High Throughput Purification

High Throughput Purification Services

Kemp Proteins has incorporated the use of IMCStips^® with our Hamilton Microlab STAR to provide clients with a flexible, high-throughput, microvolume solution to deliver purified microquantities of their proteins. We can supply up to 500µg of purified protein from several hundred transient expressions to enable our client to compare, prior to selecting the protein(s) to move forward into further studies. Using our high-quality automation to produce small volumes of proteins that provide consistent results with high accuracy, precision, and repeatability to screen your candidate proteins increases overall efficiency of your development program.

The low-down on the Tips

Designed with efficiency and user-friendliness in mind, this product uses patented dispersive solid-phase extraction to automate workflows while enhancing accuracy and consistency. Unlike fixed-bed Solid Phase Extraction (SPE) devices, IMCStips contain loosely packed resins that mix with sample solutions during aspirate and dispense cycles, ensuring maximum contact between each resin and your analytes of interest.

These tips enable Kemp Proteins to perform hundreds of microvolume purifications per day, thus enabling our clients to create efficiency in their development programs.

IMCS Tips In Action

Compared to other methods for purifying small volumes of proteins of interest the IMCStips are far superior. Using a workflow for affinity-based antibody purification method we can see a turnkey method from cell media to purified proteins in less than 2 hours per batch using a Hamilton Microlab STAR. Purification of humanized IgG1 expressed in CHO cells was carried out using MabSelect SuRe™ LX resin via IMCStips (IMCS P/N: 04T-H6R80-1A) and filtered microfuge columns (spin method). For the spin method each sample was centrifuged at 100 × g for 5 minutes; flow-through samples were then re-applied either three (3x) or five times (5x) to correspond with the number of aspirate and dispense cycles (binding cycles) carried out using IMCStips. The spin method consistently recovered less mAb than the tip-based approach, as indicated by both UV-Vis measurements (Abs @ 280 nm and SDS-PAGE.

Kemp Protein’s Specialty Purification Process

Client communication: We consider this vital to maximizing the potential for a successful project outcome. Our initial discussions are information gathering against a set of questions. The information gained enables us to generate a statement of work, a “project design”, that is shared with you. Together we refine the plan until you are in agreement that we have captured the required specifications and deliverables for the project together with and nuances that may be particular to your project. Throughout the development of the Stable Cell Line we will supply regular updates through conference calls or information download through electronic means such as “Box”.

Project Commencement: The project begins when we agree on the design specifications and you provide us any client specific deliverables. Each project is different but runs to a general set approach.

Project Approach: Our approach to specialty purification development is dependent on the project being undertaken. However, they all have certain commonalities. Using a recent project as an example we have outlined the steps for this project – other projects and resins have different steps. We have found this an efficient and effective process to achieve the desired outcome of our clients.

The process starts with the preparation of the test sample which may come from a transient or stable cell culture and may be mammalian, insect or bacterial. When the supernatant or cell pellet has been prepared ready for purification the sample is placed in an appropriate sized tube on the Hamilton STAR deck along with equilibration, wash and elution buffers together with the appropriate IMCStips.

Hamilton / IMCStips Micro-Purification Process

Small Case Study

A project demonstrating the power of this specialty purification encompassed over 100 different constructs to identify the constructs that could be expressed and purified in specified amounts. Each construct was expressed in 5-10ml test expressions. Once harvested the supernatant was purified using the IMCStips containing Ni-NTA resin.

Protocol:

Pellets recovered from harvest placed on ice.
Pellets weighed.
Freshly prepared Lysis solution;
- B-Per II Reagent (ThermoScientific, Cat#78260)
- Nuclease (#BM0025, 25,000 units) added to each pellet.
Vortex and incubate on ice. Mix well.
Transfer lysates into clean labelled tubes.
Centrifuge to separate soluble from insoluble proteins.
Load clarified lysates into 96-well plate.
Use Hamilton Star to purify the protein.
Select the appropriate purification method.
- Prepare required plates, reagents and tips as per specification.
Equilibration buffer: Phosphate or TRIS
Wash buffer: Containing high imidazole
Elution buffer: 250 or 500mM imidazole
Set sample size: up to 1500 uL; Wash 1, 2 and 3 at 600 uL.
Elution Step: 400uL with 10 times of Mixing cycles.
Set the volume to 100 uL for aliquoting into a separate plate.

Use the plate with the aliquots for QC and freeze remaining.

QC: Run SDS-PAGE and develop per SOP

Example of gel:

Result: the purification and QC of between 96 – 192 different constructs can be achieved in under a working week.

Note: All data and images on IMCStip performance was kindly supplied by IMCS – https://imcstips.com/imcstips/

Questions & Answers

What type of purification methods can be used with the loose resin filled IMCStips?

IMCS have developed specialized tips for a variety of purification techniques, including Reverse Phase, Size Exclusion, Affinity, Ion Exchange/ Phosphopeptide. In general commercially available resins are used but depending on the desired application, different resins can be used inside the IMCStips. To learn more please visit https://imcstips.com/imcstips/

What if you don’t have my protein purification method listed?

The current range of IMCS tips covers most routine chromatography methodology. However, IMCS will work with our clients to develop tips that meet their requirements, to learn more please visit https://imcstips.com/imcstips/

What is the typical workflow & timeline?

The workflow and timeline for any purification project is very application/ protein dependent and may be as rapid as 90 minutes or as long as 4 hours.

Can I use this technology for purification optimization of my protein of interest?

Yes, this technology is ideal to enable us to optimize purification techniques and resins for a variety of proteins. We can set up a matrix to test the effect of different resins, pH, imidazole concentrations to find the optimum purification run for your protein of interest.