1.1 Analytical Ultracentrifugation

The first high-speed centrifuges were mainly used to study cell "extracts". This application and the limitations of metal materials meant that the sample volume had to be kept as small as possible, so rotary heads with transparent windows were designed in order to determine the distribution of the particles in the centrifugation cell during centrifugation, and special analytical rotary heads were designed with an analytical cell that could be injected into the sample to be analyzed, and the analytical cell could be used to inject into the sample to be analyzed. The sample to be analyzed. The sample to be analyzed is a homogeneous suspension, and by accelerating the rotor head to its operating speed, the sample particles sink at a certain rate, which is determined by their size, shape and centrifugal force. Therefore, if the sample to be analyzed is a homogeneous suspension containing only one type of particle, a clear interface will move slowly towards the bottom of the cell as the particles continue to settle in the cell. If the analyzed sample is a mixture of particles, each particle will settle at its own rate. The analytical ultracentrifugation method was quickly developed on this basic and simple basis.

1.2 Differential sedimentation

Differential sedimentation is in principle the same as separation in an analytical ultracentrifuge: the centrifuge tube is filled with a homogeneous suspension, and during centrifugation the pellet moves to the bottom of the tube, where it settles. The centrifugation time is chosen just long enough for all the largest particles to settle, so that a supernatant is obtained without such particles, and this supernatant can be centrifuged at a higher speed to separate the second largest particle, and so on and so forth. The disadvantage is that the precipitation will be inhomogeneous, and if the centrifugation time is controlled so that just about all of the largest particles are precipitated, mild mixing will result, and longer centrifugation will result in more mixing.

"Washing" the sediment improves the separation achieved by differential centrifugation, by dissolving the sediment in a homogeneous medium and centrifuging it under the same conditions as in the original sedimentation process, so that particles of widely varying sizes mixed in the sediment can be separated again, with the degree of mixing considerably reduced, but it is not possible to separate particles of a similar size, nevertheless. Therefore, if you need to get better resolution, you must use some other techniques.

1.3 Rate zone centrifugation

The rate zone centrifugation technique was originally proposed by Brakke (1951), and essentially the technique is very simple: a small suspension is placed on a gentle density gradient, and this gradient is used to stabilize the settling of the particles. As a result of centrifugation, the particles leave the starting zone and move, the rate of movement being determined by the size and shape of the particles and the centrifugal force they are subjected to. After centrifugation for a period of time, the various particles will separate into a series of zones each according to their relative speed of movement. In this method, particles with a difference in settling velocity of 20% or less can be separated without difficulty, so that rate zone centrifugation extends the range of separations possible with differential sedimentation.

As with most techniques, there were initially a number of problems that prevented its widespread use in the first decade. Rate zone centrifugation could not be performed in an angular rotor head because the sample would mix with the gradient liquid during acceleration of the rotor head, and, until now, the capacity of the throwaway rotor head was significantly limited. Since the width of the settling zone is as wide or wider than the starting zone, the volume of sample loaded into the rotary head is limited if a satisfactory separation is to be achieved. In addition, the concentration of material in the sample should not be too high or the entire band will mix with the solution at the top of the gradient. Although rate zone centrifugation was used to separate mitochondria and lysosomes by Thomson et al. shortly after the introduction of this technique, rate zone centrifugation was initially used primarily for analytical separations, such as to analyze the size, and distribution of a sample of polynucleosomes or the size and distribution of RNA. Improvements in rotor design, especially the introduction of zonal rotors, have greatly expanded the application of rate zone centrifugation, which is the main method of our application, and this method can also be used with vertical rotors.

1.4 Isodensity zone centrifugation

As Harvy first pointed out, subcellular particles differ not only in size but also in density. When a suspension containing live cells is placed on a solution of greater cell density, the cells form a band at the junction of the two solutions as a result of centrifugation. When examined under the microscope, the cellular contents appear to have been separated into several layers; this separation does not yet kill the cells, but if the speed of centrifugation is increased, the cells can be divided into two parts, with the nucleus together with the denser fragments. The nucleus can be further purified by floating it in an organic solvent, taking advantage of the difference in density between these two parts. Much later, work was done to purify the nucleus by settling it in a dense sucrose solution.

