Original author: Kunpeng Xue. Senior Engineer, Welch Materials, Inc. Posted in Shanghai

Translated from Welch Materials, Inc. Wechat official account.

Original link: https://mp.weixin.qq.com/s/WD1H-Pv5FhxIuFKKQJYw9Q

1 Chiral chromatography packing “stuck neck” problem

As an independent manufacturer of chromatographic packing materials and columns in China, Welch Materials has been involved in the R&D and manufacturing of chiral chromatography packing materials and chiral columns for a long time, and has solved many difficult problems for customers in the pharmaceutical industry. For chiral packing materials, one of the key raw materials, amylose, has been used by a lot of domestic manufacturers and scientific research institutions. However, about five or six years ago, some imported amylose reagents were suddenly taken off the shelves in the Chinese market and were not sold in the market. As a result, we, like many other companies using amylose, had to face the lack of this Key Raw Materials for chiral packing materials. You may ask, how much role do chiral chromatography media and columns play in the separation? Can a small amylose reagent become a “stuck neck” problem? Well, for everyone’s doubts, listen to us slowly.

2 Necessity of R&D of chiral chromatography packing

For drug molecules and their intermediates, when their carbon atoms connect four different atoms or groups, they will show two spatial structures under the action of covalent bonds, just like objects in a mirror and outside the mirror, The fact that an object and its mirror image cannot be completely coincident is called chirality (from the Greek word cheir, which means hands, they are like a pair of hands), as shown in Figure 1.

Figure 1: Chemical Structures of Typical Chiral Compounds Amino Acids

Chirality is an attribute of nature and one of the essential attributes of nature on which human beings depend.

The chemical processes in life phenomena are carried out in a very asymmetric environment, and biological macromolecules such as proteins, nucleic acids and polysaccharides all have chirality.

For example, proteins other than bacteria are composed of left-handed L-amino acids; sugars in polysaccharides and nucleic acids are in the right-handed D configuration.

In many cases, there are significant differences in the physiological activities, toxicity and metabolic processes of chiral compounds. Thalidomide (thalidomide) is one of the typical cases. It was widely used in the treatment of pregnant women with adverse pregnancy in the 1960s, but it was later found that it can also cause fetal malformations in pregnant women. A large number of experimental results show that , the (R)-(+)-thalidomide of tertiary has sedative and hypnotic effects, while (S)-(-)-thalidomide not only has no such effect but has teratogenic effects on the fetus; another example is the (S)-configuration Ibuprofen (Ibuprofen) is a highly effective non-steroidal antipyretic analgesic, while (R)-configuration ibuprofen has basically no pharmacological activity.

Because of this, as early as 1992, the U.S. Food and Drug Administration (FDA) issued relevant regulations on the research and preparation of chiral drugs, requiring that the pharmacological activity of a single enantiomer and the full reports of pharmacokinetic experimentsmust be provided when submitting application documents for chiral drugs. The urgent need for chiral drugs in the market and new regulations formulated by drug regulatory authorities continue to stimulate the research and development and preparation of chiral drugs. The global pharmaceutical market is expected to reach USD 1,711.4 billion by 2025[1] Sexual new drugs account for about 60% of small-molecule chemical drugs. The huge market and broad development space have attracted much attention to chiral separation technology, and chiral recognition and resolution have become the hotspot of current scientific research.

Figure 2: 2016-2025 global pharmaceutical market size forecast trend chart

In view of this, the precise resolution of chiral compounds is of great significance for pharmaceutical analysis.

At present, the resolution methods of chiral compounds mainly include chemical resolution method, enzymatic or microbial method and chromatographic resolution method. Among them, chromatography refers to the use of the different interaction forces between two single enantiomers in a racemic compound and a chiral stationary phase (CSP) to cause differences in their retention times in the chromatographic system. Finally, chiral separation is achieved.

The basic principle of chiral chromatographic separation is based on the “three-point interaction” theory proposed by Professor Dalgliesh in 1952. According to this theory, between a pair of enantiomers and a chiral selector, in order to achieve chiral separation by forming diastereomeric molecular complexes with different stabilities, the presence of at least three simultaneous intermolecular interaction forces is required. Moreover, at least one of the three-point forces must be a stereochemical interaction, and the requirement of “three-point interaction” inevitably considers mutual chiral recognition from the three-dimensional spatial structure.

Chromatography has strong chiral separation performance, and the biological activity of chiral compounds can remain unchanged after chromatographic separation. As the core of the chromatographic column for separating chiral compounds, the development of chiral packing materils undoubtedly plays a vital role in improving the selectivity of chiral chromatographic separation and realizing the separation of chiral compounds.

