Impact of Lot-to-Lot Variability of Cation Exchange Chromatography Resin on Process Performance - A case study to understand the impact of lot-to-lot variability of a cation exchange resin on process

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Impact of Lot-to-Lot Variability of Cation Exchange Chromatography Resin on Process Performance
A case study to understand the impact of lot-to-lot variability of a cation exchange resin on process performance.


BioPharm International
Volume 21, Issue 5

Operation of the Cation Exchange Columns


Figure 2. Operation of the cation exchange step
All the cation exchange runs were performed on AKTA Explorer systems, which were controlled by Unicorn software. At the beginning of each run, equilibration buffer was pumped through each column for five column volumes. Following equilibration, anion exchange product from a single cell culture harvest was loaded onto the columns to a capacity of 19 mg of antibody per mL of resin. The antibody was manufactured and purified through the anion exchange step in-house. When the target loading capacity was reached, the load-line was chased with equilibration buffer for half a column volume to ensure that all the load material in the line reached the column. The columns were then washed with an additional 2.5 column volumes of equilibration buffer. At the end of wash, a step change in salt concentration was applied to the column to elute the antibody.2,3 Eluate collection was started when UV absorbance reached 0.5 AU on the ascending portion of the peak and stopped when the absorbance on the descending portion of the peak decreased to 0.5 AU. Elution buffer continued to be pumped onto the columns until the absorbance read 0.1 AU. The columns were subsequently sanitized for 30 minutes, flushed with storage buffer for four column volumes, and stored at 2–8 C when not in use. Linear velocity was maintained at 200 cm/hr for the entire duration of each run. The operation of the cation exchange step is shown schematically in Figure 2.

Use of High Conductivity Elution Buffer

In another set of experiments, elution conditions were changed for two of the columns. In these modified runs, the conductivity of the elution buffer was increased by a factor of two. Bound antibody was eluted with this high conductivity buffer. All the other process parameters remained the same.

Analysis of Samples

All cation exchange load and eluate samples were assayed for yield, product purity, and product quality. Yield was calculated by assaying for the concentration of IgG in load and eluate samples using absorbance obtained at 280 nm. Host cell proteins and leached Protein A concentrations in the load and eluate samples were measured using an in-house ELISA method, whereas DNA was quantified using a Q-PCR method. Charged isoforms of the antibody was measured with an in-house method using an analytical CEX column.

RESULTS AND DISCUSSION

Elution Volumes and Profiles


Figure 3. Lot-to-lot variation in elution volume
More than 50 initial cation exchange development runs performed using resin lot A consistently delivered eluates of less than 1.5 column volumes. In this study, lot A had an eluate volume of 1.4 CV. Cation exchange resin lot D had an unusually wide eluate peak of 2.6 CV. Lots B and C also obtained elution volumes larger than 1.5 CV. The eluate from lot B was 1.6 CV, and the eluate produced by lot C was 2.1 CV. These volumes are shown graphically in Figure 3.


Figure 4. Lot-to-lot variability in shape of elution peak
There were also significant visual differences in shape of elution profile. The UV traces of the elution profiles for lots C and D appear to have a flat portion followed by a trailing portion. This feature is not apparent for lots A and B, as shown in Figure 4.

The discussion into the probable root cause of the wide product volumes and unusual elution profiles continues below.


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