Challenges in cell culture platform development of mAb production with site-specific incorporation of non-natural amino acid for ADC generation

Ambrx’s mammalian expression platform (EuCODE) enables non-native amino acids (nnAAs) through an expanded genetic code to both generate novel bio-therapeutics and to optimize the performance of antibody drug conjugate (ADC), therapeutic proteins, monoclonal antibodies (mAbs), and, bi- and multi-specific medicines. While the ability to control the defined Drug-to-Antibody Ratio (DAR) and payload site can provide an advantage to an ADC, the site-specific incorporation of the NAAs into the antibody heavy chain introduces a unique challenge for antibody production. To enable higher performance benchmarks in time and resources for process development with stringent product quality requirements, a proprietary cell culture platform is being developed and demonstrated fast-track development of high-quality, high-titer processes for producing recombinant proteins from CHO cells. We successfully generated a CHO-K1 cell line, stably expressing engineered amber suppressor tRNA and its cognate tRNA synthetase specific for non-natural amino acid para-acetyl phenylalanine (pAF), to achieve high production of monoclonal antibodies (mAbs) containing nnAAs. The stable cell lines were further evolved using CRISPR/Cas9 genome editing technology to sequentially knock out selected genes in glutamine synthesis, and, apoptosis pathways to improve selection efficiency and prevent loss of viable cell mass in production cultures, respectively. Inhibition of apoptosis pathway leads to dramatic increase in viable cell mass and results in extended production time and increased productivity. In this presentation, we will discuss the challenges in cell culture platform development including cell line engineering, systematic DoE-based approaches on optimal chemically defined media and cell culture processes, and, strategies for scale up to clinical and commercial scales

Genetic Diversity of Salmonella enteric serovar Typhi and Paratyphi in Shenzhen, China from 2002 through 2007

<p>Abstract</p> <p>Background</p> <p>Typhoid and paratyphoid fever are endemic in China. The objective of this investigation was to determine the molecular features of nalidixic acid-resistant <it>Salmonella enteric </it>serovar Typhi (<it>S. typhi</it>) and Paratyphi (<it>S. paratyphi</it>) from blood isolates in Shenzhen, China.</p> <p>Results</p> <p>Twenty-five <it>S. typhi </it>and 66 <it>S. paratyphi </it>were isolated from 91 bacteriemic patients between 2002 and 2007 at a hospital in Shenzhen, Southern China. Fifty-two percent (13/25) of <it>S. typhi </it>and 95.3% (61/64) of <it>S. paratyphi </it>A were resistant to nalidixic acid. Sixty-seven isolates of nalidixic acid-resistant <it>Salmonella </it>(NARS) showed decreased susceptibility to ciprofloxacin (MICs of 0.125-1 μg/mL). All 75 NARS isolates had a single substitution in the quinolone resistance-determining region (QRDR) of GyrA (Ser83→Phe/Pro/Tyr, or Asp87→Gly/Asn), and 90.7% of these isolates carried the substitution Ser83Phe in GyrA. No mutation was found in the QRDR of <it>gyrB</it>, <it>parC</it>, or <it>parE</it>. Plasmid mediated quinolone resistance genes including <it>qnr </it>and <it>aac(6')-Ib-cr </it>were not detected in any isolate. Twenty-two distinct pulsed field gel electrophoresis (PFGE) patterns were observed among <it>S. typhi</it>. Sixty-four isolates of <it>S. paratyphi </it>A belonged to one clone. Eighty-seven investigated inpatients were infected in the community. Six patients infected by <it>S. paratyphi </it>A had a travel history before infection.</p> <p>Conclusions</p> <p>Nalidixic acid-resistant <it>S. typhi </it>and <it>S. paratyphi </it>A blood isolates were highly prevalent in Shenzhen, China. PFGE showed the variable genetic diversity of nalidixic acid-resistant <it>S. typhi </it>and limited genetic diversity of nalidixic acid -resistant <it>S. paratyphi </it>A.</p

CRISPR-Cas9 mediated cell line engineering of apoptosis pathways increases antibody expression with site-specific modifications for antibody drug conjugation

New generation of antibody drug conjugates (ADCs) have expanded the repertoire of antibody drugs in the clinic and the market for cancer and inflammation indications by using highly stable linkers to attach potent small-molecule drug to various targeting antibodies. The drug and site of drug linkage to the antibody can have profound impact on the physiochemical properties and pharmacological profile of the ADC. Ambrx has developed a technology, Eukaryotic Chemical Orthogonal Directed Engineering (EuCODE), which allows non-natural amino acids with diverse physicochemical and biological properties to be genetically encoded and site-specifically incorporated into proteins/antibodies in mammalian cells. The non-natural amino acid provides a handle for the attachment of a small-molecule drug to generate homogenous ADC with a defined Drug-to-Antibody Ratio (DAR). To establish a CHO expression system for high production of monoclonal antibodies (mAbs) containing non-natural amino acids, we successfully generated a EuCODE platform cell line stably expressing engineered amber suppressor tRNA and its cognate tRNA synthetase specific for non-natural amino acid para-acetyl phenylalanine (pAF). When transfected with antibody of interest engineered with amber nonsense codon (TAG) at selected sites suitable for drug conjugation, this EuCODE platform cell line generates stable cell lines producing pAF containing mAbs for site-specifically conjugated ADC. In order to improve production titers of pAF containing antibody and achieve a robust platform, the platform cell line and stable cell lines were further evolved using CRISPR/Cas9 genome editing technology to sequentially knock out selected genes in glutamine synthesis and apoptosis pathways to improve selection efficiency and prevent loss of viable cell mass in production cultures, respectively. Inhibition of apoptosis pathway leads to dramatic increase in viable cell mass and results in extended production time and increased productivity. Phenotypic and genetic properties of these CRISPR engineered cell lines and product quality of the antibody will be discussed in the context of using the platform to develop a commercial manufacturing cell line

