlated with minimal breakage upon exposure to turbulent shear. Precipitates were easily clarified at the USD scale (> 95% clarification), but less so at pilot‐scale (< 80% clarification). CONCLUSION: Precipitation is a rapid process where the final precipitate properties are controlled by the flow conditions. Therefore, the process can be manipulated to acquire a certain particle size range. A high‐throughput precipitation process is also possible. However, further investigation into large‐scale precipitate recovery is required. © 2020 Society of Chemical Industr

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

<p>Abstract</p> <p>Background</p> <p>Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements.</p> <p>Methods</p> <p>We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in <it>DMD </it>gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis.</p> <p>Results</p> <p>We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the <it>DMD </it>gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65.</p> <p>Conclusion</p> <p>The analysis of our patients' sample, carrying point mutations or complex rearrangements in <it>DMD </it>gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.</p

Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients

Removal of Eroded Gastric Bands Using a Transgastric SILS Device

Background. Laparoscopic adjustable gastric banding (LAGB) is a popular method for the treatment of morbid obesity. One of the most feared complications is gastric band erosion which occurs with a reported incidence of 0.3 to 14%. Intragastric migrated bands are best managed by endoscopic removal. Recent case studies reported successful endoscopic removal of intragastric migrated bands, but it is not always possible. We report our first experience with a transgastric removal of eroded bands using a Single Incision Laparoscopic Surgery (SILS) device. Methods. A patient who underwent gastric banding in the past (2007) presented with symptoms of epigastric pain and weight gain. Preoperative gastroscopy revealed stomach wall erosion with the gastric band partially (2/3) migrated into the gastric lumen. Attempts to remove the band by endoscopy were not successful. A laparoscopy was performed and multiple adhesions with evidence of inflammation was seen in the upper abdomen around the band. A SILS port was inserted through a 2 cm incision in the left hypochondrium with the internal ring of the port placed into the stomach through a small anterior gastrotomy. The band was cut in the stomach and removed. The anterior gastrotomy was closed. We had a perfect intragastric view of the gastric banding. Results. There were no intra- or postoperative complications. The patient was discharged on the fifth postoperative day on a gastric adapted diet. Conclusion. Removal of a gastric band after gastric erosion by SILS is feasible, safe, and effective. This is the first reported case of transgastric removal of eroded bands using an SILS device

Renal response to an acute oral protein load in healthy humans and in patients with renal disease or liver cirrhosis.

This article analyzes 57 reports published in the years 1983 through 1964 that addressed the issue of the renal hemodynamic response to an oral protein load. Seventy-three groups are reported in those studies: 52 were healthy subjects (n = 627) and 21 had renal disease (n = 256); 47 were studied using inulin (n = 407 healthy people and 112 renal patients); 26 groups were studied using creatinine (n = 220 healthy people and 144 renal patients). Patients with liver cirrhosis were also analyzed. There was great heterogeneity in methodology used, emphasizing the need for standardization. The role of plasma amino acids, glucagon, insulin, growth hormone, PGE2, 6- ketoPGA1α, brain-gut peptides, ANP, AVP, dopamine, and kinins in promoting the renal hemodynamic response to an oral protein load is discussed