Process robustness in chromatography relies heavily on the selection of the matrix. A matrix-screening program can be efficiently achieved using automated 'scouting' systems such as the BioCad (Applied BioSystems) or the Akta (GE Healthcare, previously Amersham Biosciences). These machines can be connected to several columns. The machine can then run a variety of methods through each column in turn. Different methods at different pHs can be run, gradients are used to elute the product and fractions are collected. Many permutations can be tested on the
At pH 7.5 the recovery is at its highest and the impurities near their lowest. However, a range on the pH must be allowed for. A slight rise in pH above pH 7.5 could cause a significant drop in recovery. pH 7.5 should, in this example, be the high end of the range. From this data it would be useful to define a set-point for pH of 7.0, with a tolerance of ± 0.5. Thus, a range from pH 6.5 to 7.5 would be allowed within which neither recovery nor impurity levels would vary greatly. However, it would be useful to investigate the effect of pH between pH 7 and pH 8 at intervals of 0.2 to investigate this more thoroughly since pH can be controlled to a tolerance of ± 0.2. It may then be possible to define a set-point for pH of 7.3 with a range of ± 0.2, i.e. from pH 7.1 to pH 7.5, depending on how the recovery performs between pH 7.5 and 8.0. Optimisation for robustness is key to the success of a process in manufacture, but this should be done at the method screening stage so that the other steps in the process are developed coherently and re-work can be avoided.
Figure 18.6 The possible effect of pH on recovery and impurity removal, showing the selection of a setpoint that allows for process control and enables a robust method.
different columns and this is achieved automatically. This highly efficient method of chromatography screening will generate a huge number of fractions for analysis, and this will very quickly generate a great deal of data, to facilitate the comparison of different matrices.
It is important during the screening phase to build in robustness. Do not necessarily select the conditions that give the highest yield or best purification. The problem with the best, or 'optimal', conditions is that they may only be achieved under a narrow range of conditions. These conditions may be relatively easily achieved in the laboratory where high resolution techniques on sophisticated equipment and perfectly packed columns are available, but at a large scale they may be difficult to achieve. It is preferable to rely on conditions that are easily controlled; find methods where broad ranges of pH or conductivity, for example, can be used. Figure 18.6 shows how the optimal conditions for recovery or impurity removal may not necessarily be optimal for robustness, but that the robust process need not be poor in performance.
During method screening, select the conditions where a consistent result can be achieved assuming that the parameter will vary within a specified range. A set point within this range may not give the maximum yield or best purification, but, as long as sufficient yield and purification are achieved, such a set point should be viewed as one optimized for robustness.
It is probably better to rely on a greater number of robust process steps rather than fewer steps that involve control of critical parameters. If the control of a given parameter is easy to achieve within the necessary limits then the process will be robust and readily validated. Manufacturing expertise should be consulted during process development activities to determine what level of control is practicable at large scale.
Most chromatography steps will be developed using a gradient elution on an automated system and then converted to a step elution method more suitable for large-scale implementation. However, it is possible to run gradients at large scale, collect fractions and perform in-process assays before pooling appropriate fractions. This may make a critical separation sufficiently robust for biologicals manufacture.
There is an expectation in science for innovation and in the field of biological medicines this may be particularly true as a result of the innovative nature of the products. However, whilst innovation can be useful in a process where it is needed, it should not be an aim in itself. Tried and tested methods, if they will do the job, should be used; not only are they likely to be quicker and easier to get working, and probably less costly to implement, they will be more readily accepted by regulatory authorities.
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