The chiral nature and availability of many natural products, especially carbohydrates, have long been recognized as materials for the separation of chiral enantiomers. Polysaccharide compounds are optically active natural polymers that are abundant in nature. Among the CSPs currently being developed and used, polysaccharide derivatives CSPs have gradually become the most widely used and most widely used due to their wide recognition range, high loading capacity and rich sources. One of the valid CSPs.

Polysaccharide chiral packing materials mainly include cellulose, amylose and cyclodextrin and polymers based on their derivatives, which have excellent chiral recognition due to their highly ordered helical structure. The ability of polysaccharide derivatives to directly resolve enantiomers is more and more widely used in liquid chromatography. Table 1 shows the specific classification of chiral chromatography packings. And its derivative CSP occupies a pivotal position in chiral fillers.

Table 1: Classification of chiral chromatographic separation materials

At present, the chiral stationary phases of polysaccharide derivatives are the most widely used chiral stationary phases of cellulose and amylose phenylcarbamate derivatives. Among them, the research group of Professor Okamoto of Osaka University in Japan has the most in-depth research on it.
As early as 1984, the research group of Okamoto[2,3] applied cellulose derivatives on the surface of silica gel by physical methods to form a polymer-coated silica gel film, which overcome the poor mechanical stability of microcrystalline cellulose triethyl ester (MCTA) as a CSP. In the subsequent work, Professor Okamoto’s research group synthesized a series of cellulose and amylose derivatives, and uniformly coated them on silica gel to obtain CSP, which completely solved the low mechanical strength and poor stability of chiral packing materials.
So far, the most frequently used polysaccharide chromatographic columns on the market are all from a famous Japanese chiral column company. The specific models are normal phase AD-H, AS-H, OD-H and OJ-H, reversed-phase AD-RH, AS-RH, OD-RH and OJ-RH, it can probably solve the problem of separation of about 90% of chiral compounds on the market, showing a very excellent separation effect.

So far, the global chiral chromatography packing material is basically monopolized by this Japanese chiral chromatography column company. When other conventional reversed-phase C18 columns are sold for only a few thousand yuan each( 400-500USD) , one chirla column contains about 3 grams of chirality packing materials can be sold exceeds 10,000 yuan(1600USD). Scientific research institutions and companies conducting asymmetric organic synthesis spend a lot of money on the purchase of chiral chromatographic columns every year.

In addition, if it is necessary to use a chiral preparative column for separation and purification to obtain a single enantiomeric target product, it will cost more, because the price of a chiral preparative column often exceeds 100,000 yuan(16000USD). For chromatographic packing materials manufacturers, one kilogram of chiral chromatographic packing materials can often be sold for several million yuan after being packed into a column, and the added value of its products is very high.

However, the life of chiral chromatographic columns is far less than that of conventional columns, especially the coated chiral separation materials, which are particularly “delicate”! It is precisely because of the high profits of chiral chromatographic packing materials that many chromatographic companies and scientific and technological elites in the world have challenged these technologies. However, due to the high technical barriers of chiral chromatographic separation and the huge difficulty of product industrialization, it has not been able to shake the monopoly position of this Japanese chiral chromatographic packing company.

3 Localization of Chiral Chromatography Packings – The Only Way to Solve the “Stuck Neck” Technology

Polysaccharide-based chiral chromatography packing materials are mainly made by coating or bonding polysaccharides with chiral recognition sites on fully porous silica microspheres, such as cellulose, amylose and cyclodextrin and other high molecular polymers. In order to achieve the purpose of enantiomeric separation, the coated or bonded cellulose and amylose must maintain a chiral structural environment, so that there is a “three-point interaction” between the enantiomers and the chiral separation material.

Unfortunately, the chiral structures of polysaccharide derivatives such as cellulose, amylose, and cyclodextrin are easily destroyed during coating or bonding. Therefore, the preparation of polysaccharide chiral chromatography packing materials is not only important for silica microspheres. The requirements for the substrate are high, the requirements for the coating or bonding process are often higher, and the structure of the polysaccharide itself, such as particle size, degree of polymerization, molecular weight and derivative functional groups, have extremely high requirements. Therefore, The technical barriers for the preparation of polysaccharide chiral chromatographic fillers are extremely high. At present, the chiral separation fillers and chromatographic columns in the Chinese market are mainly imported brands, and few domestic manufacturers can prepare them on a large scale.

China has the world’s largest chromatographic technology research and application team. The SCI papers we published using chromatographic technology have surpassed the United States as early as 2011 and ranked first in the world.

Unfortunately, most of the chromatographic packing materials and columns used in these scientific research papers are imported brands. The main reasons are the high technical barriers of chromatographic packing materials, the long industrialization cycle, and the extremely high requirements for the stability and reproducibility of the material itself. Since chromatographic technology is mainly used for accurate qualitative, quantitative or separation and purification of some high value-added targets, a very real problem is that the value of customer samples is often much higher than the value of a small column chromatography column, and, Once a deviation occurs, the customer needs to conduct a large number of OOS (Obviousness of Inspection Result) deviation investigations, which is time-consuming and labor-intensive. Therefore, customers are easily afraid to change the brand of chromatographic packing materials and chromatographic column.

