High-throughput screening tools are needed to triage product quality attributes of molecules and clones. We demonstrate high-throughput RapidFire-mass spectrometry analysis (i.e., 96-samples in ~ 20 mins) integrated with cloud-based protein informatics (~ 30 min/96 samples), to report and aggregate product quality attributes. This to our knowledge is the first demonstration of a complete end-to-end and scalable mass analysis solution that is hands-off, automated, and rapid (96 samples < 1 hr.).

To determine viral clearance efficacy of biomanufacturing steps, viruses are “spiked” into in-process solutions, processed and analyzed for reduction.  Due to the infectivity of these viruses, studies are conducted in BSL-2 facilities.  Costs and logistics limit analysis during process development.  Discussed in the presentation are results from several studies that utilized CHO-endogenous RVLP and  non-infectious MVM VLP’s as viral surrogates. The results demonstrate the feasibility and value of adding viral clearance predication to downstream process development and optimization. 

Accurate prediction of protein stability changes resulting from amino acid substitutions is of utmost importance in medicine to better understand which mutations are deleterious, leading to diseases, and which are neutral. Since conducting wet-lab experiments to get a better understanding of protein mutations is costly and time-consuming, computational tools based on machine learning are becoming promising alternatives to tedious and highly costly mutagenic experiments. We will review them and will present a robust machine learning-based methodology to predict the energy changes of proteins upon mutations.

Data from LC-MS analyses of host cell proteins focusing on quantitation aspects and application to recombinant proteins and AAV-based gene therapy will be presented. Additionally, combination with ribosomal footprint profiling (Ribo-seq) to improve the depth and quality of the database used for MS data searching will also be discussed. Using this approach, we identified microprotein-based HCPs present in commercial drug products and investigated their behaviour during cell culture.

The implementation of new MS-compatible standards for robust and accurate quantification of Host Cell Proteins (HCP) will be presented. The potentialities of Data Independent Acquisition approaches for HCP profiling will be highlighted and benchmarked against commonly used targeted and Data Dependent Acquisition methods. Finally, benefits of the addition of an ion mobility separation in the LC-MS/MS workflow will be assessed on various instrumental platforms including High-Field Asymmetric-Waveform Ion-Mobility Spectrometry (FAIMS) and Trapped Ion Mobility Spectrometry (TIMS). Results will illustrate how the methods can support bioprocess understanding and development and ultimately be applied for final drug product purity assessment.

The HTS group collaborated with formulation and analytical departments to build from scratch platform HT formulation screening workflows that covers study design, sample analytical plan, and data visualization. This ongoing workflow development synergizes cutting-edge robotic liquid handling system, HT study setups, HT analytical instruments, DOE JMP design, and data analysis to deliver richer information for faster and robust decision-making with less material requirement.

Low material consumptive and rapid analytics performed on early-stage engineering panels of protein therapeutic candidates can inform on protein stability and developability. Herein we examine sensitive and robust mass spectrometry-based methods using online capillary SEC and HIC – LC/MS to inform on protein attributes such as aggregation and mispairing. We will also discuss a sensitive N-terminal chemical tagging and rpLC-MS/MS-based approach to accurately identify low-level proteolytic clips.

• Overview of drug product development from formulation, fill-finish, storage, to delivery 
•  Formulation and process considerations to improve end-to-end drug product stability 
•  Integrated drug product design to suit clinical and commercial supply chain needs
  

Polysorbates are commonly used excipients in biotherapeutic formulations. These molecules may be degraded by process-related impurities, ultimately resulting in negative impacts on quality, efficacy, safety, and stability of the biopharmaceutical. Here we present work using two mass spectrometry techniques – 1) LCMS proteomics for identification of impurities and 2) RP-HPLC coupled with charge-reduced HRMS for rapid characterization of PS-80 degradation – used to identify sources of and track PS-80 degradation.

This presentation will highlight surfactants, e.g. polysorbate or poloxamer, as an important part of drug formulations responsible for stabilizing the active ingredient against interfacial stresses caused by shaking, stirring, or freezing. The stability of the surfactants within the shelf-life of the drug product is critical to ensure the quality of the final drug product since their degradation could lead to aggregation and even compromise the stability of the active ingredient.

The impurities in therapeutic protein drug products can originate from raw material, bioprocess, product itself, and during patient dosing. This presentation will highlight potential impurities of therapeutic biotech products during upstream process and downstream purification and share the best strategy to mitigate and control such impurities. Some common queries from health authorities will be exemplified as case studies. 

Regulatory guidelines such as ICH Q3D provide Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes of a biologic may also take place. To this aim, an Fc-fusion protein was treated with increasing concentrations of Ni2+, Cu2+, Zn2+ andFe3+ and analyzed under normal storage conditions, after 6 and 44 weeks of incubation at different temperatures (+5°C, +25°C, +40°C). The potential changes in conformation, oxidation, aggregation and fragmentation were monitored.

Polysorbate, a stabilizer within biologics formulations, is known to degrade via active, low-concentration lipases and esterases. The need for a rapid assay probing this specific HCP mechanism is required to improve the use of proper control strategies. A high-throughput, rapid activity assay using a fluorescent substrate allows for quantitation of lipolytic activity. This enabled an improved approach for the measurement of impurity impact and control.

Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role in polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Host cell proteins (HCPs) are a class of process-related impurities during the manufacturing of biologics. HCPs must be removed in biologics manufacturing to ensure final product purity, manufacturing robustness, and safety. HCP analysis is often performed using an enzyme-linked-immunosorbent-assay (ELISA) due to method sensitivity and ease of use. When using an ELISA kit for HCP monitoring best practices involve determining kit sensitivity (quantification and coverage), product dilutional linearity, and robustness.