Figure 4. Relationship between pH and Specie 1 concentration during diafiltration. The two centerpoint runs are for replication
purposes and start at a concentration of 50 mg/mL. The 2x concentration starts at 85 mg/mL and the 0.5x starts at 20 mg/mL.
The initial pH is consistent at 6.5.
This relationship suggests the possibility of using pH, which is easy to measure on-line, as an indirect indicator for completion
of the diafiltration process. This possibility was further examined by performing experiments at different Specie 1 concentrations.
The data are presented in Figure 4. The data support the above-mentioned correlation with both the pH and Specie 1 profiles
reaching the plateau earlier when the Specie 1 concentration is low, and later when the Specie 1 concentration is high. Thus,
for this application, pH could be used for a PAT-based control study to trigger the end of diafiltration. We can stop diafiltration
when pH reaches 8 at 5 diavolumes instead of at 8 diavolumes as presently specified. In Figure 5 we show a normalized fit
for diavolumes as a function of concentration of Specie 1 in the feedstock. This is a version of Equation 1, and the fit is
excellent (R2 = 0.98). If the feed concentration of Specie 1 is known, an operator can use this graph to estimate the number of diavolumes
required to complete the diafiltration process.
Figure 5. Relationship between number of diavolumes required and Specie 1 concentration
Similar to the case study for protein refolding, this PAT-based control scheme also has several problems with respect to implementation
in a manufacturing environment. While the pH-based control scheme is simple to implement, the empirical model presented in
Figure 5 would require well-trained manufacturing operators. Also, pH probes can be quite unreliable. Therefore, if the process
control strategy is pH-based, then it may be necessary to have redundancy (e.g, duplicate probes) built into the control scheme
to ensure accuracy of the data.
Implementation of a PAT-based control scheme for a DF step is feasible and will offer several benefits. This is particularly
attractive when an expensive diafiltration buffer is used, or when the duration of diafiltration needs to be minimized due
to product stability concerns.
CATION EXCHANGE CHROMATOGRAPHY
Figure 6. JMP analysis of data from chromatography experiments. Term refers to the variables that were reviewed in the analysis,
with Intercept as the mean value. Scaled estimate is the value for the effect of each parameter. Error is the absolute error
for each parameter. The t-ratio is the ratio of the parameter estimated to its standard error. A t-ratio greater than 2 in
absolute value is a common rule of thumb for judging significance. Prob. > |t| is the probability of getting, by chance alone,
a t-ratio greater (in absolute value) than the computed value. Given a true hypothesis, a value below 0.05 is often interpreted
as evidence that the parameter is significantly different from zero.
Chromatography steps in biotech processes often use pooling criteria that are based on UV absorbance (e.g., absorbance at
280 nm). The key advantage is the simplicity of implementing UV-based pooling criteria in a manufacturing environment. For
bind and elute applications, when the whole peak is to be collected from baseline to baseline, absorbance-based pooling criteria
offer a simple solution that ensures that all the product is collected. This works because, in most applications, the protein
concentration is linearly related to the absorbance signal.
While simpler to implement operationally, pooling by absorbance has several limitations in applications where a high-resolution
separation is being performed, and part of a peak is being collected to pool the product and pool out the impurities. Absorbance
methods are not able to differentiate between product and other proteins or other species that have similar absorbance profile.
As a result, since impurity levels vary from lot to lot due to variations in feed purity and column operating conditions,
pool purity also varies from lot to lot. Further, pooling criteria are often set conservatively in order to deliver a pool
of sufficient product quality, resulting in the loss of acceptable product for some lots.
Anurag S. Rathore, PhD, is a consultant, Biotech CMC Issues, and a member of the faculty in the department of chemical engineering at the Indian Institute of Technology. Rathore is also a member of BioPharm International's Editorial Advisory Board.
Articles by Anurag S. Rathore, PhD
