Surface and pore diffusion in macroporous and gel-filled gigaporous stationary phases for protein chromatography.

A recently introduced method [Biotechnol. Prog. 13 (1997) 429] for determining intraparticle mass transfer parameters in high speed liquid chromatography is considered in the present study for the case where the eluite adsorbs onto the stationary phase. The validity of the method was verified theoretically using simulated elution profiles and then applied to experimental data obtained using columns packed with either a macroporous or a gel-filled gigaporous stationary phase. For this purpose, experimental measurements were made using alpha-lactalbumin and bovine serum albumin as eluites at several retention factors. Apparent intraparticle diffusivities measured for the gel-filled gigaporous stationary phase were seen to increase with the retention factor, which indicates that for this material surface diffusion is a significant mechanism of mass transfer under retained conditions. Data obtained on the macroporous stationary phase revealed that the intraparticle diffusivity was independent of the retention factor, which suggests that pore diffusion remains the principal mass transfer mechanism even under conditions where proteins are adsorbed on the column packing.

Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts.

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. I. Effect of buffer composition on the gradient shape.

Numerical calculations together with simplified analytical relations based on local equilibrium theory are used to determine the factors which govern the shape of the gradient formed during chromatofocusing when simple mixtures of buffering species are employed to produce linear or concave pH gradients. The numerical and analytical development is also used to determine the relation between the gradient shape and the buffering capacities of the adsorbed and liquid phases. Experiments which verify the theoretical methods are described where internally generated, retained pH gradients of various shapes are formed using high-performance chromatography columns. The resulting experimental and theoretical basis can be employed as means for the selection of the buffer composition for use in chromatofocusing.

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. II. Separation of proteins.

The separation of proteins using high-performance chromatofocusing with linear or concave pH gradients formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically, protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed, and good resolution was achieved of protein variants differing by a single amino acid residue in separation times of 30 min or less. An especially useful elution buffer is investigated that contains only four buffering species and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when used together with a particular high-performance column packing made specifically for chromatofocusing. This elution buffer and column packing combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants. Additional applications are described in which a polyethyleneimine derivatized silica column packing and a pH gradient that is concave in shape are used for the separation of proteins in an E. coli cell lysate.

Displacement chromatography of proteins using a self-sharpening pH front formed by adsorbed buffering species as the displacer.

The preparative-scale separation of two proteins into adjoined, pure bands was accomplished using a novel, hybrid chromatography method which employs chromatofocusing using a self-sharpening pH front and displacement development. The method eliminates the use of a traditional displacer for accomplishing displacement chromatography, and was used to separate the A and B forms of beta-lactoglobulin using a strong-base anion-exchange column packing and a buffer system composed of acetic acid and either 3-(N-morpholino)propane-sulfonic acid (MOPS) or 2-(N-morpholino)ethanesulfonic acid (MES). Sample loads up to 150 mg of protein were applied to a 75 x 7.5 mm column to produce a displacement train composed of highly pure protein bands with greater than 90% recovery of protein. A discussion is given of the chromatographic behavior of proteins under concentration overloaded conditions for the case where a self-sharpening pH front formed using adsorbed buffering species is used to desorb proteins from an anion-exchange column packing.

Quasi-linear pH gradients for chromatofocusing using simple buffer mixtures: local equilibrium theory and experimental verification.

Chromatofocusing utilizes internally generate, retained pH gradients to focus proteins into narrow chromatographic bands. One of the characteristics of current chromatofocusing methods is that they use expensive polyampholyte buffers containing large numbers of ill-defined components in order to generate linear or quasi-linear pH gradients. In addition to being costly to use, polyampholyte buffers also tend to associate with proteins and often yield irreproducible gradient shapes. In order to avoid the various difficulties associated with the use of polyampholyte buffers, this study investigates the use of mixtures of simple buffering species to generate quasi-linear pH gradients on a weak-base ion-exchange column packing. The ability of these gradients to separate protein mixtures was also investigated. To optimize the conditions used, a computer simulation method using a local equilibrium model developed that predicts the shape of the pH gradient. Several experiments were performed that demonstrate the usefulness of the method and the accuracy of the model.

Intraparticle mass transfer in high-speed chromatography of proteins.

