When we think of the simplest way to produce a biopharmaceutical, the most natural conclusion is to use a cell line that will produce a drug which is as similar as possible to the naturally occurring human form of the molecule. The reason for this considered cell line selection is that cellular bioprocessing, or post-translational modification, characteristics vary considerably between cell types, especially in the area of glycosylation.
The human immune system is very good at recognizing non-human structures on proteins and glycoproteins (such as the Galα1-3Gal carbohydrate epitope) and this raises potential safety issues. However, scientists are innovative by nature and will always explore new avenues and new possibilities! This has led some to consider the use of plant cells for the production of biopharmaceuticals.
In last month's blog, we considered how glycosylation differs across different cell types. In this blog we have expanded upon that topic by concentrating on plant glycosylation and the use of plant cell lines for biopharmaceutical production.
If you would like to read our previous blog comparing glycosylation profiles in different cell lines and the analyses required, please click the button below.
It may seem strange to use plant cells when they are so clearly different in their glycoprotein production machinery. However, plant cells do offer some production advantages that manufacturers are keen to make use of.
By their very nature, plant cell systems are different to mammalian cells and thus will not contain animal derived material or indeed animal pathogens. They are also relatively quick and inexpensive to grow or culture, requiring minimal media components and simple culture conditions. However, factors such as plant pathogens, toxic alkaloids and phenols, pesticides and fertilizers impact on the subsequent purification processes.
The ability of plant cells to perform complex bioprocessing tasks such as glycosylation, folding and disulfide bridge formation also gives them an advantage over the use of bacterial systems, which may be advantageous depending on the product being manufactured.
It goes without saying that plants are significantly different, in a structural sense, from animals and this is readily manifested in their glycoprotein biosynthetic pathway. There are numerous differences between plant and human glycosylation (1). Some monosaccharides, such as sialic acids, are not found in plants but exist in human N-and O-glycans, other monosaccharides such as xylose are found in plants but not in human N-glycans and still other monosaccharides such as fucose are found in both plants and human N-glycans but are linked in different ways within the glycan structure, which results in the formation of a non-human epitope.
O-glycosylation in plants also differs from human O-glycosylation with the plants using hydroxyproline as a site of O-glycosylation and also the monosaccharide Arabinose being present in plant O-glycans. This, at first glance, may seem like the death-knell for plant-based glycoprotein production but actually this is not the case.
The presence of non-human glycans on plant glycoproteins requires due acknowledgement and consideration. As an example, taliglucerase alfa, produced by Protalix Biotherapeutics, is a recombinant glucocerebrosidase produced in carrot root cells and used to treat Gaucher’s disease. In 2012, this drug was the first plant- made biopharmaceutical to get approval from the FDA (2). The plant glycoprotein has truncated “pauci-mannose” type structures on the glycans. These are truncated structures which were shown to carry the plant specific xylose and core linked fucose moieties.
Production of this drug was achieved by developing a construct that would be vacuole-targeted within the cell prior to further glycan processing in the Golgi. These structures, whilst not found on the human form of glucocerebrosidase, were found not to affect the safety and efficacy of the product (3). Of course, this observation cannot be extended into other plant cells or other plant cell derived products so each must be analyzed on a case-by-case basis with the glycans characterized and the safety aspect investigated each time.
Another solution to the presence of plant specific glycosylation features is to use genetic engineering to remove these plant specific structures and add in more “mammalian” type glycosylation enzymes to produce more “human-type” glycans. This form of glycoengineering, along with an understanding of precise cellular localization of the enzymes, has been demonstrated for numerous enzymes and led to the production of human like glycan profiles for plant cell produced glycoproteins (4). However, depending on the protein being produced, this glycoengineering approach may not be necessary.
Just as with mammalian derived glycoprotein drug products, those produced in plant cells, whether glycoengineered or not, require full glycan characterization covering quantitative investigations of the monosaccharides present, structural identification of the glycan population (composition and linkage) and if required, an investigation into site specific glycosylation. Peptide mapping can also be used to investigate the locations and structures of N and O-glycans within the glycoprotein.
The analytical approaches we use at BioPharmaSpec for mammalian glycans are also applicable to plant glycosylation analysis. From our experience, we know that there are certain nuances that must be borne in mind when working with plant glycans, such as the inability of the enzyme used to release mammalian enzymes (PNGase F) to work on plant glycans if they express the plant core fucose epitope. We therefore use a different release enzyme (PNGase A) when working with plant glycosylation.
Structural compositions, and thus masses, elution times and some linkages will be different from what is normally seen in mammalian glycosylation patterns due to the relatively unusual nature of the plant glycan structures, notably the presence of the pentose monosaccharide, xylose. Nonetheless, a knowledge of plant glycan biosynthesis coupled with an understanding of the glycan analytical techniques and nature of the data being generated allows plant glycans to be as successfully and completely characterized, just as in mammalian systems. This knowledge facilitates the required characterization of plant glycoproteins during development and the subsequent pathway to entry into the market of more plant-based biopharmaceuticals.
Talk to a BioPharmaSpec glycan expert today about how to effectively characterize the glycosylation profile of your biopharmaceutical.
