Figure 19.HiTrap® HIC Selection Kit and RESOURCE™ HIC Test Kit.
Time and sample can be saved in the early stages of development by using small (1 ml), prepacked columns such as those in the HiTrap® HIC Selection Kit and RESOURCE™ HIC Test Kit. Media can be quickly and efficiently screened for the required selectivity. This approach is helpful since, even if the properties of the target protein(s) are known, the final selectivity, binding capacity and recovery depends largely upon the interaction of the medium with the specific protein of interest.
HiTrap® columns are prepacked with hydrophobic interaction media based on Sepharose® High Performance and Sepharose® Fast Flow while RESOURCE™ HIC columns contain media based on SOURCE™. All columns can be used for small-scale purification and are supplied with detailed protocols for use. The media in these test kits are available for large-scale production so that optimized methods can be easily transferred to the required scale of operation.
Users of ÄKTAdesign™ chromatography systems can select suitable method templates and program the system to automatically perform separations using a range of columns and a range of buffer conditions.
Figure 20. shows an example of media screening on different HIC media prepacked in HiTrap® 1 ml columns.
Buffer pH was kept at pH 5.0 to minimize the need for sample conditioning after capture. Phenyl Sepharose® 6 Fast Flow (high sub) was selected since the medium showed excellent selectivity for the target protein. The protein was eluted within the gradient and separated from the bulk contaminants. Conditions were then optimized so that a step elution could be used to maximize throughput and concentration of the target protein before scaling up. Figure 47 on page 74 shows the optimized elution scheme and subsequent scale-up.
HiTrap® columns can be used with a syringe or peristaltic pump for manual media screening, method development and method optimization. However, using a syringe limits the degree to which a HIC separation can be developed since the separation mechanism is not a simple “on/off” process and requires some degree of gradient elution to achieve a satisfactory separation.
The methods here are optimized for use with 1 mL HiTrap® columns and should be adjusted if other column volumes are used. Note that flow rates may need to be reduced due to the viscosity of sample or buffers.
The type of ligand and the nature of the target protein are highly significant parameters in determining the selectivity of a HIC medium. Consequently, the most suitable ligand must be determined empirically through screening experiments (see Screening for selectivity, page 28), preferably using the target protein. Proteins that could be assumed to have very similar properties can interact quite differently under identical experimental conditions in a HIC separation, as demonstrated by the behavior of three monoclonal antibodies in Figure 21.
Figure 21.Three monoclonal antibodies interact differently under identical running conditions using a phenyl ligand. A suitable ligand must be determined empirically.