Precipitation and Extraction Methods for Protein Purification: A Meta-Analysis of Purification Performance and Cost-Effectiveness.

For protein drug purification, packed-bed chromatography often remains both the predominant method and a bottleneck for cost and scalability. Accordingly, extensive efforts have been made to develop alternatives, such as precipitation and liquid-liquid extraction. Despite decades of development, such methods have been slow to see adoption in commercial processes. To diagnose the key barriers to implementation and guide future work, we have systematically reviewed studies of protein precipitation and liquid-liquid extraction. We classify the products, methods, and results of 168 publications representing 290 unique purification operations and analyze these operations in terms of both process economics and purification performance. Whereas it is generally assumed that precipitation and extraction methods will have lower costs than chromatography, we find that this is only the case under specific process conditions such as at a large manufacturing scale and low initial sample purity. Furthermore, we find that only a small number of the many precipitation and extraction methods reported to date have shown readiness for implementation in protein drug purification processes. Finally, we identify key factors governing both the economic and purification performance of this class of methods: first, that operating costs are almost entirely predictable by the ratio between the mass of phase-forming materials used and the mass of product protein yielded; second, that use of modern optimization techniques such as Design of Experiments is associated with significantly better purification performance and cost-effectiveness. Highlights: Alternative separation purification methods are not always cheaper than chromatographyThe use of a combination of phase separating agents remains largely underexplored/underutilizedLower initial purity and increasing production scale favor phase-separation over chromatographyThe direct material usage rate is an important predictor of alternative separation cost-effectivenessCurrent alternative separation method development has largely ignored optimization of direct material usage rate.