Leveraging Prior Knowledge to Demonstrate Analytical Competency

Analytical groups are developing more methods than ever to address mounting demand for biopharmaceuticals. Still, such teams need to work within tight timelines to help candidate therapies advance quickly through clinical trials. During a May 2022 presentation, Rajgopal Rudrarapu (senior scientist at the Almac Group) pointed out that regulatory agencies allow biomanufacturers to apply prior knowledge to facilitate analytical development and validation. He described how his company leveraged prior knowledge to develop and validate a capillary isoelectric focusing (cIEF) method for analysis of monoclonal antibody (MAb) products.

Rudrarapu’s Presentation
Several guidelines from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) encourage use of established scientific principles, peer-reviewed literature, and documented manufacturing experiences to support analytical development. Thus, when Almac sought to install new software on its PA 800 Plus capillary electrophoresis (CE) systems (Sciex), Rudrarapu’s team leveraged such information to help validate the new program.

Specifically, Almac was transitioning from 32 Karat software (Beckman-Coulter) to the Empower program (Waters Corporation), which would enable integration with Citrix cloud-based computing, centralize data, support batch processing, and facilitate data reporting. Rudrarapu and his colleagues consulted literature from the US National Institute for Standards and Technology (NIST) about its NISTmAb humanized IgG1κ reference material (8671). The document lists critical process parameters, instrument settings, and findings for multiple CE methods. The team determined that cIEF was especially advantageous to study because it enabled analysis of MAb identity and charge variants.

Almac scientists considered differences between NIST’s and Sciex’s cIEF protocols. In the NIST procedure, the capillary was rinsed with 4.3 M urea prior to MAb injection, whereas at Almac, the capillary was rinsed with sample-loading solution (SLS) before injection. Otherwise, Almac prepared samples according to directions in the NISTmAb information sheet. Almac also needed to account for NIST’s use of 32 Karat software.

Initial method feasibility studies generated electropherograms that were inconsistent with those from NIST. Rudrarapu’s team checked for instrumentation problems, including voltage leakage. Opening levers and electrodes were cleaned to prevent leakage, but subsequent runs still generated problematic data. Capillaries were inspected for internal damage. Finding none, analysts used capillaries from a different lot, but with no impact on results. Reagent expiry and contamination were considered, but fresh materials generated similar results.

By collaborating with Sciex, Almac determined that the skewed data stemmed from a polyvinylidene fluoride (PVDF) membrane used to filter the urea-gel buffer. The membrane had leached particles into the capillary, generating electro-osmotic flow (EOF) that caused shifts in the NISTmAb profiles. Almac and Sciex discovered that the problem could be prevented by applying regenerated cellulose filters or by eliminating the filtration step. In the latter case, operators would need to ensure that crystals and air bubbles had not entered the capillary.

Implementing such strategies generated acceptable cIEF profiles, but some inconsistencies remained between Almac’s and NIST’s results. Differences in data rates and peak-integration settings accounted for the discrepancies. Almac resolved those problems by matching the methods’ data rates and turning off the Empower software’s autointegration feature. After those adjustments, Almac assay results matched those from the NIST study.

Other difficulties arose during Almac’s transition from 32 Karat to Empower software. For instance, Rudrarapu’s team needed to create workarounds to enable incremental sample injection. However, leveraging prior knowledge about NISTmAb reference material enabled Almac not only to validate Empower program analyses, but also to expedite subsequent activities related to chemistry, manufacturing, and controls (CMC) portions of regulatory filings.

Questions and Answers
What difficulties arise during CE method transfers? Problems can stem from differences in instrumentation. Analysts should consider instrument performance and environmental factors such as laboratory temperature and humidity. Concerns also can arise during sample preparation. Standard operating procedures (SOPs) must be written clearly, and analysts at both sending and receiving laboratories should be trained appropriately.

What strategies should be used when implementing prior knowledge in analytical laboratories? Seek out rigorously researched literature with methods that are repeatable and relevant to a given product.

Find the full webinar online at www.bioprocessintl.com/category/webinars.