There is a scientific gap in our current understanding of how formulation and raw material variables used to manufacture poly(lactic-co-glycolic acid) (PLGA) microspheres encapsulating peptides lead to differing levels of peptide-polymer interactions and peptide acylation during encapsulation, storage, and release in vitro and in vivo. Batch-to-batch variation and unpredictable acylation levels could lead to differences in pharmacokinetics (PK) and loss of bioequivalence of generic long-acting release (LAR) formulations seeking to mimic LAR products. Such differences in bioequivalence may lead to differences in safety and efficacy. A key factor leading to the loss of parent drug is the poorly defined interaction between the peptide and the polymer, which has been shown to be a precursor to the acylation reaction. For example, salt formation between the cationic and acylation-labile octreotide, found in the Sandostatin LAR (SLAR) microsphere product, with the ionized carboxylic acid end-groups of PLGA has been shown to be necessary for acylation to occur. We hypothesize the key underlying time-dependent factors responsible for differences in peptide-polymer interactions and acylation for microspheres of similar compositions include: (a) microclimate pH in the microspheres affecting ionization of polymer carboxylic acids necessary to bind to a cationic peptide; (b) the extent to which the polymer allows penetration of the peptide into the polymer phase, and (c) the architecture of the polymer (star vs. linear) and end-capping that affect the strength and type of intermolecular forces responsible for peptide- polymer interactions. The following tasks will be developed to test our hypothesis and close the aforementioned scientific gap: 1. Develop techniques for assessing peptide-polymer interactions and peptide acylation impurities with the reference product, SLAR; 2. Reverse engineer the composition of SLAR; 3. Create Q1/Q2 formulations for the SLAR as a function of microencapsulation conditions, and determine product attributes relative to SLAR. 4. Create microsphere formulations with virtually the same composition of SLAR but with different PLGA architecture and molecular weight, and determine product attributes; 5. Compare peptide-polymer interactions, acylation, erosion behavior and release kinetics in all formulations to identify key formulation and polymer architecture controlling differences in peptide acylation and release kinetics in vitro; and 6. Verify in vitro differences observed in Task 1 and 5 by assessing key formulations for PK after intramuscular injection and direct measurements after recovery from a novel in vivo cage model. We will apply a wide range of novel experimental approaches developed by our group over the last 20 years to interrogate the peptide-polymer interactions necessary to initiate octreotide acylation in PLGAs. We will further combine this approach with a large number of rigorous and new in vitro assays to test formulations with relevance to future regulatory guidance to generics and verify their relevance to the not yet predictable in vivo environment.