The upstream process in the manufacture of the biopharmaceutical drug substance involves living cells—whether it is living recombinant cells that express a recombinant protein or monoclonal antibody, or living cells that assemble and then propagate a viral vector, or living cells that generate the DNA plasmid that is transcribe into a mRNA non-viral vector, or living patient cells that are to be genetically modified. Tight control and cGMP adherence over the handling of these living cells determine the outcome of the upstream production—the yield of the intended biopharmaceutical, the type and amount of biomolecular structural variants formed, and the type and amount of process-related impurities that must be managed in subsequent purification. Regulatory authorities have high expectations, as well as major requirements for the upstream manufacturing process of the biopharmaceutical drug substance, which will be examined in this chapter. Application, where appropriate, for the minimum CMC regulatory compliance continuum risk-based approach will be discussed.