Supermacroporous hydrophobic affinity sorbents for penicillin acylase purification

Penicillin acylase (PA, EC 3.5.1.11) is used as a raw material in the production of semi-synthetic penicillins. Although there are many methods for PA purification, affinity chromatography is advantageous as it provides efficient one step purification. In this study, poly(2-hydroxyethyl methacrylate) based cryogel column containing hydrophobic N-methacryloyl-L-tryptophan (MATrp) functional monomer as a ligand was prepared. Interaction of MATrp with amino acids in PA structure is the basis of hydrophobic interaction chromatography in this study. PHEMA and PHEMATrp cryogel columns were characterized by surface area measurements, infrared spectroscopy, swelling tests, elemental analysis and scanning electron microscopy (SEM). Initial PA concentration, pH, effect of temperature, amount of ligand, flow rate, ionic strength and time on PA adsorption on PHEMATrp cryogel were investigated. Optimum pH was determined as 5.0 for PA adsorption and maximum adsorption capacity was obtained as 6.40 mg/g. It was observed that adsorption capacity increased with the increasing of temperature. Also, PA adsorption increased up to 0.25 M salt concentration and decreased in higher salt concentrations. Data obtained in this affinity system suggests that hydrophobic interactions are dominant. In the last stage of the study, PA was purified from Penicillium chrysogenum with 76.3% yield and 332.3 purification factor.     

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)‐based anion‐exchange cryogel

Bovine milk whey contains several bioactive proteins such as α‐lactalbumin, β‐lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2‐hydroxyethyl methacrylate)‐based anion‐exchange cryogel. The monolithic cryogel was prepared by grafting 2‐(dimethylamino) ethyl methacrylate onto the poly(2‐hydroxyethyl methacrylate)‐based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high‐purity IgG from bovine milk whey.     

Integrated Downstream Processing of Lactoperoxidase from Milk Whey Involving Aqueous Two-Phase Extraction and Ultrasound-Assisted Ultrafiltration

The present work involves the adoption of an integrated approach for the purification of lactoperoxidase from milk whey by coupling aqueous two-phase extraction (ATPE) with ultrasound-assisted ultrafiltration. The effect of system parameters of ATPE such as type of phase system, polyethylene glycol (PEG) molecular mass, system pH, tie line length and phase volume ratio was evaluated so as to obtain differential partitioning of contaminant proteins and lactoperoxidase in top and bottom phases, respectively. PEG 6000-potassium phosphate system was found to be suitable for the maximum activity recovery of lactoperoxidase 150.70% leading to 2.31-fold purity. Further, concentration and purification of enzyme was attempted using ultrafiltration. The activity recovery and purification factor achieved after ultrafiltration were 149.85% and 3.53-fold, respectively. To optimise productivity and cost-effectiveness of integrated process, influence of ultrasound for the enhancement of permeate flux during ultrafiltration was also investigated. Intermittent use of ultrasound along with stirring (2 min acoustic and 2 min stirring) resulted in increased permeate flux from 0.94 to 2.18 l/m² h in comparison to the ultrafiltration without ultrasound. The use of ultrasound during ultrafiltration resulted in increase in flux, but there was no significant change in activity recovery and purification factor. The integrated approach involving ATPE and ultrafiltration may prove to be a feasible method for the downstream processing of lactoperoxidase from milk whey.     

