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Table 1 Material cost comparison for five different protein purification methods based on 1 kg product protein yield†

From: Inteins and affinity resin substitutes for protein purification and scale up

 

pMAL fusion w/tag removal

His-tag w/o tag removal

IMPACT-CN

PHB System

ELP System

Supplier

NEB

Novagen

NEB

WOOD LAB

WOOD LAB

Typical yield

40 mg/L

74 mg/L

4.5 mg/L

36 mg/L

85 mg/L

Resin capacity

3 mg/ml

8 mg/ml

2 mg/ml

-

-

Growth medium

$ 44,198.38

$ 23,101.67

$ 379,894.17

$ 126,257.54

$ 58,699.27

Induction cost

$ 35,373.35

$ 19,101.61

$ 314,115.30

$ 39,304.03

-

Buffer cost

$ 51,900.11

$ 5,921.45

$ 103,596.75

$ 27,212.63

$ 15,810.47

Resin cost

$ 624,375.00

$ 790,000.00

$ 192,000.00

-

-

Denaturation cost

$ 428,835.99

-

-

-

-

Protease cost

$ 8,480,000.00

-

-

-

-

Total cost per kg of product

$ 9,664,682.82

$ 838,124.73

$ 989,606.23

$ 153,470.17

$ 74,509.75

  1. † Cost calculations are based on a simple scaling-up of published protocols for laboratory-scale processes. Bulk prices of chemicals and growth media components were obtained from the best available rates per unit mass from Fisher Scientific. Items specific to individual methods were priced by the supplier (i.e. the protease cost was obtained from NEB, His-tag resin cost was obtained from Novagen, etc). Required quantities of all components (i.e. growth media, buffers, resin) were calculated based on the typical yields of each process for a final product yield of 1 kg. In addition, the following assumptions are included for each method: pMAL method – Yield estimate and material usage requirements were based on the supplier, NEB, recommendations in the pMAL Protein Fusion and Purification System manual [21]. In place of the additional DEAE sepharose ion exchange chromatography step for separating the protease and maltose tag, it was assumed that the same amylose resin would be used twice with regeneration. Therefore, one round of purification uses the amylose resin twice. Therefore with an assumed regeneration of four times, the same bed can be used for two separate purifications. Amylose resin binding capacity is 3 mg of fusion protein per ml of resin. This translates to 1 to 2 mg of target protein per ml of resin depending on the molecular weight of the target protein. Even so, it was assumed that 3 mg of maltose tag binds to each ml of resin, hence, underestimating the amylose resin cost. The recommended amount of protease (Factor Xa) is 1% (w/w) of the fusion protein, hence, if an average product protein 1/3 the size of the maltose binding domain tag is used, 4000 g of fusion is needed to produce 1000 g of target protein assuming perfect recovery. Despite this recommendation, the quantity of protease used was based on 1000 kg of fusion protein as opposed to target protein, hence, underestimating the protease cost. His-tag method – Typical yield was based on the published yield using Novagen Standard HisBind for purification of HisTag GST expressed in E. coli [22]. Material requirements were based on supplier (Novagen) recommendations for the HisBind kit [23]. In these calculations the protease step is not factored into the cost and the target protein retains the HisTag after purification. The regeneration is not taken into account because Novagen recommends using a different resin for each different protein. However, a routine regeneration single-step procedure or complete resin regeneration (16 steps) are available even though not considered here. IMPACT-CN method – The yield estimate was based on the average yield published by the supplier, NEB, in the IMPACT-CN manual [24]. Purified protein examples included in this estimate were [24]: Maltose-binding protein, McrB, T4 DNA ligase, Bst DNA polymerase large fragment, BamH I, Bgl II, CDK2, CamK II, T4 Gene 32 product, FseI GFP, CamK II, Invertase, and T4 Endo VII. It was assumed that the chitin beads can be regenerated 5 times as recommended by the manual. PHB method – Excess DTT in buffers for this method is only necessary for specific target proteins and has not been factored into the cost. In addition, cheaper phosphate substitute buffer is used in place of Bis-Tris in the calculation. IPTG induction does not significantly change the yield and has not been used in the calculation. Cost of purification for this method prior to these three modifications (DTT and IPTG elimination as well as Phosphate for Bis-Tris substitution) is $391,265.84 as opposed to the listed $153,470.17. ELP method – This method does not involve sonication and cell lysis can be achieved with 0.2 mg/ml lysozyme content in the lysis buffer (unpublished data) as opposed to the 1 mg/ml published [4]. Furthermore, glycerol was not included in the TB media as noted in the publication. In addition cheaper phosphate buffer is substituted for the published Bis-Tris buffer in this calculation. Cost of purification for this method without this modification (Phosphate for Bis-Tris substitution) is $122,754.12 as opposed to the listed $74,509.75.
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Microbial Cell Factories

ISSN: 1475-2859

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