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J Chem Technol Biotechnol 74 :293–299 (1999)
An Expert System for selection of protein purification processes: experimental validation¹
M Elena Lienqueo,* J Cris tian Salgado and Juan A As enjo
Centre for Biochemical Engineering and Biotechnology , Department of Chemical Engineering , Univers ity of Chile , Beauchef 861 , Santiago , Chile
Abstract : A hybrid Expert System has been implemented for selecting the optimum sequence of operations for puriücation of proteins. Two criteria were implemented to select the optimum sequence of puriücation. One of these uses the selection separation coefficient (SSC criterion) and the other uses the ünal level of purity (Purity criterion). The sequences suggested by the Expert System have been tested experimentally in two cases : with a model protein mixture and with the puriücation of b-1,3glucanase from recombinant Bacillus subtilis culture. In both cases the sequences suggested were tested experimentally and gave very good agreement with the predicted puriücation. With the model protein mixture it was shown that the sequences suggested by the Purity criterion have fewer steps than those suggested by the SSC criterion. ( 1999 Society of Chemical Industry
Keywords : Expert System ; protein puriücation ; b-1,3-glucanase ; bioseparation
1 INTRODUCTION The development of modern biology and recombinant DNA technology has made possible the production of a number of new biotechnological protein products, such as bioproducts for therapeutic uses (human growth hormone, antibodies, blood factors, vaccines and tests for diagnostics amongst others) and enzymes for industrial use (amylases, proteases and lipases). Advantages in production of a bio-product depends not only on innovations in molecular biology but also on the downstream process innovations and optimisation.1 Downstream processing of proteins can be divided into two stages : Recovery : includes the unit operations used for the transformation of the broth into a solution ready to undergo high resolution puriücation sequences. It may consider harvesting, cell disruption if the product is intracellular, separation of solid debris, protein solubilisation and refolding if the product is in inclusion bodies and precipitation of nucleic acids if disruption was needed. Puriücation : takes a multi-protein solution and puriües the protein of interest. It may consider preconditioning, several high resolution puriücation steps and in cases of therapeutical applications, ünal polishing (purity range 99–99.9%). The
¹ Paper delivered at the Separations for Biotechnology IV Conference organized by the Biotechnology and Separation Science & Technology Groups of the Socieity of Chemical Indus try, held at the Univers ity of Reading, UK, on 29–31 March 1999. * Corres pondence to : M Elena Lienqueo, Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering, Univers ity of Chile, Beauchef 861, Santiago, Chile Contract/grant s pons or : Fundac• on Andes and Vicerrectoria Aca-
puriücation process of clinical biotechnological products accounts for 70–80% of the total production costs. The basic heuristic rules for the downstream processing design are2,3. (1) Choose the separation based on the diþerent physicochemical properties, such as, surface charge (titration curve), surface hydrophobicity, molecular weight and pI values and biochemical properties such as biospeciücity with diþerent ligands, stability at diþerent temperatures and pH values. (2) Eliminate those proteins and compounds that are found in greater percentage ürst. (3) Use a high resolution step, as soon as possible. The chromatographic techniques ranked according to their efficiency are : affinity ; ion exchange ; hydrophobic interaction ; gel ültration. (4) Do the most arduous puriücation step at the end of the process (ünal polishing). The selection of an efficient puriücation process is the limiting step in downstream processes. The steps are not usually chosen in a rational manner or using the basic heuristic rules for protein separation. To overcome the solution of these problems, it has been proposed to use artiücial intelligence tools, such as
de mica, Univers idad de Chile ; Contract/grant number : Beca P6/ 080/97 Contract/grant s pons or : Proyecto Fondecyt ; Contract/grant number : 1950620 Contract/grant s pons or : Proyecto Catedra Pres idential en Ciencias . (Received 13 July 1998 ; accepted 24 November 1998 )
( 1999 Society of Chemical Industry. J Chem Technol Biotechnol 0268-2575/99/$17.50