Characterization of a novel continuous supermacroporous monolithic cryogel embedded with nanoparticles for protein chromatography

A novel continuous supermacroporous monolithic cryogel embedded with nanometer-size particles was prepared by the radical cryogenic co-polymerization of acrylamide (AAm), N,N'-methylene-bis-acrylamide (MBAAm), allyl glycidyl ether (AGE) and the dispersed surfactant-stabilized Fe3O4 nanoparticles under the freezing-temperature variation condition in a glass column. This special separation matrix has interconnected supermacropores with pore size of 10-50 microm, which permit the free-passage of microbial cells or cell debris in the culture fluids and then is interest in downstream processes. The axial liquid dispersion coefficients of the new continuous supermacroporous monolithic bed at different liquid flow rates were obtained by measuring residence time distributions (RTDs) using tracer pulse-response method. The experimental results showed that the axial liquid dispersion within the bed was weak in a wide water flow rate of 0.5-15 cm/min. The axial dispersion coefficient was found to be increased exponentially with the increase of liquid flow rate. Chromatographic process of bovine serum albumin (BSA) in the cryogel monolithic bed was carried out to reveal the protein breakthrough and elution characteristics. Compared with other reported cryogel beds in literature, the protein adsorption capacity of the present cryogel bed was improved due to the embedded nano-sized solid adsorbents in the gel matrix. Microstructure morphology of the embedded nanoparticles in the cryogel and the gel matrix structure were also analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in this paper.     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

In-situ graft-polymerization preparation of cation-exchange supermacroporous cryogel with sulfo groups in glass columns

Graft polymerization of monomer chains with expected functional groups onto the matrix pore surfaces by initiator is an effective approach for introducing ion-exchange groups to cryogel matrix to get anion- or cation-exchange supermacroporous cryogels. In this work, a novel cation-exchange cryogel with sulfo binding groups was prepared by grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) onto polyacrylamide-based cryogels in glass columns. The grafting polymerization was achieved in an in-situ manner which was performed by pumping the initiator and the reactive solution of graft monomer with sulfo binding groups directly through a cryogel bed pre-produced in a glass column under frozen condition. The axial liquid dispersion characteristics within the monolithic cryogel beds before and after the in-situ polymerization were compared by measuring residence time distributions (RTDs) at various liquid flow rates using tracer pulse-response method. Microstructure morphology of pores within cryogels was analyzed by scanning electron microscopy (SEM). Chromatography of lysozyme was carried out to reveal the protein breakthrough and elution characteristics in the obtained cryogel beds.     

Application of Supermacroporous Monolithic Hydrophobic Cryogel in Capturing of Albumin

Supermacroporous poly{2-hydroxyethyl methacrylate-co-[N,N-bis(2,6-diisopropylphenyl)-perylene-3,4,9,10-tetracarboxylic diimide]} [poly(HEMA-co-DIPPER)] monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with DIPPER as functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50 μm size. Poly(HEMA-co-DIPPER) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA-co-DIPPER) cryogel was 14.7 g H₂O/g dry cryogel. Poly(HEMA-co-DIPPER) cryogel was used in the adsorption/desorption of albumin from aqueous solutions. The nonspecific adsorption of albumin onto plain poly(HEMA) cryogel was very low (3.36 g/g polymer). The maximum amount of albumin adsorption from aqueous solution in acetate buffer was 40.9 mg/g polymer at pH 5.0. It was observed that albumin could be repeatedly adsorbed and desorbed with the poly(HEMA-co-DIPPER) cryogel without significant loss of adsorption capacity.     

Thiacarbocyanine as ligand in dye‐affinity chromatography for protein purification. II. dynamic binding capacity using lysozyme as a model

A constant development of dye‐affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye‐affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30°C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K_d) obtained was 2.61 ± 0.36 × 10^–5 m , proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid and efficient one‐step purification of a serralysin family protease by using a p‐aminobenzamidine‐modified affinity medium

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p‐aminobenzamidine‐modified affinity chromatography. The affinity medium was synthesized by using p‐aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant K _d and theoretical maximum adsorption Q _max of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high‐performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.     

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.     

An improved capillary model for describing the microstructure characteristics, fluid hydrodynamics and breakthrough performance of proteins in cryogel beds

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.     

Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels

The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 microm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)6-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques.     

