Monoclonal antibodies (mAbs) are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute (CQA) that has ramifications for patient safety and physiological efficacy – one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-f-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from days 4 to 7 in a batch process used for IgG1 production from CHO cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by approximately 30%. The supplements caused significant changes in gene expression by day 4 of the cultures where 2FP addition significantly reduced FUT8 and nucleotide sugar transporter gene expression (by approximately 2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1-6 genes (by 1.5-3-fold). These gene expression data alongside the glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.