The stability and half-life of proteins vary considerably. Antibodies, for example, tend to be very stable whereas growth factors are labile. The short-lived nature of growth factors is important to maintain localized effects but hinders their exploitation, and the exploitation of other proteins, for research and medical applications.
The stability of proteins in plasma varies significantly. Immunoglobulins are amongst the most stable whereas growth factors have much less durability
PODS™ (POlyhedra Delivery System) is a protein production technology which dramatically increases the stability of proteins. The science underlying PODS™ is based on a survival mechanism used by the Bombyx mori cytoplasmic polyhedrosis virus (CPV): In late stages of infection, crystalline cubes develop within insect cells. These crystals are typically one to five microns across and are formed from the polyhedrin protein expressed by CPV. As the crystals are formed, mature CPV virions specifically attach via a protein motif on their surface and become encased within the crystal. This protects the virus resulting in much greater durability with an extended window of opportunity for infection of a new host.
PODS™ technology adapts this stabilization strategy. We have generated improved immobilization tags facilitating the incorporation of essentially any protein into the crystal structure. To date, the technology has been used to generate PODS™ containing twenty-five different growth factors and numerous other proteins including 17 vaccine candidates. Compared with standard growth factors, PODS™ growth factor have much higher stability in storage and an extended half-life in use. For example, PODS™ LIF protein can be stored at 25˚C for several months without any changes in bioactivity. However, in the presence of proteases, present in sera or released by cultured cells, the PODS™ slowly disintegrate releasing their cargo. The slow release characteristics allow PODS™ proteins to maintain >50% peak levels in cell culture after 5 days. Is contrast, the activity of standard growth factor is completely abolished (see below). In animal models of bone repair, PODS™ growth factors have also demonstrated their therapeutic potential. For example, BMP-2 generates significantly better bone growth than standard BMP-2 and activity is retained for over 10 weeks. Key advantages of the technology include:
- Slow release depot formulation
- Efficacious for weeks
- No denaturation or refolding of the protein during production resulting in 100% bioactivity
- May be attached to substrates for localized effect
- Minimal batch-to-batch variation
- Produced in insect cells for important post-translational modifications
- Animal-free production
- Long shelf life – no wastage
- Stable at 4oC - no need to freeze and aliquot
- Available in small, medium and large pack sizes
- Cost effective
The concentration (pg/ml) of a recombinant growth factor or a PODS™ growth factor in solution was measured using an ELISA assay over a period of 6 days. At the start of the experiment, on day 1, recombinant growth factor (rhLIF) was maximal but was completely depleted by day 6. In contrast, >50% of peak levels were still present for PODS™ LIF proteins on day 6.
- Reduced frequency of cell culture media changes
- Significantly stabilized growth factor concentrations available in media for more predictable cell behavior: Standard growth factors' short half-lives result in dramatic drops in availability after a few hours or days. The concentration is never ideal and a compromise between peak concentrations that are tolerated and minimal concentrations that are required for the biological effect.
- 100% levels of bioactivity and high reproducibility of product manufacture providing greater consistency of results
- Ready attachment to substrates allowing localized deposition within a culture system
- Development of physiologically relevant concentration gradients for patterning
PODS™ crystals offer a very simple approach to generating physiologically relevant protein gradients. An SEM image showing a granular field of PODS™ crystals containing neurotrophic growth factors. The crystals have been applied to the culture surface using a pipette. PC12 neuronal precursor cells were subsequently plated. The cultures were incubated under static conditions for 96 hours. During this time, the PODS™crystals secrete their cargo creating a uniform, high concentration of growth factor over the crystals with a gradient around the edge. PC12 cells move up the gradient towards the edge of the crystal field. At close magnification, it is possible to see neurites (arrowed) extending from the neuronal cells. These neurites are perpendicular to the gradient and connect with the neighboring cell allowing the neuronal cells to form a single connected chain. Data Courtesy of Prof Hajime Mori, Kyoto Institute of Technology.
PODS™ crystals are generated in cells in which the polyhedrin protein is expressed at high levels under the control of the polyhedrin promoter. PODS™ crystals containing growth factors are formed when a second protein, also under the control of a polyhedrin promoter, is co-expressed. This second protein is termed the cargo protein. The second protein is tagged with a short peptide sequence which causes the cargo protein to bind to the growing polyhedrin crystal. As the cubic crystal continues to grow, the cargo protein becomes encased.
Cargo proteins are incorporated into PODS™ crystals when co-expressed with the polyhedrin protein.
How big are PODS™ crystals?
PODS™ crystals are cubic and typically 1-15 microns in size with a modal size of 3-4 microns. During the manufacturing process, some PODS™ crystals may fracture resulting in smaller PODS™ crystal fragments.
What is a PODS™ crystal's buoyant density?
PODS™ crystals are heavier than water and will settle on the surface of a culture vessel. Care should be taken when aliquoting since PODS™ crystals will sink to the bottom of a tube within a few minutes. The majority of PODS™ crystals will remain in suspension for up to 60 min in a 20% glucose solution (or a solution of similar density).
How stable are PODS™ crystals?
