What are the benefits of introducing multimodal ion exchange chromatography as a guard column and could they include a significant increase in mAb purity? This article presents a study in which two protein A affinity chromatography resins were compared to see whether the introduction of a guard column could improve mAb purity and to discover which of the protein A resins produced the best results.
Optimizing yield and purity while keeping costs under control
When designing a purification process for monoclonal antibodies (mAbs) you want to include as few steps as possible. Each step leads to additional costs and will usually reduce the overall yield – since no step has 100 percent recovery – unless it contributes to improved purity, economy or yield in other steps.
The clarified supernatant generally goes through 2 to 3 chromatography steps after harvesting, often as well as a polishing step to achieve the desired final level of purity. Each step must be developed, tested, optimized, and documented.
With each step leading to additional costs and reduced yield, the product becomes more valuable the longer you proceed in the downstream process. The most costly step is affinity chromatography. Affinity chromatography is the fundamental backbone of your purification process, as, with only one step, you will generate high purity with, in most cases, high yield. However, the protein A column will not only be your most expensive tool, but also it will typically be used at the start of the purification process – when the feed is full of impurities – so a wise strategy would be to try to find ways of prolonging its life span.
The logic of an optimized purification process
Generally, this means designing a purification process that maximizes both yield and purity, whilst at the same time controlling costs. This requires a process that removes a lot of bulk impurities during the first steps in the downstream process, and focuses on high yield steps particularly at the end of the process, when every gram is precious. It might also make sense to remove a lot of impurities before affinity chromatography in order to protect the protein A column and reduce the need for cleaning in place (CIP). This could all be achieved by using a guard column.
Removing bulk impurities using multimodal ion exchange chromatography
Introducing a guard column immediately after the harvesting steps can remove critical substances, such as DNA and host cell proteins (HCPs). It will thereby render the rest of the downstream process more robust and it will also protect the protein A column from factors that impact its performance. Multimodal ion exchange chromatography is a rather uncomplicated step in flow-through mode, which will not make your purification process unnecessarily cumbersome.
Moreover, adding a guard column early in the downstream process should not only protect the protein A resin and reduce costs in the affinity chromatography step; it should also result in a higher purity of the mAbs, and potentially higher overall yields.
Putting it to the test
We designed a study where WorkBeads™ 40 TREN was introduced upstream of affinity chromatography. WorkBeads 40 TREN is a multimodal ion exchange chromatography resin, which is positively charged below pH 9. For affinity chromatography, WorkBeads affimAb was used. WorkBeads affimAb is a protein A resin developed for industrial mAb purifications. WorkBeads affimAb is alkali-stable and has a high dynamic binding capacity. For comparison, we also included a market-leading resin, MabSelect SuRe™ from GE Healthcare, in our study.
Four combinations were tested in the study, where different materials were used:
- WorkBeads affimAb
- WorkBeads 40 TREN + WorkBeads affimAb
- MabSelect SuRe
- WorkBeads 40 TREN + MabSelect SuRe
20 mL of clarified Chinese Hamster Ovary (CHO) cell extract was loaded onto each of the two protein A columns or onto the WorkBeads 40 TREN columns, in the combinations where these guard columns were used. The impurities were analyzed and measured by SDS-PAGE, CHO HCP ELISA and PicoGreen™ DNA analysis. The results are summarized in the table below and you can download a full version of the study at the bottom of this page.
Table 1. Analysis of different collected flow through and eluate from four combination run on WorkBeads affimAb, WorkBeads 40 TREN + WorkBeads affimAb, MabSelect SuRe, and WorkBeads 40 TREN + MabSelect SuRe.
|Resins included in flow chart for experiments 1-4||HCP (ng/mL)1 *||HCD (ng/mL) 2 *|
|1a. WorkBeads affimAb (flow through)||NA3||10000|
|1b. WorkBeads affimAb (mAbs eluate)||775||40|
|2a. WorkBeads 40 TREN (guard); WorkBeads affimAb (flow through)||NA3||130|
|2b. WorkBeads 40 TREN (guard); WorkBeads affimAb (mAbs eluate)||610||40|
|3a. MabSelect SuRe (flow through)||NA3||10000|
|3b. MabSelect SuRe (mAbs eluate)||>1000||500|
|4a. WorkBeads 40 TREN (guard); MabSelect SuRe (flow through)||NA3||130|
|4b. WorkBeads 40 TREN (guard); MabSelect SuRe (mAbs eluate)||950||40|
1 CHO HCP ELISA analysis.
2 PicoGreen DNA analysis.
3 Above detection limit.
* The values obtained in this application are based on the experimental conditions and setup as described above. Different sample batches under different conditions will generate variation in absolute numbers obtained.
Improved purity with WorkBeads 40 TREN
The experiments showed a relatively low level of HCP in the mAb eluates from WorkBeads affimAb with and without WorkBeads 40 TREN included upstream. The eluted mAbs from MabSelect SuRe contained more than 10-fold higher levels of HCP (> 10000 ng/mL) compared to WorkBeads affimAb. When WorkBeads 40 TREN was added upstream MabSelect SuRe, the HCP level was reduced to a 10-fold lower level, indicating its ability to remove protein impurities from high bioburden samples.
Host cell DNA, HCD, was analyzed using a PicoGreen assay. The study showed that WorkBeads affimAb bound less DNA non-specifically than MabSelect SuRe, as low as 40 ng/mL for WorkBeads affimAb compared to 500 ng/mL with MabSelect SuRe. However, by using WorkBeads 40 TREN as a guard column upstream of MabSelect SuRe, the HCD level in the eluate after the MabSelect SuRe column was reduced to 40 ng/mL.
The different flow-throughs were also analyzed, showing that approximately 99 percent of the HCD was removed by WorkBeads 40 TREN used upstream to protect the protein A columns.
Summary of study results
The study showed a threefold improvement in the reduction of HCP and a 99 percent reduction in HCD when using WorkBeads 40 TREN, prior to WorkBeads affimAb. In addition, our study showed that WorkBeads affimAb produced higher mAb purity than the market-leading resin.
In other words, the study showed that multimodal ion exchange chromatography with WorkBeads 40 TREN, used upstream of affinity chromatography, is an efficient strategy to protect the protein A column and expand its life span. Introducing WorkBeads 40 TREN will both save costs as well as achieve higher mAb purity.
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Posted by The Bio-Works Team
The Bio-Works staff are seasoned and enthusiastic (yes, even a bit nerdy) experts in purification resins, methods and strategies with many years of experience of the ins and outs of this field. We love our job and we’d love to help you do yours by sharing some good solid facts, principles and best practice. In short – “purification made simple”.Facebook LinkedIn Twitter Google+