Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling

Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

The Possible Use of Preoperative Natriuretic Peptides for Discriminating Low Versus Moderate-High Surgical Risk Patient

Perioperative risk scores for patients undergoing noncardiac surgery are generally considered inaccurate, poor, or, at best, modest. We propose estimating a patient’s pretest and posttest probability of cardiac morbidity and death using the preoperative scoring system plus the negative likelihood ratio from brain natriuretic peptide (BNP) or N-terminal proB-type natriuretic peptide (NT-proBNP) plasma levels. Our clinical challenge scenario showed a pretest probability of postoperative major cardiac complications with the patient risk factor as 6.6% for the Revised Cardiac Risk Index and between 1% and 5% (intermediate risk) per the recent European Society of Cardiology and the European Society of Anesthesiologist guidelines for surgical risk estimation. In fact, the American College of Cardiology and the American Heart Association guidelines consider the same surgical procedure for elevated risk. The posttest probability takes advantage of a negative likelihood ratio from BNP plasma levels, with patient risk factor reduced to 0.8% and surgical risk to 1.1%. In the same way, the pretest American College of Surgeons National Surgical Quality Improvement Program score decreased from 18.8% to 3.5% for severe complications and from 0.9% to 0.1% for death at ≤90 days. Following noncardiac surgery, postoperative complications and mortality are often cardiac in nature. The negative likelihood ratio of BNP and NT-proBNP plasma levels provides a quick, low-cost tool for recognizing and reclassifying the cardiovascular risk of those undergoing noncardiac surgery, thereby singling out low- versus moderate-high-risk surgical patients.