Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control.

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

A View on the Importance of "Multi-Attribute Method" for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy.

Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5):881-90, 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.

Cost-effectiveness analysis of predischarge monitoring for apnea of prematurity.

OBJECTIVE: It is standard practice to defer discharge of premature infants until they have achieved a set number of days without experiencing apnea. The duration of this period, however, is highly variable across institutions, and there is scant literature on its effectiveness or value-for-money. Our objective was to establish the economic impact of varying durations of predischarge observation for apnea of prematurity. METHODS: Using computer simulation, we compared the alternatives of hospital monitoring for 1 to 10 days, after apparent cessation of apnea, with no monitoring and with the next longest period of monitoring. The daily probability of apnea requiring stimulation after a given number of apnea-free days was obtained from chart review of 216 infants, beginning on the day they attained both full feeds and temperature stability in an open crib. Baseline rates of survival or impairment, utilities for calculation of quality-adjusted life years (QALYs), outcomes for respiratory arrest at home, and long-run costs for neurodevelopmental impairment were derived from the literature. Hospital expenditures were obtained from itemized billing records for infants on each of the final 10 days of hospitalization and converted to costs using Medicare cost-to-charge ratios. Costs are reported in 2000 US dollars. RESULTS: For infants born at 24 to 26 weeks' gestation, each additional day of monitoring cost from $41000 per QALY saved for the first day to >$130000 per additional QALY gained for the tenth day. Cost-effectiveness was poorer for infants who were born at gestational ages >30 weeks. Results were sensitive to the proportion of charted apneas requiring stimulation that would actually progress, without intervention, to respiratory arrest. CONCLUSIONS: In this model, the cost-effectiveness of predischarge monitoring for apnea of prematurity declined significantly as the duration of monitoring was increased. Consideration should be given to alternative uses for resources in formulating neonatal discharge guidelines.