We have tested a variety of conditions to assess their impact on the integrity of PODS™ crystals. PODS™ crystals are stable in high temperatures and remain intact after boiling for 1 hour. They are highly stable when stored in solutions between pH 6-8. Above pH 10, PODS™ crystals lose their stability and dissolve rapidly (in a few hours). PODS™ crystals are stable in standard cell culturing temperatures for extended periods of time (> 10 days).
How is the cargo protein released?
In addition to pH, PODS™ protein crystals are broken down in solutions containing proteases. Proteases may be derived from components of the solution (e.g. serum) or secreted by cells. Therefore the culture system affects the amount of growth factor available in solution. In contrast to gel-encapsulated proteins (using hydrogels such as PLGA) PODS™ crystals do not produce an initial burst release of cargo proteins. For naked PODS™ crystals in cell culture, peak release has been shown to occur at day 2, then gradually diminishes.
Is it possible to modify the release profile?
The release of cargo proteins into cell culture occurs over a period of 2-3 weeks. This release period can be extended to several months, if the PODS™ crystals are combined with a scaffold, such as a collagen.
Will PODS™ crystals adhere to plastics?
PODS™ crystals will adhere to untreated plastics. For sub-aliquoting, we recommend using low adherence plastics, such as low protein binding centrifuge tubes.
Why are PODS™ proteins sold by numbers of crystals?
PODS™ cargo proteins are incorporated into the polyhedrin crystal within the insect cell. Consequently, it is not possible to obtain a direct measurement of the amount of cargo protein that is present in a sample. When PODS™ crystals are expressed, both polyhedrin proteins and cargo proteins are expressed from within a single vector and both utilize the same promoter sequence. Therefore, the ratio of cargo to polyhedrin protein will be constant. The size distribution of PODS™ crystals is also constant. Consequently, the number of crystals has been adopted as the unit of quantification for PODS™. It has been estimated that 50x106 PODS™ crystals typically generates a peak concentration in the medium equivalent to 3.3 µg of standard recombinant protein.
How many PODS™ crystals should be used?
PODS™ crystals provide a depot of proteins which are steadily secreted. As stated above, it has been estimated that the biological activity of 50x106 PODS™ crystals generates the same peak dose as 3.3 µg of standard recombinant protein. However, at 5 days following the start of seeding the PODS™ crystals, more than 50% of these peak levels are still present in the culture system. Ultimately, the amount of PODS™ crystals that is optimal for a particular experiment should be determined empirically, using 50x 106 PODS™ crystals equivalence to 3.3 µg of standard growth factor as a good starting point.
How often should media and PODS™ crystals be replaced in the culture system?
The frequency of media change depends on (1) the speed with which nutrients are exhausted or degraded and (2) the speed with which toxic metabolites accumulate. In most cell culture systems, the stability of growth factors is an over-riding issue which drives media replenishment. However, particularly in cells with high levels of metabolic activity, other factors will eventually become important once the stability issue has been addressed.
Are PODS™ crystals transparent?
PODS™ are crystalline and will refract light. A large number of PODS™ crystals may interfere with the generation of images using imaging techniques such as phase-contrast.
Can PODS™ crystals have a physical impact on cell behavior?
Physical features on a culture surface may impact the behavior of certain cells although we have not observed any noticeable effects in the cultures we have tested. If PODS™ crystal topology is a concern, PODS™ crystals may be incorporated into a hydrogel surface coating. Custom made Matrigen™ Softwell plates (flat hydrogels of defined elasticity) containing PODS™ crystals are available from Cell Guidance Systems.
Do PODS™ proteins have important post-translational modifications?
PODS™ crystals are made in insect cells. PODS™ cargo proteins, therefore, contain most of the post-translational modifications that are found in mammalian cells. For example, PODS™ Wnt-3a, which requires palmitoylation and glycosylation for biological activity, is functional.
Are PODS™ crystals immunogenic?
The polyhedrin protein has been tested in-vivo and an inflammatory response has not been observed. Lack of immunogenicity for foreign proteins is not without precedent: silk fibroin protein from the silkworm B. mori is commonly used for surgical stitching and Botulinum toxin is widely injected in cosmetic procedures to reduce the appearance of wrinkles.
Are PODS™ proteins equivalent to standard growth factors?
The stability of standard recombinant growth factors varies significantly. The most labile, such as FGF-1, have a half-life measured in minutes limiting their utility. The stability of PODS™ growth factors that are encased in PODS™ crystals is much longer but once released will be the same as their standard recombinant counterparts.
The amount of available growth factor in a culture system is a function of the speed of release from the crystal and the subsequent stability. In the first few hours of a culture containing only newly added PODS™ crystals, there is little growth factor protein available for the cells. Significant amounts of growth factor protein are available after one day. The initial lag of protein availability may be corrected if necessary by adding a small amount of standard recombinant growth factor.
Why are PODS™ proteins 100% bioactive?
PODS™ cargo proteins such as growth factors are captured and purified without denaturation. Unlike standard recombinant proteins, they do not require refolding or successive column purification steps. Consequently, biological activity is inherent.
Do you offer custom services?
Yes, we can produce your protein in PODS™ crystals. Please see our PODS™ service page.
We are interested in developing therapeutic applications, can you help us?
We already have several therapeutic programs in place. Please contact us if you plan to develop a therapeutic application
For further information, please contact: firstname.lastname@example.org