Monolithic molecularly imprinted cryogel for lysozyme recognition

The application of molecularly imprinted polymers in the selective adsorption of macromolecules such as proteins by monolithic protein‐imprinted columns requires a macroporous structure, which can be provided by cryogelation at low temperature in which the formation of ice crystals gives a porous structure to the molecularly imprinted polymer. In this study, we applied this technique to synthesize lysozyme‐imprinted polyacrylamide cryogels containing 8% w/v of total monomers and 0.3% w/v of lysozyme. The synthesized cryogel was sponge‐like and elastic with very fast swelling and reshaping properties, showing a swelling ratio of 24.5 ± 3 and gel fraction yield of about 72%. It showed an imprinting effect of 1.58 and a separation factor of 1.37 for cytochrome c as the competing protein. Adsorption studies on the cryogel revealed that it follows the Langmuir isotherm, with a maximum theoretical adsorption capacity of 36.3 mg lysozyme per gram of cryogel. Additionally, it was shown that a salt‐free rebinding solution at low flow rate and pH = 7.0 is favorable for lysozyme rebinding. This kind of monolithic column promises a wide range of application in separation of various biomolecules due to its preparation simplicity, good rebinding characteristics, and macroporosity.     

Biosorption of Chromium(III) by Biomass of Seaweed Sargassum sp. in a Fixed-Bed Column

This work aimed at modeling chromium biosorption using the biomass of seaweed Sargassum sp. in a fixed-bed column. The mathematical model used was obtained from the mass balance of the component in the liquid phase and in the biosorbent material. The effects of both axial dispersion in the column and the resistance to mass transfer in the solid were considered for the solution of the partial differential equations of the model, using the Galerkin method on finite elements. To represent the equilibrium data of the batch system the Langmuir isotherm were used. The chromium ion adsorption capacity of the seaweed Sargassum sp., at a temperature of 30°C and pH 3.5, was 2.61 mmol/g. The model performance was evaluated from experimental data obtained at 30°C for flow rates of 2, 6 and 8 mL/min. The parameters of the model, mass transfer and axial dispersion coefficients, were adjusted from these experimental data. The model proved adequate to describe chromium biosorption dynamics in fixed-bed columns.     

Organized cryogel composites with 3D hierarchical porosity as an extraction adsorbent for nucleosides

Macroscopic monoliths are highly desirable in many fields of application. Herein, well organized organic–inorganic cryogel composite with a three‐dimensional hierarchical meso‐ and macroporous structure are presented, which were produced by in situ copolymerization of mesoporous multifunctional silica (size: 1–20 μm; pore: 2–20 nm mostly) and monomers (hydroxyethyl methacrylate and diallyldimethylammonium chloride) in water below the freezing point. This copolymerization method effectively adjusted the macropores of the basic cryogel, and the nanosilica was more homogeneously dispersed in the basic cryogel. The specific surface area of the cryogel composite was increased 17 times versus than that of the basic cryogel. The abundant meso‐ and macroporous pores on the cryogel composite provided sufficient reactive sites favorable for the efficient mass transport of target compounds. When the cryogel composite, as solid phase extraction adsorbent, was coupled with high‐performance liquid chromatography, an analytical tool, the nucleosides were quantified with good selectivity, lower detection limits (0.9–1.3 ng/mL) and satisfactory recoveries of greater than 80% from spiked human serum.     

Humic acid particle embedded super porous gum Arabic cryogel network for versatile use

Super porous gum Arabic (GA) cryogels were synthesized by crosslinking of natural GA with divinyl sulfone at cryogenic conditions, −20°C for potential biomedical applications. Humic acid (HA) nanoparticles were also prepared by using degradable and biocompatible crosslinkers such as trimethylolpropane triglycidyl ether, poly(ethylene glycol) diglycidyl ether, and trisodium trimetaphosphate in a single step and then entrapped within GA cryogel network as GA/HA particle cryogel. Furthermore, GA/HA cryogel was used as a template for Ag, Cu, and Fe nanoparticle preparation, and their antimicrobial properties were tested against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis strains. The minimum inhibition concentration values of Ag and Cu nanoparticle‐loaded GA/HA cryogel composites were determined as 10 mg mL⁻¹. Furthermore, the blood compatibility tests such as hemolysis and blood clotting indexes were determined for GA cryogels and found to be more compatible with 0.08 ± 0.01% hemolysis and 89.4 ± 6.1 blood clotting values, whereas the hemolysis of the Ag, Cu, and Fe nanoparticle‐loaded GA/HA Ag, Cu, and Fe metal nanoparticle cryogel composites decreased in the order of Fe > Cu > Ag nanoparticles.     

