Extracellular polysaccharides found on the outermost layer of bacteria, such as capsular polysaccharides or micro capsular polysaccharides, are key virulence factors responsible for invasive bacterial infections. They also serve as the primary antigenic components in vaccines designed to prevent and control such infections.
The capsule is a layer of mucous substance located outside the bacterial cell wall, consisting primarily of polysaccharides. These polysaccharides are haptens, they cannot effectively elicit a helper T-cell response and typically induce an immune response via a non-T-cell-dependent mechanism. However, as the immune systems of infants under two years of age are not yet fully developed, they are unable to mount an adequate immune response to polysaccharide vaccines. This issue can be addressed by using glycoconjugate vaccines: these are formed by covalently linking polysaccharide units—which vary in structure and quantity—to non-sugar units (such as proteins, short peptides or lipids), with protein-polysaccharide conjugates being the most common. The non-sugar component acts in a manner similar to an adjuvant, activating helper T cells and subsequently promoting the maturation and differentiation of B cells into memory cells, thereby providing long-term immune protection.
Factors affecting vaccine immunogenicity
Several parameters can influence the immunogenicity of polysaccharide-conjugated vaccines, including the size and structural modifications of the polysaccharide, the carrier protein used, and the ratio of polysaccharide to protein. Traditionally, to synthesize polysaccharide-conjugated vaccines, the polysaccharide is first extracted from bacterial cultures and isolated through a series of purification steps. Depending on their genetic characteristics, bacterial strains can produce polysaccharides of varying chain lengths. Furthermore, the fermentation and purification conditions employed have a profound impact on the length of the polysaccharide chains. Polysaccharides are polymers composed of identical repeating units, ranging in length from ten to several thousand units. Whilst protein synthesis involves a specific termination codon to ensure that all protein molecules produced are identical, no such signal exists in bacterial polysaccharide production; consequently, polysaccharide molecules are characterized by a wide range of sizes, meaning they are polydisperse. The molecular size distribution of each batch of polysaccharide must be determined and monitored, as any variation may indicate a lack of production consistency during the purification steps or a loss of structural integrity of the polysaccharide.
Shorter polysaccharides are generally preferred to enhance binding yield and consistency, and to facilitate the purification of the conjugate. Furthermore, working with smaller-sized polysaccharides may facilitate characterization of the entire process, including sugar activation and conjugation, and may lead to the production of conjugates with a more defined structure. Consequently, polysaccharides are typically fragmented and size-reduced using chemical or mechanical methods to generate a homogeneous population of lower molecular weight fragments prior to conjugation with the carrier protein.
High pressure homogenization controls the size of polysaccharides
There are various methods for reducing the size of polysaccharides, but not all methods are necessarily suitable for specific polysaccharide structures, and some techniques cannot be scaled up for industrial applications. Generally speaking, cleavage methods exhibit varying degrees of specificity; some techniques are capable of targeting specific chemical bonds within the polysaccharide structure, such as deamination and sodium periodate oxidation. However, other methods are relatively random，for example, acidic and alkaline hydrolysis can affect different types of chemical bonds，potentially resulting in a product with more dispersion compared to the original polysaccharide. Therefore, it is essential to monitor the cleavage process and halt the reaction at the predetermined average chain length.
Mechanical size control using high-pressure homogenizers is one of the most widely used methods for the production of polysaccharide-conjugated vaccines. The high-pressure homogenization process is particularly well-suited to reducing the size of polysaccharides containing non-sugar substituents, such as O-acetyl, glycerophosphate and acetyl groups commonly found in Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus and Group B Streptococcus. Homogenization is an ideal method for size reduction because, once the polysaccharide concentration, pressure and number of cycles have been established, the process can be highly reproducible. Temperature control is crucial for minimizing adverse side effects. This method reduces the polydispersity of polysaccharide molecules in solution, as only larger molecules are reduced in size，rather than smaller ones. However, each polysaccharide must be evaluated independently to ensure that the composition and structure of the repeating units do not undergo adverse changes.
In summary, high pressure homogenization is a simple and scalable technique that offers good reproducibility and is free from side reactions. This technique has been employed in the processing of capsular polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, Group B Streptococcus and Staphylococcus aureus, amongst others.
YOCELL can provide various models and specifications of high pressure homogenizers to meet the process requirements at different stages.