The effect of intraparticle mass-transfer resistances on the peak shape at high flow velocities in the range currently used in high-speed protein chromatography was investigated both theoretically and experimentally. The asymmetry of the protein bands under these conditions was quantified by the difference between the first moment and the retention volume of the peak apex, this being much easier to determine than the peak skewness. A general method is introduced for the evaluation of the mass-transfer characteristics of a given chromatographic sorbent from the variation in peak asymmetry with reduced velocity. The method is shown to be most useful when the number of theoretical plates is between 3 and 300, which is the regime where peak asymmetry is prevalent. Measurements by isocratic elution under nonretained conditions were made on three chromatographic sorbents, each representing a general class of stationary phase configuration, i.e., gigaporous, mesoporous, and gel-filled gigaporous particles. Mass-transfer parameters were evaluated using the new method based upon the variation of the peak asymmetry with the fluid velocity. For the purpose of comparison, column mass transfer parameters were also evaluated from the variation in the reduced plate height with reduced velocity, a method most useful when the peak asymmetry is small and remains constant in the velocity range investigated. It is shown that the two methods are complementary and yield, within experimental error, the same intraparticle diffusion parameters. It was demonstrated using these methods that the diffusional behavior and the first moments of unretained eluites for the gel-filled gigaporous column packing correspond to a sorbent particle where eluites diffuse through liquid-filled pores containing a uniform distribution of solid cylinders, with the cylinders representing the polymer chains in the gel material. Similarly, the methods were used to verify that, at high flow rates, intraparticle convection can contribute substantially to the rate of intraparticle mass transfer in gigaporous column packings.

Experimental and numerical studies of the chromatofocusing of dilute proteins using retained pH gradients formed on a strong-base anion-exchange column.

The separation of dilute protein mixtures was achieved using simple monovalent buffering species to form retained, internally produced pH gradients on a strong-basic anion-exchange column. Highly focused proteins bands localized on stepwise pH transitions were produced experimentally under trace and volume overloaded feed conditions. Numerical simulations were performed that accurately predict the pH profile and protein band shapes in the column effluent. Experimental results were combined with numerical investigations to explore strategies for designing efficient preparative-scale chromatofocusing systems using simple, inexpensive buffers and adsorbents.

Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks.

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc.

A body powered prehensor with variable mechanical advantage.

The purpose of this research was to improve body powered, voluntary closing (VC) prosthetic prehension. A prototype prehensor with variable mechanical advantage was fabricated and tested. The device operates at low mechanical advantage during sizing of an object to reduce cable excursion requirements. It shifts to high mechanical advantage during gripping to allow high prehensile forces to be generated with reduced cable tension. The prototype provides a mechanical advantage of 2.4, nearly five times that of conventional VC devices. The prototype also acts as a holding assist; after grip forces are applied, they can be maintained with a cable tension of only 3 lb (13.34N). Field testing indicated that the device performs well in many tasks. The mechanism allows greater range of motion while an object is grasped than standard voluntary closing prehensors. However, the device performed poorly in grasping very compliant objects. To address this problem, a switch has been incorporated into the prototype to allow it to be used in a free-wheel mode.

Effect of intraparticle convection on the chromatography of biomacromolecules.

The effect of intraparticle convection in chromatographic columns packed with gigaporous particles (i.e., where dpore/dparticle > 10(-2)) on the band spreading of unretained biomacromolecules is investigated both experimentally and theoretically. A model is developed for the analysis of mass transfer in spherical particles of bidisperse pore structure when both convection and diffusion take place in the larger pores but only diffusion occurs in the smaller pores. The predictions of the model were experimentally verified. It is demonstrated that gigaporous particles have advantages over conventional porous particles (i.e., where dpore/dparticle < 10(-3)) for applications that do not require high resolving power, to bring about fast separation. This is because columns packed with gigaporous particles can be operated at high flow velocities without significant loss of efficiency due to the enhancement of mass transfer by intraparticle convection. The results of the model are used to examine the effectiveness of gigaporous column packings for rapid analytical chromatography and for the concentration and recovery of a dilute solute in a saturation-regeneration cycle utilizing frontal chromatography.

Feedback regulation in preparative elution chromatography.

Input-output models utilizing discrete variables are developed for elution chromatography and used together with control theory to investigate behavior patterns between output variables (e.g., yield, purity, and production rate) and input variables (e.g., cut point locations and feed slug size). Variable interactions and controllability characteristics for isocratic and stepwise gradient elution in linear and nonlinear chromatography are investigated by using the relative gain array, singular value decomposition, and direct simulation. Inverse-based regulatory systems are evaluated for updating process inputs by using information on process outputs such that product quality and system performance parameters are efficiently maintained for the case where the column is overloaded and the solute peaks overlap with each other. Strategies for feedback optimizing control are also discussed.

Evaluation of a regional resource center for multiply handicapped retarded children.

A modified before--after, control-group evaluation design was employed to assess the impact of a demonstration program for multiply handicapped retarded children. Child development, parent and therapist attitudes, and knowledge and use of services were evaluated. Findings revealed no significant positive impact of the program on development, attitudes, and knowledge; the treatment group used more services. A significant change to a more negative attitude on the part of the treatment-group parents with regard to some aspects of normalization was found. Major impact of the program was in the area of coordination of services.