Unravelling the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.

We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms.

BACKGROUND: Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the holehole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide certain resolution between these two conformational isoforms. OBJECTIVE: The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms. METHODS: The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC). RESULTS: SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms. CONCLUSION: Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions.

Validation of the Chinese Version of the Coronary Artery Disease Education Questionnaire - Short Version: A Tool to Evaluate Knowledge of Cardiac Rehabilitation Components.

Background: Patient education is the first step in implementing a cardiac rehabilitation (CR) program and a powerful tool for promoting behavioral changes in cardiac patients. In China, the clinical workload is so heavy that a short and reliable tool for assessing disease-related knowledge is needed for targeted patient education. Objective: The aim of this study was to translate, adapt and validate the Chinese version of the Coronary Artery Disease Education Questionnaire - Short Version (CADE-Q SV). Methods: The CADE-Q SV was translated to simplified Chinese and culturally adapted to the Chinese context. The translated version was reviewed by a committee of seven experts in cardiovascular disease, and the content validity of the questionnaire was established. The psychometric properties of the questionnaire were analyzed considering the responses of 240 CR patients. The Kuder-Richardson-20 (KR-20) coefficient and Cronbach's alpha were used to assess internal consistency. The intraclass correlation coefficient (ICC) was used to assess test-retest reliability. The criterion-related validity was evaluated by determining whether there were differences in the total scores of patients with different educational levels. Confirmatory factor analysis (CFA) was used to assess the factor structure. Results: Three items from the original version were adapted to reflect Chinese culture. The content validity index was 0.94. The KR-20 score was 0.856. All ICC values were > 0.70. The knowledge scores of patients with different educational levels were significantly different, indicating that the criterion-related validity of the Chinese CADE-Q-SV was acceptable. CFA validated the five-factor structure of the Chinese CADE-Q-SV. Conclusion: The Chinese CADE-Q SV questionnaire has good reliability and validity. This short, efficient tool can be completed quickly, assess disease-related knowledge in cardiovascular patients and serve as a reference for individualized patient education in China. It can also be used to evaluate the effectiveness of CR-related patient education interventions.

Polygonatum sibiricum polysaccharides (PSP) improve the palmitic acid (PA)-induced inhibition of survival, inflammation, and glucose uptake in skeletal muscle cells.

Polygonatum sibiricum polysaccharides (PSP) can decrease the levels of fasting blood glucose, total cholesterol, and triglyceride (TG) in hyperlipidemic and diabetic animals. It can also reduce inflammatory cytokines and promote glucose uptake in adipocytes. However, the underlying molecular mechanisms of PSP in improving insulin resistance (IR) in skeletal muscle remain unclear. In this study, palmitic acid (PA) induced an IR model in L6 myotubes. After treatment, cell proliferation was measured using the CCK8. miR-340-3p, glucose transporter 4 (GLUT-4), and interleukin-1 receptor-associated kinase 3 (IRAK3) expression was measured by qRT-PCR. IRAK3 protein levels were measured by Western blotting. Glucose in the cell supernatant, TG concentration in L6 myotubes, and the levels of IL-1ß, IL-6, and TNF-α were measured by an ELISA. We found that cell survival, glucose uptake, and GLUT-4 expression in L6 myotubes were significantly suppressed, while lipid accumulation and inflammatory factor levels were enhanced by PA stimulation. Furthermore, PSP treatment markedly alleviated these effects. Interestingly, PSP also significantly reduced the upregulated expression of miR-340-3p in the L6 myotube model of IR. Furthermore, overexpression of miR-340-3p reversed the beneficial effects of PSP in the same IR model. miR-340-3p can bind to the 3'-untranslated regions of IRAK3. Additionally, PA treatment inhibited IRAK3 expression, whereas PSP treatment enhanced IRAK3 expression in L6 myotubes. Additionally, miR-340-3p also inhibited IRAK3 expression in L6 myotubes. Taken together, PSP improved inflammation and glucose uptake in PA-treated L6 myotubes by regulating miR-340-3p/IRAK3, suggesting that PSP may be suitable as a novel therapeutic agent for IR.

Poly (Ethylene Oxide)-Based Block Copolymer Electrolytes Formed via Ligand-Free Iron-Mediated Atom Transfer Radical Polymerization.

The Br-terminated poly (ethylene oxide) (PEO-Br) is used as a green and efficient macroinitiator in bulk Fe-catalyzed atom transfer radical polymerization (ATRP) without the addition of any organic ligands. The polymerization rate is able to be mediated by PEO-Br with various molecular weights, and the decrease in redox potential of FeBr2 in cyclic voltammetry (CV) curves indicates that an increased coordination effect is deteriorated with the depressing reaction activity in the longer ethylene oxide (EO) chain in PEO-Br. In combination with the study of different catalysts and catalytic contents, the methyl metharylate (MMA) or poly (ethylene glycol) monomethacrylate (PEGMA) was successfully polymerized with PEO-Br as an initiator. This copolymer obtained from PEGMA polymerization can be further employed as a polymer matrix to form the polymer electrolyte (PE). The higher ionic conductivity of PE was obtained by using a high molecular weight of copolymer.