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 removed 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 taken into consideration of residual HCP profile in the product, the dose, dosage form, and administration route etc. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of a Merck investigational stimulator of interferon genes protein (STING) agonist, MK-1454, which is a cyclic dinucleotide synthesized using E. coli cell lysate overexpressing cyclic GMP-AMP synthase (cGAS) as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools, 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 API.