Douglas D. Frey

High-performance cation-exchange chromatofocusing of proteins

Chromatofocusing using high-performance cation-exchange column packings, as opposed to the more commonly used anion-exchange column packings, is investigated with regard to the performance achieved and the range of applications possible. Linear or convex gradients in the range from pH 2.6 to 9 were formed using a variety of commercially available column packings that provide a buffering capacity in different pH ranges, and either polyampholytes or simple mixtures having a small number (three or fewer) of buffering species as the elution buffer. The resolutions achieved using cation-exchange or anion-exchange chromatofocusing were in general comparable, although for certain pairs of proteins better resolution could be achieved using one type of packing as compared to the other, evidently due to the way electrostatic charges are distributed on the protein surface. Several chromatofocusing methods were investigated that take advantage of the acid-base properties of commercially available cation-exchange column packings. These include the use of gradients with a composite shape, the use of very low pH ranges, and the use of elution buffers containing a single buffering species. The advantages of chromatofocusing over ion-exchange chromatography using a salt gradient at constant pH were illustrated by employing the former method and a cation-exchange column packing to separate beta-lactoglobulins A and B, which is a separation reported to be impossible using the latter method and a cation-exchange column packing. Trends in the apparent isoelectric points determined using cation- and anion-exchange chromatofocusing were interpreted using applicable theories. Results of this study indicate that cation-exchange chromatofocusing is a useful technique which is complementary to anion-exchange chromatofocusing and isoelectric focusing for separating proteins at both the analytical and preparative scales.

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.

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.

Characterization and purification of bacteriophages using chromatofocusing

The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: phiX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials.

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.

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO-cell derived biotherapeutics†

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380

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.

Dispersion in stacked-membrane chromatography

Abstract A study was conducted of the separation efficiency of a chromatography column consisting of layers of porous polyvinyl chloride membranes incorporating submicron silica particles. It was determined that dispersion inside the membrane was the dominant bandbroadening mechanism under most conditions. Experimental data are discussed and compared with previous work.

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.

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.