Most early separations involved suspending particles in liquids of selected densities, and centrifuging them to suspend particles smaller than the density of the medium and precipitate particles larger than the density of the medium. Segmented floatation separations were time-consuming and cumbersome, especially when separating more than one type of particle, so later researchers established isodensity gradient centrifugation. The suspension of particles to be separated is placed on a density gradient liquid, or indeed the particles are dissolved in a solution making a gradient, and by centrifugation the particles either float or sink to a liquid of the same density as they are. Here they have no weight, and no matter how long the centrifugation takes, they never move again. The particles become a series of zones, each in its own zone of density.

This technique was originally used to analyze ultracentrifuges, which had fewer problems than rate zone centrifugation, and was used by Beaufay et al. in 1959 to show that there was a group of enzymes involved in the synthesis and decomposition of hydrogen peroxide that settled with the enzyme during differential centrifugation, whereas it was actually bound to microsomal particles very different from the lysosomes (or peroxisomes).

Since then, isodensity gradient centrifugation has been widely used for the separation of biologics, however, one of its major drawbacks compared to rate zone centrifugation is the unavoidable exposure of the particles to high concentrations of the gradient solution during isodensity separation, which can cause damage to the particles and the possible emergence of a number of extra-ordinary zones equivalent to the damaged particles.

Membrane separation process uses selective permeable membrane as the separation medium, when there exists some kind of driving force on both sides of the membrane (e.g. pressure difference, concentration difference, potential difference, etc.), the components on the raw material side will be selectively permeated through the membrane in order to achieve the separation purpose. Usually, the raw material side of the membrane is called membrane upstream, and the permeation side is called membrane downstream. Different membrane processes use different membranes and have different driving forces. Membrane separation technology is mainly divided into microfiltration, ultrafiltration, nanofiltration, reverse osmosis.

The term "chromatography" was first coined by the Russian chemist Zwaiter in 1903. He used petroleum ether as a solvent on a white flag column to separate the pigments in green leaves into zones of different colors, which he named "chromatography". Later Martin and Synge published a paper on liquid partition chromatography in 1941, which laid the foundation for the development of liquid chromatography, gas chromatography, paper chromatography and thin-layer chromatography, for which they won the Nobel Prize. In our country in the 20th century after the 1950s carried out the work in this area, but the terminology used is not consistent. Chemical workers like to use "chromatography", and biochemical workers are accustomed to use "color layer method" or "chromatography". At present, the names of these methods have not been standardized in China.

Chromatography is a method used to separate various components of a mixture. A chromatographic system consists of two phases, a stationary phase and a mobile phase. When the mobile phase flows through the stationary phase spiked with a sample, the components are separated by moving at different speeds due to their different concentration ratios between the two phases. The stationary phase can be a solid or a liquid supported by a solid or gel. The stationary phase can be packed into a column, spread into a thin layer or coated into a film called a chromatographic "bed". The mobile phase can be a gas, called gas chromatography, or a liquid, called liquid chromatography.

3.1 Gel Filtration Chromatography

It is also known as exclusion chromatography, gel permeation, gel chromatography, molecular sieve chromatography and so on. It is a technique of liquid chromatography to separate by molecular size. It is mainly applied to component separation: desalting, replacing buffer, removing harmful reagents, purifying protein, peptide, polysaccharide and other biomolecules, calculating molecular size and homogeneity of molecules. It has the advantages of rapid buffer change, mild, high yield, any buffer system, removal of aggregates, separation by size, etc., but the disadvantages are low selectivity and limited sample volume. If you want to improve the resolution, then you can choose to detect the column efficiency, detect the separation, reduce the up-sample volume, reduce the flow rate, use smaller media particles of media, connect 2 chromatography columns and other methods.