Two criteria for selecting the best sequence of operations for puriücation have been implemented. With this new database the system calculates the purity level and this value is compared with the level of purity required. The variable & corresponds to the peak width (Table 1). It will aþect the selection criteria.22 0. #i \ Concentration of contaminant i Concentration of all contaminants (3) Deviation factor (DF). Efficiency factor (g). Prot–Ex–Puriücation is a hybrid Expert System that combines rules stated by experts and mathematical relationships using physicochemical databases of the proteins to be puriüed. These relationships are shown in Table 1. Expres s ions and parameters us ed for SSC and Purity criteria Efficiency Factor (g) 1.46 Where : Q repres ents abs olute value of s urface charge (coulomb moleculeÉ1).66 Peak width (&) 0. this value represents the relative concentration of each contaminant.47 log mw Dimensionless retention time (Kd). The best process will be that which has the highest SSC value. this variable represents the behavior of the proteins in a separation carried out by gel ültration. Values of & are independent of the protein concentration for the normal conditions used in the protein puriücation process. JC Salgado.00 0.15 0. JA Asenjo Expert Systems (ES). and / repres ents s urface hydrophobicity.6 In Table 1 the results obtained for a number of proteins are shown.86 0. (ii) Purity criterion Considering that the most important value is the ünal purity level and that now we had developed an algorithm to calculate the purity after a puriücation step. the system Retention time (Kd ) 7383 (Q 1025/mw ) 1 ] 15844 (Q 1025/mw ) 5972 (Q 1025/mw) 1 ] 17065 (Q 1025/mw) / 2. The optimal sequence of steps is chosen until the required level of purity is reached. it is necessary to calculate the new concentration of all contaminants after the chromatographic technique selected has been applied and construct a new database of contaminants concentration. This is done as shown in Fig 1 for each protein contaminant present for the actual chromatographic step chosen by the system using the SSC criterion. we implemented this criterion as a possible selection criterion. 294 J Chem Technol Biotechnol 74 :293–299 (1999) .ME Lienqueo.00 1. Finally the system creates a list with the deüned sequence of operations. Chromatographic Techniques Anion exchange Cation exchange Hydrophobic interaction Gel filtration Table 1. the dimensionless retention time in a speciüc chromatographic technique) and the same property of the contaminant : DF \ o Kdproduct [ Kdcontaminant o (2) The amount of contaminant eliminated after a chromatographic step is given in Fig 1 for diþerent situations.6 After determining which chromatographic technique gives the maximum SSC value.6 The present work discusses the implementation of two algorithms for selecting the optimal sequence of puriücation in Prot–Ex–Puriücation and it tested experimentally the puriücation operations predicted by the Expert System. Mathematical relationships for predicting Kd have been derived using the physicochemical properties of proteins. This criterion compares the ünal purity level obtained after a particular chromatographic technique has been applied. The use of Expert Systems for solving biotechnological problems in the selection downstream process has been documented. this variable relates the diþerence between a property of the target protein (for example. Its value is constant for each type of separation and the chromatographic material used and has to be measured experimentally.4 Reactivate Planing P8. The relationship developed for this purpose was : SSC \ DF g#i (1) Concentration factor (Hi). The purity concept is deüned as : Purity \ Concentration of the target protein & Concentration of the proteins present (4) After determining which chromatographic technique gives the highest purity level.5 FPLC assistant (Pharmacia) and Prot– Ex. ie Protein Puriücation Advisor. In this way contaminants which are always present in a high concentration have to be eliminated ürst. mw repres ents molecular weight (Da). (i) SSC criterion (separation selection coefficient) The Expert System will select the best process using the values of the SSC (separation selection coefficient) calculated for each chromatographic technique and each contaminant protein. this parameter gives account of the unequal capability of the diþerents separation processes to separate diþerent proteins.15 0.39–0. ion exchange or hydrophobic interaction chromatography.
