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Inhalation February 2020 13 aureus (MSRA) in the lung. 2 We conducted an inhala- tion study of one of these novel compounds, LGP-A, in healthy rats to assess the pharmacokinetic profile and we quantified formation of its metabolite. Based on the results from several animals, the concentration of the metabolite generated over the PK time course was higher than the dose of drug initially deposited in the lung, which was an impossible result. e investiga- tion of the methodology provided assurance that the problem was not related to ion suppression or incom- plete extraction of LGP-A from the tissue samples. A remaining possibility was that the reference standard for LGP-A and/or its metabolite were unsuitable. is could arise if the reference standard contained impuri- ties or was compromised in some other way. To address this situation, our synthesis group manufactured new, higher-quality, supplies of reference standards of LGP-A and its metabolite and the resulting reanalysis of the PK samples using the new standard curves led to a complete resolution of the mismatch. Another example of rapid issue resolution occurred in a study of INS1009, a prodrug of treprostinil that is in development for treatment of pulmonary arterial hypertension. 3 Due to its short half-life, the approved inhaled formulation of treprostinil (Tyvaso ® , United erapeutics) is administered four times daily; more- over, side effects are often dose-limiting. erefore, a long-acting prostanoid analog like INS1009, which maintains the positive attributes of treprostinil and is amenable to once-daily or twice-daily dosing, but has fewer treprostinil-related side effects, would be of clinical benefit. In a Phase I study in healthy human subjects, a lipid nanoparticle formulation of INS1009 had a lower plasma treprostinil C max and fewer respiratory-related side effects compared to inhaled treprostinil. 4 We have used an internally developed liquid chroma- tography–mass spectrometry (LC-MS/MS) method over a t wo-year period to support the INS1009 research program and the methodology has been robust. However, in a recent study it was observed that the MS signal for a newly prepared standard curve was about 10 times lower than historical observations, resulting in concentrations of INS1009 appearing ten times higher than projected. We investigated whether the "apparent" loss in signal was due to a reduction in the performance of the mass spectrometer over time by testing the same analyte in solution rather than in the biological matrix. After confirmation that the mass spectrometer was working properly, we evaluated whether the signal loss might be coming from the specific lot of lung tissue used as a matrix for preparing the standards. Ionization enhancement or suppression can occur when com- pounds that co-elute with the drug interfere with the ionization process in the MS detector and can dramat- ically affect the sensitivity of the target analytes. We resolved this issue by re-preparing the standard curve in a more appropriate lot of matrix. samples, to receipt of the final report were often longer than in-house timeframes would have been if we had the internal capability to conduct those activities. For example, if the project team had developed multiple formulations for evaluation in an inhalation pharma- cokinetic (PK) study, they would have to wait for one CRO to conduct the in vivo study and ship the biolog- ical samples to a second CRO, and then wait for the second CRO to quantify the levels of drug in the tissue and blood samples in order to determine whether any of the formulations had the appropriate release profile, or if the release profile of a given formulation would have to be modified before moving into pre-clinical efficacy studies. Also, if the PK results were variable or inconsis- tent, investigation and troubleshooting would need to be initiated, which would further delay the start of the efficacy study. erefore, outsourcing the bioanalysis activity would become a bottleneck and result in a delay in the advancement of our research programs. For these reasons, Insmed decided to develop internal capability in biological sample analysis. Ever since the laboratory was established, sample analysis time has been greatly reduced. When the project team questions the veracity of data, there is immediate and direct contact between the analytical and research team members, allowing for an expedited investiga- tion effort, leading ultimately to resolution. Currently, all biological sample analysis for non-GMP (good manufacturing process) studies are done by our own bioanalytical laboratory. (Figure 1). In-house benefits Having internal bioanalytical capability has also resulted in more rapid resolution for issues that were perceived to be associated with the analytical method- ology. For example, Insmed is developing novel lipo- glycopeptides (LGPs) that may have superior antibiotic properties against planktonic, intracellular or biofilm infections of methicillin-resistant Staphylococcus Figure 1 Evaluation of bioanalytical samples at Insmed, using a Sciex LC-MS/MS (Concord, Ontario, Canada)