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MabSelect SuReTM: A quick start to the development of monoclonal antibody capture steps
MabSelect SuReTM: A quick start to the development of monoclonal antibody capture steps
In the purification of monoclonal antibodies, in order to obtain the desired high quality antibodies, a two or three step chromatography step is typically employed. Typically, the chromatographic medium used in the three-step process is Protein A→cation exchange→anion exchange; the chromatographic medium used in the two-step process is Protein A→Capto adhere (multimodal ion exchange). All monoclonal antibodies (Mabs) have some similar properties, so they can be purified using the same platform method. Please note that the platform does not imply the same process, it simply means that certain aspects of the process do not need to be developed from scratch, as previous monoclonal antibody process development experience can be utilized.
The purpose of this discussion is to propose a purification process that can be used in the purification of most Mabs produced by mammalian cells. Here we present a general starting suggestion (not optimized) for process development based on MabSelectTM SuRe chromatography media.
MabSelectTM SuRe is a Protein A chromatography medium that is resistant to the demanding and cost-effective CIP program, 0.1-0.5 M NaOH.
The recommended approach for process development is to use High Throughput Process Development (HTPD) - a large number of chromatographic parameters can be evaluated in a short time using PreDictorTM 96-well plates (Reference 1). The optimal process parameters are then scaled up, ie HTPD→lab scale→ pilot plant→production. This approach provides a lot of data that is especially useful for the Quality by Design (QbD) approach. Even when using HTPD, some of the information in the next table can be used as a starting point.
system
control
Chromatographic medium
Column
Bed height (cm)
CV (ml)
ÄKTATM avant 25 or 150
UNICORNTM 6
MabSelect SuRe
TricornTM 10/200
20
15.71*
Chromatography method for MabSelect SuRe capture step
step
Volume or time
Buffer composition
Retention time, minutes (linear flow rate, cm/hr)
0. Only after saving - balance
3 CV
20 mM sodium phosphate, 0.15 M NaCl, pH 7.4
7.5 (160 cm/hr)
Balance
0.25 CV
20 mM sodium phosphate, 0.15 M NaCl, pH 7.4
3.4 (350 cm/hr)
2. Loading
80% dynamic load at 5% penetration
As needed
2.4-4.8 (500-250 cm/hr)
Long retention time → high binding capacity
3. Cleaning
3 CV
20 mM sodium phosphate, 0.15 M NaCl, pH 7.4
2.4-4.8 (500-250 cm/hr)
4. Intermediate cleaning
2 CV
25 mM sodium phosphate, 0.5 M NaCl, pH 7.0,
(5% isopropanol, optional)
3.4 (350 cm/hr)
5. Clear
3 CV
20 mM sodium phosphate, 0.15 M NaCl, pH 7.4
3.4 (350 cm/hr)
6. elution
Controlled by a UV detector or at a pre-determined volume
0.1 M acetic acid, pH 2.9**
3.4 (350 cm/hr)
7.CIP
2 CV=15min
0.1M NaOH
7.5 (160cm/hr)
8. Rebalance
3 CV
20 mM sodium phosphate, 0.15 M NaCl, pH 7.4
3.4 (350 cm/hr)
9. Only after the last round of running - save
4 CV
20% ethanol
7.5 (160 cm/hr)
Note: In addition to the loading step, the flow rate can be increased to 500 cm/hr for other steps;
Approximate total cycle time *** ~ 17CV x 2.4 min/CV + 11CV x 3.4min/CV + 2 CV x 7.5 min/CV ~ 95min ~ 1.6 hours. Even if you do it twice, the total time is about 3.2 hours. There are no speed limit steps in production.
Basic simplified process development
Sample Preparation - Samples must be filtered before loading onto the MabSelect SuRe column. At least a sterile filter must be used; in addition, a depth of adsorption filter can be used prior to sterile filtration. After the cell culture is harvested, the run should be completed as soon as possible. The conditions that need to be preserved before the cell culture is run should be sterile filtered and stored at 4 ° C or frozen (if possible).
Run #0 – Blank Run - In order to remove non-covalently fixed ligands, a blank run should be performed prior to the first run on the new MabSelect SuRe chromatography media to reduce ligand leakage during chromatography. Two changes should be used for all stages of the chromatographic methods listed above - first, equilibration buffer (ie no protein) should be used during the loading phase, and second, the elution phase of the blank run should be set to 3 column volumes. And is not controlled by the monitoring function.
Run #1 - Loading Conditions - The next experiment should be run to determine the dynamic binding capacity (DBC) of your Mab for the MabSelect SuRe column for a retention time of 2.4-4.8 minutes. According to the above procedure, the column was overloaded to 50 g/L, the penetrating component was collected, and then the concentration of Mab in the penetrating component was measured, and 5% penetration was calculated.
Run #2 – Set the loading condition to 80% dynamic load at 5% penetration, then follow all the steps above. If this run produces acceptable levels of purity, quality and throughput, the process used here can be locked.
Analysis - Analysis of the purity and quality of the Protein A step sample after adjustment to the next loading conditions and filtration through a sterile filter. In addition, purity and quality must be analyzed after 2 or 3 step-based chromatography steps based on the platform process. If the above results in low yield or high host cell protein residues (HCP) in the eluted fraction, further cleaning and elution conditions must be optimized to complete further process development.
Cleaning conditions - GE Healthcare has completed a pilot study of various cleaning conditions. To learn more about these studies, ask your local GE Healthcare representative for a scientific poster discussing the Intermediate Cleaning Steps for Protein A (Reference 3).
Elution conditions - Various elution conditions can be considered, such as citrate buffer (10-100 mM) or glycine. When optimizing the elution conditions for better impurity removal, the highest pH at which the antibody is effectively desorbed is determined, however, this may increase the elution component volume. Additionally, elution conditions are designed to match the pH required for virus inactivation, as discussed below.
Step Duration - All of the step durations mentioned here are only symbolic, and if the chromatograms are collected in a particular step and the indicated time is collected, the duration of the actual steps can be shortened.
Virus inactivation - The pH in the eluted fraction should be maintained at 3.6 or lower for at least 30 minutes for proper virus inactivation. If the eluted fraction has a higher pH, the pH needs to be lowered by the addition of acid titration or by further optimization of the elution buffer volume. The pH was then adjusted by the addition of 0.1 M NaOH and the pH had to be adjusted immediately to match the next loading conditions. (eg for pH 5-6 for cation exchange or pH 6-8 for Capto adhere). Precipitation may occur during elution and generally occurs during low pH virus inactivation after pH titration. It is possible to precipitate lipids and trace amounts of Mab, HCP and Protein A. This precipitate can be removed using a sterile filter - the correct pore size of the filter membrane must be chosen - although the membrane pore size normally used in production is already large enough for this step.
in conclusion
The recommended process development approach is to use HTPD and analyze multiple conditions. The information provided in this document is based on prior experience and can serve as a starting point for step development. For further assistance with step or process development, please contact your local GE Healthcare sales representative or GE Healthcare's Fast Trak division.
Note:
* The correct packing method should be used - see Reference 2 (compression factor 1.15) for details;
** 0.1 M acetic acid will have a pH of approximately 2.9 and no buffering capacity. The resulting pH of the eluted fraction will depend on the elution component volume and the buffer capacity of the previous wash buffer. In most cases, the pH of the eluted component will be between 3.5 and 4.0;
*** For cycle time: Assuming a material concentration of 2.5 g/L, the loading capacity is 35 g/L. The save time and the balance time after saving are not counted (~53 minutes).
references