Demonstrating analytical similarity of a biosimilar HLX04 to bevacizumab with a panel of state-of-the-art methods and tiering of quality attributes.

Therapeutical monoclonal antibodies are structurally and functionally complex, whereas the innovator's manufacturing processes are proprietary. With respect to the similarity assessment, a proposed biosimilar product needs to demonstrate a side-by-side comparison between the reference product (RP) and candidate product in terms of physicochemical properties and biological activities, as well as nonclinical and clinical outcomes. Here, a comprehensive analytical similarity assessment was performed for in-depth comparison of HLX04, China-sourced Avastin® (CN-Avastin®), and Europe-sourced Avastin® (EU-Avastin®) following a tier-based quality attribute (QA) evaluation. A series of orthogonal and state-of-the-art analytical techniques were developed for the assessment. Ten lots of HLX04 were compared with 29 lots bevacizumab RP. Referred to the characterization results, HLX04 is highly similar to the RPs with respect to physicochemical properties and biological functions. In addition, HLX04 was found with similar stability and degradation behaviors upon multiple stressed conditions to bevacizumab. Minor differences were observed in glycosylation, aggregates, FcγRIIIa(F), and FcγRIIIa(V) binding activities; nevertheless, they were evaluated and demonstrated not to impact clinical outcomes. According to the reported clinical results, the totality of evidence, including the pharmacokinetic, efficacy, safety, and immunogenicity, further shows that HLX04 is similar to CN-/EU-Avastin®.

Quality by Design-Based Assessment for Analytical Similarity of Adalimumab Biosimilar HLX03 to Humira®.

Quality by design (QbD) is an efficient but challenging approach for the development of biosimilar due to the complex relationship among process, quality, and efficacy. Here, the analytical similarity of adalimumab biosimilar HLX03 to Humira® was successfully established following a QbD quality study. Quality target product profile (QTPP) of HLX03 was first generated according to the public available information and initial characterization of 3 batches of Humira®. The critical quality attributes (CQAs) were then identified through risk assessment according to impact of each quality attribute on efficacy and safety. The anticipated range for each CQA was derived from similarity acceptance range and/or the corresponding regulatory guidelines. Finally, a panel of advanced and orthogonal physicochemical and functional tests and comparison of 6 batches of HLX03 and 10 batches of the reference standard demonstrated high similarity of HLX03 to Humira®, except for slightly lower percentage of high mannosylated glycans (%HM) in HLX03 which had no effect on FcγRIII binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity in human peripheral blood mononuclear cell (PBMC). All above demonstrated the feasibility and efficiency of QbD-based similarity assessment of a biosimilar monoclonal antibody (mAb).

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology.

BACKGROUND: A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. OBJECTIVES: The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. METHODS: A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. RESULTS: The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. CONCLUSION: Besides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.

[Effects of Electroacupuncture on Expression of Myocardial Chloride Channel-2 and CLCA Proteins in Mice with Acute Myocardial Ischemia].

OBJECTIVE: To observe the influence of electroacupuncture stimulation (EA) of "Neiguan" (PC 6)/"Lieque" (LU 7) on ST segment of electrocardiogram (ECG) and the expression of myocardiac chloride channel (CLC)-2 and calcium (Ca2+)-activated chloride channel accessory (CLCA) proteins in acute myocardial ischemia (AMI) mice, so as to explore its mechanisms underlying improvement of AMI. METHODS: Thirty ASIC 3-/- knock out mice were randomly divided into control, AMI model, EA-Neiguan (PC 6), EA-Lieque (LU 7) and EA-non-acupoint groups. The AMI model was induced by multiple subcuta-neous injection of isoprenaline (ISO, 0.5 g/L, 20 mg/kg). EA was applied to bilateral PC 6, LU 7, or non-acupoint[the mid-point between "Tianshu" (ST 25) and "Shenque" (CV 8)] for 20 min, once daily for 7 days. The ST segment of ECG of the standard Ⅱlimb-lead was recorded using a PowerLab data acquisition device, the change of myocardial histology observed by using microscope after HE staining, the activity of serum superoxide dismutase (SOD) detected using Colorimetric method, and the expression of CLC-2 and CLCA proteins of the left ventricle myocardium detected by Western blot. RESULTS: Outcomes of HE staining showed that the ischemic injury (sarcoplasm swelling and necrosis, etc.) of the left ventrical myocardial tissue after mo-deling was relatively milder in the EA-PC 6 group, not in the EA-non-acupoint group. The SOD activity was significantly lower in the model group than in the control group (P<0.01), and obviously increased in the EA-PC 6 group(P<0.01), but not in the EA-non-acupoint group (P>0.05). The expression levels of myocardial CLC-2 and CLCA proteins were significantly up-regulated in the model group compared with the control group (P<0.01) and markedly down-regulated in the EA-PC 6 group (not in the EA-non-acupoint group) in comparison with the model group(P<0.01), suggesting a specificity of effects of EA-PC 6 in improving myocardial injury and down-regulating CLC-2 and CLCA protein expression. CONCLUSIONS: EA of "Neiguan" (PC 6) can effectively suppress the increase of expression of CLC-2 and CLCA proteins in the left ventricle myocardium, which may contribute to its effect in relieving myocardial injury in mice.