Measuring the bioactivity of anti-IL-6/anti-IL-6R therapeutic antibodies: presentation of a robust reporter gene assay.

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.

A novel alternative for pyrogen detection based on a transgenic cell line.

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

Improve the product structural robustness based on network motifs in product development.

The stability and safety of products will be reduced if product structures are vulnerable to failures of key components. Existing methods for improving product structural robustness mainly focus on some key components, but they cannot provide designers with universal and explicit structure optimization strategies. From the viewpoint of product structural networks, the motif is the fundamental meta-structure, and it is efficient to analyse product structural properties. Motivated by this, strategies to improve product structural robustness are explored by considering relationships between typical motifs and product structural robustness. First, product structural networks are constructed by collecting the structural information of a series of product generations. Second, typical (anti-) motifs are identified based on an enumeration algorithm, and the robustness is measured considering the largest connected cluster. Then, relationships between the frequency of different motifs and product structural robustness are obtained through principal component regression. The results of a case study on the smartphone show that anti-motifs are negative for product structural robustness. Motifs with loop structures are positive for product structural robustness. Accordingly, relevant strategies to improve product structural robustness in product development are developed.

A Novel Reporter Gene Assay for Pyrogen Detection.

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signaling molecule that causes the excessive secretion of various pyrogen-induced pro-inflammatory factors. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. The RGA could detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 µg/ml, and 1 µg/ml, respectively. The method had good precision and accuracy and could be applied to many molecules (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, Haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, group A and group C meningococcal conjugate vaccine, diphtheria, tetanus, pertussis [acellular, component], poliomyelitis [inactivated] vaccine, and imject alum adjuvant). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.

A simple and robust reporter gene assay for measuring the bioactivity of anti-RANKL therapeutic antibodies.

RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL-RANK-NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.