Zencore Analytical Sciences (AS) team are highly experienced in providing analytical support for biologics R&D, manufacturing, and documentation support for IND, BLA/NDA submission to FDA & China NMPA. Zencore AS team offers a comprehensive analytical strategies and solutions for biomolecules at different stages, ranging from pre-clinical to post approval stage.
Developability refers to the likelihood of a candidate protein drug becoming a viable therapeutic based on its structure and stability. High throughput assays, as well as the cutting-edge structural characterization tools are offered to support the developability study.
Critical qualities attributes (CQAs), which include size heterogeneity, charge heterogeneity, glycosylation, primary and higher-order structure and biological activities, are typically monitored for the candidate protein drug at this stage.
Zencore AS team has extensive experiences in analytical method development and method qualification/validation.
DS/DP release testing
Reference standard qualification
Characterization of product related impurities, quantification of the process related impurities, elucidation of degradation pathway
Critical quality attributes assessment and testing strategy
Work with customers to set up specifications for DS and DP.
Perform the release testing of drug substances and drug products.
Perform stress testing, accelerated stability, and long-term stability studies.
Transfer, qualify, and validate analytical methods.
Comparability study for IND/BLA filings.
Comparability study to support process change at different stages.
Similarity assessment of biosimilar products.
Comparability study required by the Prior Approval Supplement (PAS).
Leveraging our well-established analytical platform and cutting-edge technologies, Zencore AS team offers efficient and high-quality analytical services. A comprehensive in-house platform methods covering compendial, physicochemical, biochemical, biophysical and biological method have been well established and project specific analytical methods can be developed based on the project needs.
Background: Binding assays for both individual target and dual-targets have been developed for bispecific antibodies.
Challenges: The binding affinity of the two distinct targets varies significantly, which presents challenges in the development of the dual-target assay.
Results: First, two individual binding assays were developed, followed by the selection and optimization of the 1st antigen-antibody-biotinylated and 2nd antigen-enzyme labeled streptavidin for the dual assay. Several conditions, such as the choice of the coating antigen, antigen concentrations, incubation time, and chromogenic reaction conditions, were optimized to improve the robustness of the dual-target assay. Both the individual and dual-target binding assays were fully validated and used in batch release testing and stability studies.
Background: Zencore AS team has developed a competitive ELISA for detection of fusion protein binding activity.
Challenges: The fusion protein has no Fc tag.
Results: A biotinylated fusion protein was generated and mixed with unlabeled fusion protein before mixing with the anti-fusion protein antibody. Then, the streptavidin-labeled detection donor was bound to the biotinylated fusion protein specifically, bringing the donor closer to the fluorescent acceptor and enabling fluorescence resonance energy transfer (FRET). The FRET signal intensity was inversely related to the concentration of unlabeled protein sample. In this assay, homogeneous time-resolved fluorescence (HTRF) readout was achieved by FRET between donor of europium-XL665 complex and fluorescent acceptor. This method was validated with high-sensitivity and robustness.
For a peptide drug candidate, Zencore AS team generated the functional cell line by electroporating two vectors simultaneously, followed by clone screening and evaluation. The final cell line was capable of expressing the specific target protein to which the peptide could bind specifically, activating the luciferase expression in the cells. The cell-based assay was validated and used in the lot release and stability studies.
Zencore team developed an analytical method for miRNA extraction, amplification, and detection. Purified miRNA was transcribed to cDNA by reverse transcription, followed by a 3-step PCR to ligate the specified fragments and the amplification of the target sequence. Real-time fluorescence quantitative PCR was implemented to determine the concentration of the miRNA using the cycle threshold (Ct) value. Method was subsequently validated.
Accurate determination of sialic acid distribution (Z value) is important for assessing the quality attributes of glycoproteins with high sialic acid content. Zencore utilized the rapidly labeled 2-AB N-glycan testing kit (HiTang® N-sugar chain detection kit), which resolved the systematic error caused by sialic acid shedding during sample pre-processing. This method enables accurate determination of sialic acid distribution (Z value).
Background: Higher-order structure of the insulin analogs plays an important role in the half-life of the drug. Characterization of insulin hexamer is required by regulatory authorities.
Challenges: The formulation buffer contains phenol and zinc ions, which contribute to the formation of higher-order structure of the insulin analogs. However, phenol’s absorption interferes with absorbance of analytes, while zinc ions are insoluble in the formulation buffer.
Results: The drug product in the formulation buffer at the target concentration were directly measured by AUC in the interference mode, with a hexamer content more than 90%.
