Monoclonal antibodies (mAbs) are important biopharmaceuticals for the treatment of many diseases. During manufacturing the proteins tend to form aggregates, which reduce product yields, influence drug performance and safety. Environmental conditions during production in mammalian cell culture influence the formation of high molecular weight (HMW) species. In this report, we show how mAb aggregates can be detected directly in the cell culture supernatant using size exclusion chromatography (SEC) in a high pressure liquid chromatography (HPLC) system. We have investigated the impact of batch cultivation in different culture vessels, the addition of Valproic acid (VPA) as small molecule enhancer of protein production and the influence of the cell culture environment itself on the formation of mAb aggregates in Chinese hamster ovary (CHO) cell culture. Our results prove that aggregate formation can occur already during upstream processing (USP) due to intracellular and extracellular mechanisms and is not only a problem in downstream processing (DSP).