Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production. To identify barriers, we analyzed the purification performance and economics of 290 phase separation purifications from 168 publications. First, we found that studies using Design of Experiments for optimization achieved significantly greater mean yield and host cell protein log10 removal values than those optimizing one factor at a time (11.5% and 53% increases, respectively). Second, by modeling each reported purification at scales from 10 to 10,000 kg product/year and comparing its cost-effectiveness versus chromatography, we found that cost-effectiveness depends strongly on scale: the fraction of phase separations predicted to be cost-effective at the 10, 100, and 1000 kg/year scales was 8%, 15%, and 43%, respectively. Total cost per unit product depends inversely on input purity, with phase separation being cheaper than chromatography at the 100 kg/year scale in 100% of cases where input purity was ≤ 1%, compared to about 25% of cases in the dataset as a whole. Finally, we identified a simple factor that strongly predicts phase separation process costs: the mass ratio of reagents versus purified product (the "direct materials usage rate"), which explains up to 58% of variation in cost per unit of purified product among all 290 reports, and up to 98% of variation within particular types of phase separation.
Cost-Benefit Analysis , Liquid-Liquid Extraction/methods , Proteins/isolation & purification , Proteins/chemistry , Phase Separation
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.