Starch is a kind of widely distributed polysaccharide and is also composed of D-(+)-grape units. Its structure is more complex than that of cellulose. The chemical structure is shown in Figure 3. Starch is composed of about 20% amylose (amylose) and 80% amylopectin (amylopactin), but amylose is a linear polymer, and amylopectin is a branched polymer connected by C1-C6.

Figure 3: Chemical Structure of Starch

For chiral packing materials, especially amylose-modified chiral packing materials , the biggest challenge is not the performance of the matrix silica microspheres, but the physical and chemical properties and coating or bonding process of the key raw material-amylose itself, because the resolution mechanism of polysaccharide chiral packing materials still mainly relies on “three-point interaction”, and the parameters (such as particle size, degree of polymerization, molecular weight distribution) of amylose with chiral recognition function will definitely affect the final resolution effect.

At present, more than 90% of the chiral column market in the Chinese market is monopolized by a Japanese company, and their chiral columns have even become the world standard. They even clearly write “World Standard for Chiral Column Separation” in the product information. This is something to be very proud of! However, this does not mean that domestic chromatographic column companies cannot approach or reach the products of these imported chiral chromatographic packing materials companies after years of technical accumulation.
Especially in today’s environment where China is facing the “stuck neck” technology of developed countries, it is even more necessary for our domestic technology companies to do a good job in product research and development and technical exploration. Take the key raw materials of chiral packing materials discussed in this article- amylose for example, although polysaccharides such as cellulose and amylose are very common substances, there are many types of amylose on the market, however, after in-depth research, it was found that only a Japanese company’s amylose raw material can meet the needs of chiral packing materials in the world, and its price is beyond the imagination of ordinary people. The price of amylose per kilogram is as high as 600,000 yuan(100,000USD). Moreover, it is a pity that the amylose starch has not been sold in the Chinese market in recent years, which has led to the fact that some domestic companies and research teams that make chromatographic packing materiasl are faced with the situation that no raw materials are available, which greatly affects the progress of scientific research. We also have found that even if the amylose raw material supplied by the company is used, the chiral packing materials produced cannot achieve the separation effect of the chiral packing materials of the Japanese company mentioned above. This once again proves that the key high-tech products cannot be bought by buying them. It must be done by many domestic researchers to conduct research on the ground and make breakthroughs step by step.

From the development of chiral separation materials, we can find that this Japanese chiral packing materials company has an amazing degree of control over the upstream and downstream industry chain and its core raw materials. This is also an important reason why other companies around the world have not been able to shake the monopoly position of this Japanese company in chiral materials for so many years.

Relevant data show that Japan’s manufacturing industry has always been at the forefront of the world, with more than 60% of the global share in 256 fields. Among the top 100 global innovation companies in 2018, there are 39 Japanese companies. For example, Fanuc, founded in 1956, the yellow giant at the foot of Mount Fuji, is the world’s largest robot company and the world’s largest professional CNC system manufacturer, accounting for 70% of the global market share.

On the surface, it seems that many Japanese companies are withdrawing from certain markets, but in fact these companies are transforming and upgrading to more sophisticated fields. Japanese companies have strong strength in the upstream field of the product manufacturing process, mastering key products and technologies such as chemical and electronic materials, components, precision equipment and instruments, occupying a dominant position in the upstream of the industry chain, and being the undisputed “hidden champion” !

The reason why Japan controls the key materials and technologies of many industries is not because they are smarter than people in other countries, but because the Japanese have enough patience, they have a spirit of perseverance and their craftsmanship of excellence, which enables them to put advanced Technology is the ultimate, and this is what we should learn from the Japanese.

Welch Materials conducts research based on the specific needs of chiral separation in response to the “stuck neck” problem of amylose. At the beginning, we also constantly questioned whether we could solve this problem. So many chromatographers in the world could not solve the problem of amylose raw materials suitable for chiral fillers. Why can Welch Materials solve it? We have encountered too many setbacks over the years, we have also cooperated with many well-known scientific research institutions and universities in China, thinking about relying on external forces to overcome this problem, but without exception, we have failed.

In the end, there is really no way, we can only explore step by step to solve this problem, although almost no one in our team is familiar with the knowledge of food chemistry and amylose. However, with the attitude of “stupid birds fly first” and learn the “craftsman spirit” of developed countries such as Germany and Japan, our R&D team conductss various experimental schemes to test and explore and finally, after years of continuous efforts, developed a amylose which is suitable for chiral packing materials. (as shown in Figure 4). to be honest, when we saw the perfect result, our entire R&D team was stunned, and there was such a strong feeling of “happiness came too suddenly”!