Separation of influenza virus‐like particles from baculovirus by polymer‐grafted anion exchanger

The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 10⁹ particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles.     

Buffer Standards for the Physiological pH of the Zwitterionic Compound,DIPSO, from 5 to 55 °C

The values of the second dissociation constant, pK ₂, and related thermodynamic quantities of 3-[N,N-bis (2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO) have already been reported over the temperature range 5 to 55 °C including 37 °C. This paper reports the pH values of four NaCl-free buffer solutions and four buffer composition containing NaCl salt at I=0.16 mol⋅kg⁻¹. Conventional pa _H values are reported for all eight buffer solutions. The operational pH values have been calculated for four buffer solutions recommended as pH standards, at 25 and 37 °C after correcting the liquid junction potentials with the flowing junction cell.     

Production of Recombinant Porcine Interferon alpha Using PHB–Intein-Mediated Protein Purification Strategy

Interferons (IFNs) are involved in the pathogenesis and recovery of viral and other infectious diseases. Recombinant IFNs have been used as anti-infectious agents exhibiting a broad range of antiviral and immunomodulatory properties in both human and domestic animals. In this report, we describe a highly efficient and economical approach to purify porcine IFN alpha (PoIFNα) using polyhydroxybutyrate (PHB) as the affinity carrier and intein for self-cleaving removal of the affinity tag. Additionally, the conditions of protein expression and purification have been optimized. Our results suggested that culture medium containing 1.62% (w/v) of sodium lactate dramatically increases the accumulation of PHB binding protein in Escherichia coli cells. High yields of recombinant PoIFNα (30–35 mg/L, 97% purity by high-performance liquid chromatography) were obtained using intein-mediated self-cleaving conditions using a cleavage-inducing buffer with a pH of 6.5 at 20 °C for 24–36 h. The antiviral activity of the recovered recombinant PoIFNα was up to 1.4 × 10⁶ IU/mg of protein ascertained using recombinant human IFNα1 as a standard. This report also demonstrates that large-scale production of intein-mediated purification of highly pure and active recombinant PoIFNα is feasible for the purposes of experimental studies, veterinary clinic therapeutics, and swine infectious disease control.     

Influence of acid‐base dissociation equilibria during electromembrane extraction

Electromembrane extraction is affected by acid–base equilibria of the extracted substances as well as coupled equilibria associated with the partitioning of neutral substances to the supported liquid membrane. A theoretical model for this was developed and verified experimentally in the current work using pure 2‐nitrophenyl octyl ether as supported liquid membrane. From this model, extraction efficiency as a function of pH can be predicted. Substances with log P < 0–2 are generally extracted with low efficiency. Substances with log P > 2 are generally extracted with high efficiency when acceptor pH < pK_aH ? log P. Twelve basic drug substances (2.07 < log P < 6.57 and 6.03 < pK_aH < 10.47) were extracted under different pH conditions with 2‐nitrophenyl octyl ether as supported liquid membrane and fitted to the model. Seven of the drug substances behaved according to the model, while those with log P close to 2.0 deviated from prediction. The deviation was most probably caused by deprotonation and ion pairing within the supporting liquid membrane. Measured partition coefficients (log P) between 2‐nitrophenyl octyl ether and water, were similar to traditional log P values between n‐octanol and water. Thus, the latter have potential for pK_aH ? log P predictions.