Scale-up Protein Purification
The scalability of protein purification is crucial for researchers involved in process development. While only a few micrograms of protein are needed during the screening phase, several milligrams or even grams may be required in later phases. But which type of resin (e.g., agarose resin or magnetic beads) or which format (e.g., spin columns, FPLC columns, etc.) is suitable for each scale? The following sections explain the recommended types and formats for different scales of protein purification.
Protein purification columns, such as spin columns, gravity flow columns and FPLC columns, from IBA Lifesciences, are pre-packed with agarose resin and have a defined resin bed volume of up to 10 ml. As a result, they provide a limited yield and are recommended primarly for screening and laboratory-scale applications.
Both magnetic beads and agarose resin can be used for protein purification, from the screening phase to laboratory-scale protein production. For large-scale protein production, agarose resin is recommended. Both options are available as suspensions in various filling sizes, allowing the resin bed volume to be individually selected and easily adapted to specific purification conditions.
The following sections describe the optimal use of these formats for protein purification, ranging from screening to large-scale production.
Recommended application range of IBA Lifesciences protein purification products depending on the protein purification scale.
Upscaling of agarose resin
Agarose resins are suitable for applications ranging from screening to large-scale protein purification. However, unlike magnetic beads, they are not recommended for batch procedures because agarose resin is easily lost during pipetting, and the supernatant cannot be completely removed.
The best results are achieved when agarose resin is packed in tips or columns designed for centrifugation, gravity flow, or liquid chromatography systems.
For screening applications, spin columns pre-filled with agarose resin are ideal when only a few samples need to be purified in parallel. However, for high-throughput protein purification involving many small samples, IMCS resin-filled tips provide a simple and efficient solution, especially when used with robotic systems.
Learn how Strep-Tactin®XT 4Flow® high capacity resin works in combination with IMCStips® to achieve excellent results in this application note.
Small-scale high-througput protein purification with Strep-Tactin®XT 4Flow® high capacity resin filled in IMCStips®. A) Schematic representation of automated protein purification with IMCStips®. B) SDS-PAGE gel showing purification of TST-CD34 mAB from CHO-S supernatant with IMCStips® on a Hamilton Microlab® STAR™. The purification process resulted in a purity of >95%.
For laboratory scale, IBA Lifesciences offers both pre-packed gravity flow columns (0.2 ml to 10 ml resin bed volume) and FPLC columns (1 ml and 5 ml resin bed volume), all available with 4Flow ® agarose resins. Gravity-flow columns are ideal for small sample volumes and for purifying a limited number of samples in parallel. Additionally, no special laboratory equipment is required. For larger sample volumes, FPLC columns used with chromatography systems, such as an Äkta ®, are recommended. FPLC columns can also be connected in series to increase the resin bed volume and protein binding capacity. If FPLC columns are not an option, large sample volumes can also be applied to gravity-flow columns with the help of the application aid WET FRED.
FPLC columns are also suitable for large-scale protein purification. For this purpose, IBA Lifesciences offers agarose resins in 1 L bottles, allowing users to fill large FPLC columns themselves. The best price-performance ratio is achieved with Strep-Tactin®XT 4Flow® high capacity resin, which features a very high protein binding capacity of 31 mg protein per ml of agarose resin (determined using a a 52 kDa Twin-Strep-tag fusion protein). Other agarose resins, Strep-Tactin® 4Flow® high capacity and Strep-Tactin®XT 4Flow® offer lower protein binding capacities but maintain the same specificity for the Strep-tag® and deliver equally high protein purity.
The applications of agarose resins and magnetic beads are summarized in the table below. For a detailed comparison of their specifications, you can find an overview here.
Upscaling of magnetic beads
During the early screening phase, magnetic beads can be used in combination with pipetting robots or in small reaction tubes. They are highly scalable and suitable for direct use in batch processes. An example of automated high-throughput purification using the Tecan Fluent® system can be found in the poster “From screening to large scale purification: adaptability of MagStrep® Strep-Tactin® XT beads”.
As the demand for purified protein increases to laboratory-scale, the amount of MagStrep® Strep-Tactin® XT beads can simply be increased, maintaining the same yield per milliliter and protein purity. However, magnetic beads are rarely used for laboratory-scale protein purification because their cost per milliliter is generally higher than that of agarose-based resins. Despite this, MagStrep® Strep-Tactin® XT beads can be frequently regenerated without any loss in protein binding capacity. This reusability, combined with their high binding capacity and purity, can significantly reduce process costs. Additionally, no specialized laboratory equipment or expertise is required - only a suitable magnet, such as those provided by Sepmag®.
Learn more about the scalability of MagStrep® Strep-Tactin® XT beads for protein purification in the application note
Easy scalability of the purification process with MagStrep® Strep-Tactin®XT beads was tested by purifying a Twin- Strep-tag® fusion protein from different sample sizes (A). Scaling up the purification results in an increasing amount of isolated protein (B) while maintaining the yield per ml of lysate (C).
