The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery.

Flocculants have been employed for many years as aides in the clarification of wastewater, chemicals and food. Flocculants aggregate and agglutinate fine particles resulting in their settling from the liquid phase and a reduction in solution turbidity. These materials have not been widely used in the clarification of mammalian cell culture harvest. In this paper we examined chitosan as a flocculent of cells and cell particulates in NS0 culture harvest and the subsequent further clarification of this material by continuous flow centrifugation followed by depth and absolute filtration. Chitosan is an ideal flocculant for biotechnology applications as it is produced from non-mammalian sources (typically arthropod shells) and is also available in a highly purified form that is low in heavy metals, volatile organics and microbial materials. Chitosan is a polymer of deacetylated chitin. The deacetylation imparts limited solubility on insoluble chitin and the amino groups on the polymer result in a polycationic material at acidic and neutral pH that can interact with polyanions, such as DNA and cell culture debris (typically negatively charged). Likely the interaction of chitosan with cell culture particulate forms a germinal center for further interaction and agglomeration of particulates thereby reducing the solubility of these materials resulting in their settling out into the solid phase. Chitosan improved the clarification throughput six to seven folds without a deleterious effect on monoclonal antibody recovery or purity. The procedure for utilizing chitosan is facile, easily implemented, and highly effective in improving material clarity and increasing material throughput.

New concepts in the chromatography of peptides and proteins.

Although chromatography using a variety of novel bed configurations (e.g. fluidized beds, expanded beds, simulated moving beds, annular rotating beds, etc.) has been of recent interest, the majority of practical applications of analytical and preparative chromatography employ a stationary adsorbent bed into which a feed slug is charged periodically, similar to the technique first described by Mikhail Tswett over 100 years ago. However, new concepts in both the practice and theory of fixed-bed chromatography are continuing to expand the available range of applications for separating peptides and proteins.

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.

Monoclonal antibody heterogeneity analysis and deamidation monitoring with high-performance cation-exchange chromatofocusing using simple, two component buffer systems.

The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions.

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion-exchange adsorbents.

Although it is commonly believed that a column packing used for chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.

Chromatofocusing using micropellicular column packings with computer-aided design of the elution buffer composition.

Micropellicular, anion-exchange column packings are used in chromatofocusing to demonstrate the resolution and speed achieved when proteins are separated under these conditions. Linear or concave pH gradients are produced with simple mixtures containing four or fewer individual buffering species instead of the more commonly used polyampholyte buffers. Computer-aided design methods are demonstrated for selecting the composition of the elution buffer to produce a pH gradient of a desired shape. The method is applied to high-resolution, analytical- and preparative-scale separations involving horse myoglobin, human hemoglobin variants, and bovine carbonic anhydrase. A useful selection of buffering species is described capable of producing pH gradients of a variety of shapes in the range between pH 9.5 and 5.5.