This experience also tells us profoundly: even in the small chromatography industry, what developed countries such as Europe, America, and Japan can do well, we can do it well, even if we are a little stupid and slow, but as long as we persist unremittingly, never give up to climb the peak of chromatographic separation material research, there is nothing impossible to achieve! The overcoming of this “stuck neck” problem has also made our entire team realize: to do a seemingly trivial and very common thing well; to actually solve a very common problem is the biggest innovation! Innovative things must serve human beings, must give full play to its value, and truly contribute to the country and the people.

Just like a case that we are all familiar with in the past few years: in the manufacture of ballpoint pens, China has become a well-deserved pen-making country, producing tens of billions of pens every year, but behind a series of proud numbers, it is the core technology and materials are highly dependent on Import, a large number of “balls” and “ball seat” of the ballpoint pen tip still need to be imported, whether it is production equipment or raw materials, it has long been in the hands of Switzerland, Japan and other countries. However, after a lot of efforts, China’s related industry companies and scientific research institutions finally broke the monopoly of imports and achieved great success! Therefore, more and more cases now show that as long as Chinese enterprises are based on scientific and technological innovation and overcome difficulties one by one down-to-earth, we will certainly be able to contribute to the construction of the motherland, and the Chinese nation will surely achieve great rejuvenation!

Figure 4: The chiral filler developed by Welch Materials
Core raw material amylose sample map

The ability of Welch Materials to break through the production technology of amylose chiral chromatography packing materials fully demonstrates the importance of perseverance and perseverance. No matter how far-fetched the goal may seem, as long as we follow the spirit of “the foolish man moving mountains”, it is possible to achieve it. What does it matter if we are stupid?

With this persistent spirit, the R&D team of Welch Materials broke through the “stuck neck” problem of industrial preparation of the key raw material amylose for chiral packing materials, completely solved the problem of independent supply of amylose, and solved the problems of uneven coating process and unstable column packing resulted in the production of high-performance amylose chiral chromatographic packing materials and chromatographic column. At present, we can not only provide high-performance amylose chiral chromatography packing materials, but also provide a complete set of solutions for customers who need chiral preparation and purification. We also have the production capacity of chiral packing materials from milligrams to kilograms or even 100 kilograms.

Figure 5 shows the four coating chiral separation materials developed by Welch Materials based on cellulose and amylose, and tested according to the quality inspection standards of Japanese import companies. Their separation performance is basically close to the separation performance of Japanese companies. The resolution reaches 18-20 (take trans-stilbene oxide as an example), and the chromatogram of its separation effect is shown in Figure 6. Figure 7 is tchromatograms of the Welch chiral column applied to the resolution of some chiral compounds. It can be clearly seen from the chromatograms that the separation effect is excellent, and the baseline separation is achieved.

Cellulose derivatives:
High-purity spherical silica gel coated with cellulose derivatives

Amylose derivative type:
High-purity spherical silica gel coated with amylose derivatives

Figure 5: Four types of coated columns developed by Welch Materials
Figure 6: Standard test chromatogram of Welch amylose chiral column

Mobile phase: n-Hexane/Isopropanol=90/10; Fow rate: 0.5ml/min; Temperature: Ambient (Nominally 23℃); Detector: 254nm; Injection Volume: 10µl

Figure 7: Separation result of some chiral racemates on Welch chiral columns

Welch Materials Chiral Columns Product Information

Order information

SpecificationCellu-DAmy-DCellu-JAmy-SCellu-DRAmy-DRCellu-JRAmy-SR
5μm,4.6×150mmH00219-31041H00221-31041H00218-31041H00220-31041H00262-31041H00264-31041H00261-31041|H00263-31041
5μm,4.6×250mmH00219-31043H00221-31043H00218-31043H00220-31043H00262-31043H00264-31043H00261-31043H00263-31043
5μm ,10×150mmH02682-21101H02684-21101H02681-21101H02683-21101H02686-21101H02688-21101H02685-21101H02687-21101
5μm ,10×250mmH02682-21102H02684-21102H02681-21102H02683-21102H02686-21102H02688-21102H02685-21102H02687-21102
5μm,21.2×150mmH02682-21105H02684-21105H02681-21105H02683-21105H02686-21105H02688-21105H02685-21105H02687-21105
5μm,21.2×250mmH02682-21106H02684-21106H02681-21106H02683-21106H02686-21106H02688-21106H02685-21106H02687-21106

References

  • China Business Information Network. https://www.askci.com/news/chanye/20211224/1015011701874.shtml
  • Ichida A., Shibata T., Okamoto I., et al. Resolution of enantiomers by HPLC on cellulose derivatives [J]. Chromatographia, 1984, 19(1): 280-284.
  • Okamoto Y., Kawashima M., Yamamoto K., et al. