GEN Exclusives

More »

Tutorials

More »
Feb 15, 2006 (Vol. 26, No. 4)

Optimizing Bacterial Cell Processing

Defining the Steps Involved in Protein Isolation and Refolding to Provide High-Yield

  • Click Image To Enlarge +
    Fig.1: Recombinant proteins produced within a bacterial cell.

    Bacterial cells are widely used for production of various therapeutic proteins. Isolating these valuable proteins from the other material in the culture media and refolding them into their active configuration are critical steps in manufacturing recombinant biopharmaceuticals. The challenge is to obtain these proteins in high-yield. The type of protein and how it is stored within the cell determine the isolation and purification methods that biotherapeutic manufacturers need to use in production processes.

    All proteins are synthesized at the ribosome in the cytoplasm of the cell and then transferred to their operational areas. Recombinant proteins that are synthesized in bacterial host cells are often stored within the cell. The intracellular proteins in the cytoplasm or the periplasmic space may exist in soluble form or accumulate in insoluble aggregates called inclusion bodies, or IBs. Less frequently, proteins are secreted by the cells into the culture media (Figure 1). It is the location and state of the desired protein that determine the procedures and strategies necessary for its isolation and purification.

  • Isolation

    Click Image To Enlarge +
    Fig.2: Isolation of intracellular and extracellular (secreted) products.

    One of the first goals in bacterial cell processing is the separation of cells and cell debris from the proteins of interest. In the case of recombinant proteins that are secreted into the culture media, the bacterial cells can be separated from the fermentation broth and discarded. Cell removal can be achieved by centrifugation, tangential flow filtration (TFF), or normal flow filtration (NFF). After separation, the desired proteins are located in the supernatant, permeate, or filtrate and can be further purified from that solution in another process step (Figure 2).

    If the desired products are intracellular, the cells have to be harvested, or separated from the media, before protein removal (Figure 2). Centrifugation or TFF are commonly used for this step. After cell harvest, the cells are disrupted, or broken, in a process called cell lysis, to cause the release of the cell contents. Cell debris and contaminants have to be removed in a subsequent processing step. The nature of the expressed proteinsoluble or aggregateddetermines the approaches for further isolation after cell breakage.

  • Recovering Proteins From Bacterial Lysates

    Since IBs and soluble proteins exhibit different properties, the procedures to recover them from bacterial lysates are distinct. When working with soluble products like proteins, centrifugation, TFF, and NFF are commonly used for removing the bulk of cellular components. However, each method presents certain limitations.

    Cell lysates are typically characterized by a high-load of particles with a small size distribution. The NFF filter media may not have a sufficient capacity to cope with the high-particulate load, and many small particles are able to pass through the filter. Even with tight filters, the filtrate turbidity can be high. TFF and centrifugation can handle a wider range of solids and are usually better choices for recovering proteins from bacterial lysates. The quality of the permeate or supernant as judged by turbidity will depend on the membrane used or the centrifuge conditions applied.

    IBs are insoluble aggregates of denatured and inactive recombinant proteins. They have a relatively high density and can be easily pelleted by centrifugation. TFF may be a valid option for the concentration of IBs along with contaminating cellular debris, while removing the cellular and media proteins.

  • IB Processing

    Click Image To Enlarge +
    Fig.3: Processing of inclusion bodies

    Recombinant proteins aggregated into IBs are usually inactive and denatured. The major challenge in processing IBs is being able to recover biologically active and/or soluble protein in high-yield. To accomplish this, the protein in these IBs must be isolated, solubilized, and refolded in vitro to its active state prior to downstream processing (Figure 3).

    After isolation the IBs are resuspended and solubilized in buffer containing a strong denaturant (6-M Guanidinium Hydrochloride, G-HCl/Urea).

    The solution at this stage will contain the solubilized IB along with the cellular debris and residual media and cellular proteins. The solubilized IB can then be separated from the cell debris using NFF, TFF, or centrifugation, where the IB will be contained in the filtrate, permeate, or the centrate. This solubilized IB then needs to be refolded.

    To reduce the protein aggregation, the refolding process is usually carried out at low-protein concentrations (10100 g/mL). The process begins with the gradual removal of the denaturing agent (Urea/G-HCl) that can be achieved by methods including dialysis, rapid or slow dilution, or chromatography. Filtration steps to remove residual aggregates or contaminants are included at intervals appropriate to the particular process.

    Clarification using TFF or NFF requires careful selection of filter or membrane type. Specific feed information, such as particle size and properties, must be considered along with the composition of the feed solution. Various vendors offer a portfolio of products that are suitable for these applications.

  • Upstream Savings=Downstream Savings

    Whether proteins expressed by bacterial cells are intra- or extracellular, they must be efficiently removed from the cells and cell debris and returned to their active form, before they are useful as biotherapeutics. Optimizing the clarification step reduces waste of valuable product and makes downstream processes more efficient, saving time, equipment, and materials.



Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...
More »

GEN Poll

More » Poll Results »

Alzheimer's Therapies

Do you think an effective treatment for Alzheimer’s will be found within the next 10–15 years?