1 Characterization of proteins 2. Details about the programming of the instrument and running the methods can be found in ‘Separation Technique File No 100. The triangle on the left is the product protein and the triangle on the right repres ents each contaminant. The b-1. All buþers were degassed with helium for 10 min.3.1. J Chem Technol Biotechnol 74 :293–299 (1999) . (b) C \ C *2. as described in ‘Development Technique File No 200’ (Pharmacia Biotechnology).1.0 (Buþer A).5 Isoelectric point and titration curves Isoelectric points and titration curves were obtained with a Phast System (Pharmacia Biotechnology. 2 1 2 1 2 1 where C is contaminant concentration after a chromatographic s tep and C is contaminant concentration before a chromatographic s tep.1.7 2. Uppsala Sweden).1. 2.4 b. 2. The gels were developed using Coomassie Blue. mw. (c) C \ C *1.3. UK. soybean trypsin inhibitor (SBTI).1 Model protein mixture Anion exchange chromatography : A Q-sepharose Fast Flow column with Bis(tris)propane 20 mmol dm~3 pH 7. 2 1 chooses this as the technique to use at this step. The fermentation broth was centrifuged to remove bacterial cells and debris and then it was concentrated by using cross-ýow ultraültration with a 5000 M cut-oþ ülter (Sartorius).1.02* (&2-2*DF 2)/&2.3glucanase from Bacillus subtilis ToC46 (pPFF1) culture. The components of the r fermentation broth (b-1.1. Sweden) and the peaks were characterized (pI. Mo. 2.Selection of protein puriücation processes Figure 1.02.3-glucanase activity was assayed by measuring reducing sugar equivalents released from laminarin.1. Thaumatin was a gift of 4F Nutrition. The buþer was Trizma base 20 mmol dm~3 pH 7. Uppsala. Finally the system creates a list with the deüned sequence of operations.2. titration curve. A sequence of steps is chosen until the required level of purity is reached.04* (&-DF )2/&2.1 Model protein mixture Four proteins were used : thaumatin (Tau). The s haded area s hows the amount of contaminant left with the protein after each s eparation in threee different s ituations : (a) C \ C *0. USA).3-glucanase and contaminants) were fractionated by ion exchange Qsepharose FF (Pharmacia Biotechnology.2 b. serum from bovine albumin (BSA) and ovalbumin (Ova). It will then compare the purity with that required.3 Protein concentration Protein concentration was assayed by using the Bradford method with BSA as the standard.2 lm pore size membrane. All proteins and chemicals were from Sigma Chemical Co (St Louis.1.1.3-glucanase activity was also measured in each fraction.6 Determination of molecular weight Molecular weights were obtained with Phast System using gradient Phast Gel 8–25 in agreement with the protocol SDS-PAGE described in ‘Separation Technique File No 110’.glucanase from B subtilis ToC46 (pPFF1) culture B subtilis ToC46 (pPFF1) was grown in continuous culture in a BioFlo II bioreactor (New Brunswick).0 buþer was used.7 Determination of hydrophobicity Hydrophobicity was measured by hydrophobic interaction chromatography using a Phenyl-superose gel. IEF and electrophoretic titration curve analysis’ (Pharmacia Biotechnology). Criteria to determine the percentage of contaminant eliminated after a chromatographic s tep.2 Chromatography 2. Each protein was dissolved in buþer A (2 mg cm~3). It is expressed as the concentration (ml dm~3) of ammonium sulfate at the protein elution point. The gels were developed with Coomassie blue as has been described in Development Technique File No 200’ (Pharmacia Biotechnology). 2. using Phast Gel IEF 3–9 (linear pH gradient from 3 to 9). In this paper two examples have been tested experimentally : a model protein mixture and b-1. 2. 295 2 Methods 2. Northallerton. All proteins and buþer solutions were sterilized by ültration through a 0.glucanase activity b-1. hydrophobicity and protein concentration). 2.