3.2 Ion exchange chromatography

Ion exchange chromatography is a type of adsorption chromatography that separates molecules by differences in their charge. It is widely used for its suitability for all stages of purification and all scales of production, its controllability, its high selectivity, its high loading, its ability to concentrate samples and its high recovery. In addition, no ion exchange is perfect, and choosing the right ion exchange media is critical; different samples and different purification purposes require different ion exchange media. It is also worth noting that the sample should be treated before loading: removal of particulate matter (by centrifugation or filtration), adjustment of pH and ionic strength (by desalting or buffer exchange), unlimited volume of sample but less protein than the column load, and attention to the effect of nucleic acids (anion exchange).

3.3 Hydrophobic chromatography

Hydrophobic chromatography is a technique in liquid chromatography that separates biomolecules according to their hydrophobic nature, which is complementary to ion exchange, gel filtration and affinity chromatography. It has mild, non-denaturing conditions for purification; it is also a concentration technique; it is characterized by high selectivity and high yield.

3.4 Affinity chromatography

Affinity chromatography is a technique for separating biomolecules through specific interactions between them. It is a particularly easy-to-use method characterized by simplicity, high purity, and concentration of the sample. It is especially common to purify proteins because it is easy to use, one-step purification can result in purity greater than 95%, removal of specific impurities, and rapid separation. Widely used in the separation of monoclonal and polyclonal antibodies, fusion proteins, enzymes, DNA-binding proteins, any protein can bind its ligand.

In recent years, the application of advanced isolation and purification techniques in the preparation of traditional and new vaccines has been an effective means of improving vaccine efficacy and reducing side effects. At present, the widely developed viral subunit vaccines, polysaccharide vaccines for encephalitis, Haemophilus influenzae conjugate vaccines and nucleic acid (DNA) vaccines for various types of viruses, etc., all have greater or lesser differences in the methods of their development, composition and physical and chemical properties, as well as in the original sources, etc., and their isolation and purification methods have relative specificity.

Different routes of separation and purification should be chosen for different vaccines, but generally speaking, they all include two basic stages: primary separation and refined purification. The main task of primary separation stage is to separate the cells and culture fluid, break the cells to release the product (if the product is in the cells), concentrate the product and remove most of the impurities, etc., the separation methods available at this stage include cell crushing technology, centrifugal sedimentation, salting out and ultrafiltration concentration technology, etc.; Refined purification stage is to use a variety of high-resolution technology, so as to make the purpose of the protein and a small number of interfering impurities as far as possible to separate, to achieve the required quality standards, ultracentrifugation technology and a variety of chromatographic technology, to achieve the quality standards. In the purification stage, various techniques with high resolution are used to separate the target protein from the small amount of interfering impurities as much as possible, so as to achieve the required quality standard.

From the domestic and foreign new vaccine and genetic engineering products, in its separation and purification technology application development status can be seen:

①Membrane separation and ultracentrifugation purification technology has a broad development prospect in the purification process of vaccines, protein fractions, peptides and biomolecules. Vaccines prepared by this process have the purity and properties required by regulations, but the relatively low antigen recovery rate, cumbersome and long operation period, and high technical equipment requirements have been gradually replaced by the increasingly mature chromatographic technology in recent years, especially in the development of genetic engineering vaccines;

②Chromatographic separation technology is showing increasing benefits in terms of simplified operations, improved efficiency, energy savings and cost reduction. The introduction of chromatographic media with multifunctional matrices and fully automated high-efficiency chromatographic systems has broadened the range of applications and made it easier to implement standardization. Among the many chromatographic methods, affinity chromatography may become the most effective purification method for efficiently separating target proteins from low concentration cultures;

③Purification technologies for both traditional and new vaccines must be developed by combining related technologies from various disciplines, and no single technology has yet been able to undertake the entire process of isolation and purification alone. Innovative separation and purification processes are often the result of combining the strengths of several new and existing technologies. Traditional centrifugation, filtration and sedimentation technologies are more often used as the starting step of the whole vaccine separation and purification process, and are used in the preliminary separation process, and the combination of chromatography and sedimentation, centrifugation and other traditional separation technologies has gradually become the mainstream of vaccine separation and purification.