Plant cell culture stability

Bramble, J. L., Graves, D. J., and Brodelius, P., Plant Cell Culture Using a Novel Bioreactor The Magnetically Stabilized Fluidized Bed, Biotechnol. Prog.,... 

The addition of PVP 360,000 at a concentration of 0.75 g L 1 has been reported to yield a 35-fold increase in the level of extracellular foreign protein in suspended plant cell cultures [66]. The effectiveness of PVP in stabilizing secreted proteins depends on both the polymer molecular weight and its concentration. Low-molecular-weight (10,000 and 40,000) PVP was found to be less effective than PVP 360,000... 

Similar results were observed for the stabilization of an antibody produced in hairy root culture [11]. In contrast, some proteins are more resistant to stabilization efforts. As shown in Fig. 3, plant-produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4) are only moderately stabilized with the addition of bovine serum albumin (BSA). The use of known stabilizers such as PVP and gelatin was ineffective for these proteins. This difference highlights the need for more general protein stabilizers for use in plant cell cultures. 

Tsoi BM, Doran PM. Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures. Biotechnol. Appl. Biochem., 2002 35(Pt 3) 171-180. 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

Fig. 2.2 Stability of IgCi monoclonal antibody added to sterile plant and animal cell culture media. ( ) Murashige and Skoog (MS) medium (A) Dulbecco s minimal essential medium (DMEM) with 10% serum and (A) serum-free Ex-cell 302 medium. The error bars indicate standard errors from triplicate flasks. (Reproduced with permission, from B. M. -Y. Tsoi and P. M. Doran, Biotechnol. Appi. Bio-chem. 2002, 35, 171-180. Portland Press on behalf of the IUBMB.)...

For other plant-derived antibodies, stability was shown to be similar to mammalian counterparts. For instance, a humanized anti-herpes simplex virus monoclonal antibody (IgGl) was expressed in soybean and showed stability in human semen and cervical mucus over 24 h similar to the antibody obtained from mammalian cell culture. In addition, the plant-derived and mammalian antibodies were tested in a standard neutralization assay with no apparent differences in their ability to neutralize HSV-2. As glycans may play a role in immune exclusion mechanisms in mucus, the diffusion of these monoclonal antibodies in human cerival mucus was tested. No differences were found in terms of the prevention of vaginal HSV-2 transmission in a mouse model, i.e. the plant-derived antibody provided efficient protection against a vaginal inoculum of HSV-2 [58]. This shows that glycosylation differences do not necessarily affect efficacy. 

As it is imperative that the plant-derived hiopharmaceutical product must be obtained repeatedly and on a consistent basis, a master cell culture bank, seed bank for transgenic plants, or virus seed stock for transient expression systems must be constantly maintained. Storage conditions must therefore he optimized to prevent contamination and ensure viability. Both transgene stability (e.g., reversion to wild type or sequence drift of plant virus expression vectors) and protein expression levels must be monitored in a representative plant of a given bank or stock to minimize any possible variation in expression levels that may affect safety and consistency of the hnal product. A program that monitors lot-to-lot consistency of the hiochemical and biological properties by comparing the product with appropriate in-house reference standards could he implemented as a fundamental component of product development. 

Alfonso, M., J.J. Pueyo, K. Gaddour, A.-L. Etienne, D. Kirilovsky, and R. Picorel (1996). Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB- states, and increased sensitivity to light stress. Plant Physiol., 112 1499-1508. 

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