76 [0.03 1. 2.0 1.0 plus (NH ) SO 42 4 1.1 Purification of BSA Assessments were done using both criteria (SSC and Purity) implemented in Prot–Ex–Puriücation for puriücation of BSA from a mixture of four proteins (BSA.3 Criteria implementation Both algorithms were implemented using Nexpert ObjectTM. SBTI.5 plus (NH ) SO 1. Sequence s ugges ted by the Expert Sys tem to obtain a purity s uperior to 94% in the purification Purity Purity Criterion Chromatography s teps Anion exchange at pH 7.89 Charge (Coulomb molecule ) 10 É25 pH 4 . (b) Second s tep s ugges ted : hydrophobic interaction chromatography.0.5 42 4 mol dm~3 buþer was used.54 0.0 [1. (c) Third s tep s ugges ted : anion exchange chromatography at pH 7. Hydrophobic interaction chromatography : Phenylsepharose fast ýow column Trizma base 20 mmol dm~3 at pH 6.14 [0.05 [2.54 1. JA Asenjo Table 2.22 1.1.94 pH 5 . (a) Firs t s tep s ugges ted : cation exchange chromatography at pH 6.86 0.0% 63.98 pH 7 . 2.90 pH 6 .0 Hydrophobic interaction Anion exchange at pH 7.76 1.3.16 [1. Its contribution is SSC Criterion Chromatography s teps Table 3. The data on this mixture used in the consultations are given in Table 2.5 buþer was used.0 [1.glucanase from B subtilis ToC46 (pPFF 1) culture Hydrophobic interaction chromatography : Phenylsepharose fast ýow column Trizma base 20 mmol dm~3 at pH 7.5% 296 J Chem Technol Biotechnol 74 :293–299 (1999) . Steps s ugges ted by SSC criterion. The SSC criterion selects a puriücation sequence based on the elimination of the contaminant that gives the highest SSC value. Ovalbumin and thaumatin). RESULTS AND DISCUSSION 3.0 33.36 [2.20 [2. a commercial shell from Neuron Data Figure 2. Anion exchange chromatography : A Q-sepharose Fast Flow column with bis-tris 20 mmol dm~3 at pH 6.ME Lienqueo.13 0.5 mol dm~3 buþer was used.87 pH 8 .7 94.90 0.68 [2.65 [1.91 BSA Ovalbumin SBTI Thaumatin Cation exchange chromatography : S-sepharose Fast Flow column MES 50 mmol dm~3 at pH 6.0 buþer was used.1% 49.2 b.2.17 1.40 1. JC Salgado.5% 97. The results obtained for a target of 94% purity are shown in Table 3.0. Phys icochemical properties of protein mixture Proteins Initial Concentration (mg cm É3) 2 2 2 2 Molecular weight (Da ) 67 000 43 800 24 500 22 200 Hydrophobicity [(NH ) SO ] 42 4 0.0 Hydrophobic interaction Purity Cation exchange at pH 6.0 [2.2.0 [0.
73 [1. Steps s ugges ted by Purity criterion. The chromatograms for puriücation sequence suggested by the Purity criterion are shown in Figs 3a and 3b. In this example 3.04 297 J Chem Technol Biotechnol 74 :293–299 (1999) . described through the product of concentration factor (h) the efficiency factor (g) and the deviation factor (DF ).50 0. In those cases where all contaminants have the same concentration (equal concentration factor) and the efficiency is constant then DF is the variable that has the main contribution.22 [1.47 [1.2 Experimental investigation The sequence suggested by the Purity and SSC criteria were experimentally investigated.0 [2.0) to eliminate SBTI and to reach a ünal purity level of 97%. The chromatograms for puriücation sequence suggested by the SSC criterion are shown in Fig 2a.06 [2.51 [0. the second step suggested for both criteria was hydrophobic interaction chromatography which eliminates Ovalbumin.87 [2.47 [0.0 is used as suggested by the Purity criterion it is possible to eliminate thaumatin and a part of SBTI.09 41 000 32 900 35 500 62 500 40 600 69 600 40 600 69 600 1.55 [0.04 [3.0 [2. This situation takes place because the Purity criterion chooses the optimum chromatographic step considering all the contaminants present. However.0 1.32 [1.06 [1.62 pH 6 .25 0.00 0.00 0.73 [2. This has been conürmed experimentally.55 1.22 [0.74 2. The SSC criterion is based on the elimination of the contaminant that presents the most diþerent properties from those of the target protein (Fig 2a).06 [0.33 pH 8 .0 [1.42 0. The SSC criterion considers only the contaminant that gives the highest SSC value.46 1. Finally.17 [0.0. if anion exchange chromatography at pH 7.60 Molecular weight (Da ) 31 000 Hydrophobicity (NH ) SO 42 4 0. Fig 2c shows the separation of part of SBTI from the mixture.02 pH 7 . the purity achieved after the puriücation is only 33%.65 [3.25 0.00 Charge (Coulomb molecule É1) 10É25 pH 4 .0 is useful to eliminate the thaumatin protein. Nevertheless.82 [1.00 0.82 [1. For example. obtaining a purity of 64%. (b) Second s tep s ugges ted : hydrophobic interaction chromatography. Phys icochemical properties and concentration for the main proteins in Bacillus s ubtilis ToC46 (pFF1) Proteins Initial Concentration (mg cm É3) 0. Figure 3.74 0.46 [0.3-glucanas e Contaminants Low hydrophobic Contaminant 1 Contaminant 2 Medium hydrophobic Contaminant 3 High hydrophobic Contaminant 4 Contaminant 5 Contaminant 6 Contaminant 7 Contaminant 8 2.79 [0. Figure 3b shows the separation of ovalbumin from the mixture.00 0.00 0. Figure 2b shows the separation of ovalbumin from the mixture.04 [1.50 1. Finally the SSC criterion considers an additional step (anion exchange chromatography at pH 7. (a) Firs t s tep s ugges ted : anion exchange chromatography at pH 7. 2b and 2c. Table 4.Selection of protein puriücation processes the cation exchange chromatography at pH 6. Figure 3a shows the separation of thaumatin and a part of SBTI from the mixture. For this reason the chromatographic step chosen using the Purity criterion is the optimum for that stage and gives a higher purity than that obtained when the SSC criterion is used.00 [0.55 [1. as shown in Figs 2 and 3.0 [0.20 0.26 0.73 [0.52 b-1. Fig 2a shows the separation of thaumatin from the mixture.51 [1.09 0.22 [0.82 [3.70 [0.25 0.46 pH 5 .