Cylindrical illumination with angular coupling for whole-prostate photoacoustic tomography

Current diagnosis of prostate cancer relies on histological analysis of tissue samples acquired by biopsy, which could benefit from real-time identification of suspicious lesions. Photoacoustic tomography has the potential to provide real-time targets for prostate biopsy guidance with chemical selectivity, but light delivered from the rectal cavity has been unable to penetrate to the anterior prostate. To overcome this barrier, a urethral device with cylindrical illumination is developed for whole-prostate imaging, and its performance as a function of angular light coupling is evaluated with a prostate-mimicking phantom

The effects of graded levels of calorie restriction : XI. Evaluation of the main hypotheses underpinning the life extension effects of CR using the hepatic transcriptome

We would like to acknowledge the BSU staff for their invaluable help with caring for the animals.Peer reviewedPublisher PD

The effects of graded levels of calorie restriction : XIX. Impact of graded calorie restriction on protein expression in the liver

Open Access under the OUP Agreement The work was supported by grants from the National Key Research and Development program of China 2018YFA0801000, UK Biotechnology and Biological Sciences Research Council BBSRC (BB/G009953/1 and BB/J020028/1) and the National Natural Science Foundation of China (NSFC Aging program grant: 91649108). D.L. was supported by Office of Naval Research (ONR) (N000141512377) and J.R.S. was supported by a Wolfson research merit award from the Royal Society and a President’s International Fellowship Initiative (PIFI) professorial fellowship from the Chinese Academy of Sciences (CAS).Peer reviewedPublisher PD

Investigation and analysis of psychological stress and professional identity of nursing students during COVID-19 pandemic

426-432Certain diseases or infections affect large number of people in short span of time. A local endemic disease can outbreak into an epidemic affecting the whole population or region which at times extend to other countries and continents and become pandemic. Pandemics results in loss of life as well as economy. Pooled efforts and resources, effective sharing of data, parallel multiple approaches as well as physical and mental state of front line staff influence management of pandemics. The coronavirus disease COVID-19 caused by SARS-CoV-2 started in December 2019 from Wuhan in China, is now a worldwide public health emergency affecting millions of people. It affects many frontline healthcare workers too. Here, we studied psychological stress and professional identity of nursing students for possible correlations, if any, and analyze influencing factors. We used purposive sampling technique with 415 nursing students in Nanjing, China through a general information questionnaire, perceived stress scale and nursing professional identity questionnaire. Students' origin, monthly living expenses and their knowledge on epidemic prevention and treatment have shown a significant impact on their psychological stress (P <0.01). Similarly, students' gender, origin, clinical practices and knowledge of prevention and treatment, and whether they actively learn such knowledge impact significantly on their professional identity (P <0.01). The overall score of psychological stress was (24.47±7.35) and professional identity had 72.47±8.07. The stress condition exhibited negative correlation with the degree of professional identity (P <0.01，r = -0.457). Increased psychological stress, had lower sense of professional identity. Overall, analysis of data on perceived stress and professional identity pandemic suggests that stress levels are inversely proportional to knowledge in effective ways of handling the pandemic. Students with clinical practice fared better in terms of professional identity. The study suggests nursing students to stay focused on studies, clinical practice and counselling, if required

Comparison of velopharyngeal morphology of two palatoplasty techniques in patients with hard and soft cleft palate

PurposeThe study aims to compare the velopharyngeal morphology of hard and soft cleft palate (HSCP) patients after Furlow and Sommerlad palatoplasty.Patients and methodsA total of 51 patients (20 cases in Furlow palatoplasty group, 16 cases in Sommerlad palatoplasty group and 15 normal children in the control group) were included in our study. Velopharyngeal function and speech outcomes of patients with HSCP who had either Furlow palatoplasty or Sommerlad palatoplasty for cleft palate repair were evaluated by perceptual speech assessment (PSA), lateral cephalometric radiographs and nasopharyngoscopy. To assess velopharyngeal morphology of patients treated with two techqiques, we analyzed measurements such as velar length, pharyngeal depth, and the Adequate ratio (the ratio of velar length to pharyngeal depth). Furthermore, skeletal landmarks including cranial base, cervical vertebrae, posterior nasal spine which were defined as the pharyngeal triangle were measured. Finally, the position of the point U relative to the pharyngeal triangle were compared.ResultsVelopharyngeal closure (VPC) rate in Furlow palatoplasty group accounted for 90%, while that in Sommerlad palatoplasty group was 81.3%. PSA of the former group was significantly better than that of the latter group (P &lt; 0.05). Velar length, pharyngeal depth and the Adequate ratio (1.37 ± 0.14 vs. 1.41 ± 0.15) were comparable between the Furlow group and control group (P &gt; 0.05), while Sommerlad group had a shorter velar length, deeper pharyngeal depth and a smaller Adequate ratio (1.20 ± 0.18) compared to the above two groups (P &lt; 0.05). Furhermore, the point U of Sommerlad group in the pharyngeal triangle was higher than that of the other two groups.ConclusionsIn the treatment modality of patients with HSCP, both Furlow palatoplasty and Sommerlad palatoplasty seem to be effective. Furlow palatoplasty appears to have velopharyngeal morphology similar to normal control group., while Sommerlad group shows a shorter velar length, deeper pharyngeal depth and a smaller Adequate rati