How to Separate Molecules

Most fermentation broths require 3–5 unit operations: cell removal (centrifugation or MF), product capture (IEX or affinity), concentration (UF), and polishing (crystallization or SEC). The specific sequence depends on whether the product is intracellular or secreted, its MW, charge, and solubility. The untangle.bio app generates optimized routes for any product-impurity combination.

When MW difference is <2×, size-based methods (UF, SEC) are insufficient. Focus on charge difference: if the proteins have different isoelectric points, ion exchange at the right pH (between the two pI values) will bind one and let the other pass. For example, lysozyme (pI 11.35) and ovalbumin (pI 4.5) are easily separated by cation exchange at pH 7 — lysozyme binds tightly while ovalbumin flows through.

Cost-effective sequences maximize yield at each step and use inexpensive operations first. Centrifugation and depth filtration are cheap per liter processed; affinity chromatography is expensive but highly selective. The optimal sequence typically does cheap/bulk operations early (cell removal, precipitation) and expensive/high-resolution steps later on concentrated, clarified material.