7% 70.5 Knowledge discovery The development of the present expert system has allowed us to ünd and compare two diþerent criteria for the selection of the optimal puriücation stages for proteins. Figures 4a and 4b show that the scheme for puriücation suggested by the Expert System is valid for puriücation of b-1. One way to obtain this result. The chromatograms for the puriücation sequence are shown in Figs 4a and 4b. minimizing the resources used.3-glucanase from B subtilis ToC46 (pFF1) culture. JC Salgado. is to minimize the number of puriücation steps. Figure 4a shows the separation of ‘low hydrophobic proteins’ (contaminants 1 and 2) and part of the medium hydrophobic proteins’ (contaminant 3) from the b-1.3-glucanase Assessments were done using the Purity criterion implemented in Prot–Ex–Puriücation for puriücation of a b-1.4 Experimental investigation The sequence suggested by the Purity criterion was experimentally investigated.3-glucanase. 2b and 2c. it is very important to decide an appropriate initial step. Figure 4. because the ürst step is used to eliminate the main contaminants while the subsequent steps are used to eliminate those contaminants of smaller importance. 3.5 32. However. Figure 2a. 3. Thus. Steps s ugges ted by Purity criterion for the purification of glucanas e. Figure 4b shows the separation of contaminants 4.5 Purity Hydrophobic interaction Anion exchange at pH 6.3-glucanase from B subtilis ToC46 (pFF1) culture. the Purity criterion suggests an optimal puriücation sequence which is more realistic than that suggested by the SSC criterion. Furthermore this implementation has allowed us to ünd and evidently choose the Purity criteria as the optimal one for the most efficient puriücation step. Where there are many contaminants. However the sequences suggested by the Purity criterion have fewer steps than those suggested by the SSC criterion.3-glucanase from B subtilis ToC46 (pFF1) culture. 3. Sequence s ugges ted by the Expert Sys tem for Purity Criterion Purity Experimental Validation Chromatography s teps Hydrophobic interaction Anion exchange at pH 6. The results obtained for 70% purity are shown in Table 5. The data on this system are given in Table 4. ACKNOWLEDGEMENTS Financial assistance from Fundacio n Andes and Vicerrectoria Acade mica.5.3 Purification of b-1. 4 CONCLUSIONS The purity and the SSC criteria suggest valid puriücation sequences that have been experimentally validated. (a) Firs t s tep s ugges ted : hydrophobic interaction chromatography.3-glucanase. In this ürst step. On a large scale it is necessary to obtain the highest possible yield. both criteria are recommended as a ürst guessing point to start the experimental puriücation processes instead of the commonly used trial and error method. Universidad de Chile (Beca J Chem Technol Biotechnol 74 :293–299 (1999) 298 . the main contaminants were eliminated. 5–6 and 7–8 from the b-1. and 3a and 3b clearly show that both schemes for puriücation of BSA suggested by the expert system are perfectly valid for this process. (b) Second s tep s ugges ted : anion exchange chromatography at pH 6. JA Asenjo Purity Criterion Chromatography s teps Table 5. as in the case of the puriücation of b-1.ME Lienqueo.3% 33–38% 65%–70% It can be seen that a percentage of SBTI (50%) is not eliminated in the previous step (Fig 3a).
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