At Alphalyse, we’ve curated a comprehensive database comprising Mass Spec (MS) data on 34,865 uniquely quantified Host Cell Proteins (HCPs) from hundreds of HCP projects spanning various drug categories and a spectrum of sample complexities, ranging from early process samples to final purified drug substances. The extensive database provides information about commonly found HCPs in similar drug types and insights into the process clearance of problematic HCPs.

Residual host cell proteins (HCPs) are critical quality attributes in biotherapeutics, impacting patient safety and product efficacy. Mass spectrometry (MS) is gaining prominence for its precision in identifying and quantifying individual HCPs, playing a vital role in risk assessment, process optimization, and quality control of biotherapeutic products. This presentation will provide an update on USP's initiatives to enhance the quality and consistency of MS-based HCP analysis. Key developments include physical reference materials for HCP analysis by MS and the introduction of a new general chapter, Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry. This chapter, currently under review by the USP Expert Panel following public comments, outlines best practices for HCP identification and quantification by LC-MS/MS. The USP's strategy for developing and characterizing physical reference materials, including intact protein and stable-isotope labeled (SIL) peptides to support identification and quantitation of high-risk and abundant HCPs, will be discussed.

HCPs in adeno-associated virus (AAV) products can be effectively enriched by ProteoMiner beads and the detergent Pluronic F-68 can be simultaneously removed without loss of low-abundance HCPs. Up to a 34-fold increase in the enrichment of HCPs can be achieved by using ProteoMiner beads comparing to direct digestion. After applying ProteoMiner beads on AAV products, HCPs at a level as low as 0.1 ng/mL can be detected.

As gene editing technologies evolve, analytical and QC methods need to be created to determine the functional activity of different gene-editing components. This assay was created to quantify the relative activity of prime-editing mRNAs. By introducing an edit into the luciferase gene of a luciferase-expressing cell line, the activity of target mRNAs were determined by their ability to correct the base-pair insertion and restore function to the luciferase gene.

we present an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular recognition-based analyses were achieved in a single step utilizing an isocratically eluted Mobile Affinity Selection Chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation.

Residual Protein A is a process-related impurity that needs monitoring, due to potential safety considerations. A new Protein A resin was introduced into the process which uses a new commercially available ELISA (kit 1). Challenges during qualification using kit 1 led to the use of a well-established Protein A commercial kit 2. This presentation will focus on the challenges observed transitioning between the two commercial Protein A ELISA kits.

Silicone tubing is used in various unit operations during biologics drug product (DP) manufacturing. Hold of protein formulations in semi-permeable silicone tubing over time may impact product quality, particularly protein concentration. We developed a semi-empirical mechanistic diffusion-based model that predicts protein concentration change over hold time for a given formulation type and tubing size. Overall, our study suggests the significance of monitoring water loss from silicone tubing during DP manufacturing.

Polymer excipients—such as polysorbate 20/80 and poloxamer 188, used in formulation of biotherapeutics—share the same building block which is polyethylene oxide. Charge-reduction mass spectrometry coupled with two-dimensional ion density mapping has been used for rapid profiling, fingerprinting, and speciation of polymeric excipients. This approach has proven to be a fast and effective tool for the visualization of polymeric species from the intact structure.

It is crucial to assess antibody polyreactivity early on to minimize potential risks. In this presentation, I will discuss an ensemble model created within AbbVie which can accurately predict outcomes in both the baculovirus particle and bovine serum albumin assays. To train this model, we utilized a vast dataset of sequences, enriched with experimental conditions, obtained through a highly efficient application. The resulting models displayed strong and consistent performance across various antibody types.

The comparability study is to assess the effect of manufacturing changes on product quality. In this presentation, I will overview the current thinking of health authorities on the comparability in complex biotech and gene therapy products, highlight the comparability strategies, and share the industry practices to ensure continuous product quality throughout the product lifecycle.

The application of surfactants, mainly polysorbates, is a common practice to prevent surface- or agitation-induced protein aggregation in liquid formulation. However, polysorbates, despite their common application, bring along disadvantages, including chemical and enzymatic instability. This presentation will provide an overview of the protein-stabilising capability of surfactants against agitation- and interface-induced stresses, and corresponding assays for its evaluation. Furthermore, a focus is set to alternative surfactants suitable to replace polysorbates.

Introducing Sail Biomedicines' platform and discussing various methods for the analysis of circular RNAs. The presentation will identify key challenges in the analytical development of high-quality Endless RNA (eRNA). The discussion will focus on purity evaluation of circular RNAs and a novel AEX-HPLC analytical method used for eRNA.

The presentation will outline critical quality attributes of mRNA lipid nanoparticle (LNP) drug products, with a focus on early clinical phases. It will also address potential impurities and degradation pathways pertinent to mRNA LNP formulations. A selection of key analytical methods essential for quality control and analytical characterization will be showcased. 

• Quality attributes for mRNA LNP products will be outlined
• Impurities/degradation pathways of mRNA LNP products to consider
• A snapshot of some key analytical methods?