Efficient One-Step Protein Purification at Smaller Scales

Results of one-step cell culture clarification and sterilization study show the utility of dual-layer 24-well filter plates in protein purification

January 27, 2022

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Protein expression, and in particular recombinant protein expression, has been a cornerstone of drug discovery and an increasingly important contributor of front-line therapeutics since the first recombinant human protein, insulin, was approved by the FDA in 1982. Fast forward to today and there are more than 170 approved therapeutic proteins in use globally for the treatment of conditions as wide-ranging as cancer, multiple sclerosis, congestive heart failure, and Crohn’s disease. These manufactured proteins account for most of the top-selling drugs in the world today, and research to identify new proteins of interest continues. Given this interest in the development and production of new recombinant and native proteins for a wide variety of uses, from basic research all the way through to therapeutics, there is a need for new tools to streamline the process and accelerate development.

 

One of the great strengths of protein expression is also its weakness

 

Protein production relies on cloning the gene of interest into a host cell that will then express the protein of interest. Expression systems include bacterial, insect, and mammalian cells that are amenable to cultivation in suspension, i.e., they can be grown up in solution in a reaction vessel which simplifies the growth of the cells and the expression of the proteins. However, suspension cell cultures have one common problem when it comes time to harvest. The protein that we want is part of a complex mix of cell culture media, intact cells, and cellular debris, and must be purified before it can be used. Paradoxically it is often easier to purify proteins at commercial and even developmental scales where we are only working with one single protein, and in volumes that are amenable to automation or semi-automation of the purification process.

 

Problems with protein purification at smaller scales

 

Purification of proteins at the research level is compounded by two factors. Firstly, the volumes are much smaller, in most cases requiring that the protein purification process is a manual, or mostly manual, one. Secondly, at the research phase scientists are often working with multiple different variations of protein structure, with several different recombinant proteins being manufactured in parallel to support comparative studies and enable selection of a final candidate with the optimal set of characteristics. This means that researchers are often conducting multiple small volume purifications in parallel, substantially increasing the workload. Not only that but the purification process typically involves several steps, each designed to remove smaller and smaller particulate matter from the original media. This step-wise process is not only tedious but creates a compounding sample loss as some volume (the hold-up volume) is lost at each step.

 

Protein purification: clarification and sterilization in a single step

 

One solution that addresses both problems is to combine several filtration steps into one using a multilayered filtration membrane. The Pall AcroPrepTM 24-well cell clarification and sterile filtration plates (AcroPrep CCSF plates) are designed for exactly this purpose. The membranes in these plates combine a Seitz® depth filter over a Supor® EKV 0.65/0.2 mm membrane which allows the removal of particulate matter over a wide range of sizes in a single step. The filter plates can eliminate everything from expression host cells as large as 15 – 30 mm for mammalian cells, right down to cellular debris and other suspended particles as small as 0.2 mm, enabling clarification and sterilization of the raw culture media in a single step. Formatting these filters into a 24-well plate allows for filtration using a centrifuge or a vacuum manifold and allows users to process multiple samples in parallel. This combination of 24-well plate format and multilayered filter greatly reduces the time required for protein purification as well as reducing the hold-up volume of sample lost in the purification process, increasing yields.

 

Results of a study evaluating AcroPrep Cell Clarification and Sterile Filtration plates in protein purification

 

In a recent study, AcroPrep 24-well CCSF plates were tested to determine their efficiency at clarifying both raw culture (intact cells) and lysates (lysed cells). The researchers used cultures of both E. coli and S. cerevisiae that were passed through AcroPrep 24-well CCSF plates in a series of experiments using both centrifugation and vacuum to process the plated samples. Samples were evaluated before and after filtration using four quality parameters:

 

  • Turbidimetry, to measure the turbidity or cloudiness of the solution
  • pH and conductivity to assess the level of dissolved and ionic particles present
  • Optical Density (OD600) to determine the amount of suspended particulate matter
  • Total protein (protein concentration) measured before and after filtration to determine the % protein recovery
  • Processing time and hold-up volumes were also recorded for each branch of the study.

 

Results of AcroPrep CCSF protein purification

 

Reductions in measured turbidity and optical density showed that the AcroPrep plates were effective at clarifying samples of lysed and intact cells from both species, with similar results for both processing methods and a total protein recovery of >88% for all groups, with the majority of groups showing recovery in excess of 95%. Average processing time across all groups was just over seven and a half minutes but varied significantly between intact and lysed cells with the lysed cells taking longer, likely due to the high density of mid-sized particles in the form of cell debris. These results demonstrate the utility of the Pall AcroPrep plates to accelerate the process of protein purification from cell media, a task that has previously required significant time and manual effort over multiple steps to complete.

 

Read the full details of these studies in the Application note: Cell Clearance of E. coli and S. cerevisiae Cultures and Lysate Filtration with AcroPrep™ 24-well Cell Clarification and Sterile Filtration Plates.

 

Watch the video below and find more details on the AcroPrep 24-well CCSF plates.

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