Hyperosmolality has been commonly investigated due to its effects on the production and quality characteristics of monoclonal antibodies (mAbs) produced in CHO cell fed-batch cultures. However, the application of hyperosmolality at different times and its effect on biopotency have seldom been researched, especially in perfusion culture. In our study, different degrees of hyperosmolality induced by sodium chloride were investigated in anti-IgE rCHO cell fed-batch cultures and anti-CD52 rCHO cell perfusion cultures during the initial and stable phases. The results showed that the initial hyperosmolality group (IHG) in fed-batch and early phase of perfusion cultures exhibited significant suppression of the viable cell density yet an enhancement in specific productivity, whereas the stable hyperosmolality group (SHG) achieved higher mAb production in both fed-batch and perfusion cultures. Additionally, the SHG produced less aggregates and acidic charge variants than IHG in fed-batch culture, which differed from perfusion cultures. However, the contents of non-glycosylation heavy chain (NGHC) and man5 were higher in SHG than in IHG in fed-batch cultures at plus 60 and 120mOsm/kg, which was similar to perfusion cultures. Furthermore, the biopotency in the IHG was higher than in the SHG at plus 60 and 120mOsm/kg in fed-batch cultures, which is similar to complement-dependent cytotoxicity (CDC) efficacy in perfusion cultures. The biopotency of all group was acceptable, except FI3. Thus, the study shows that hyperosmolality at a certain level could be beneficial for both mAb production, quality and biopotency, which could play an important role in process development for commercial production.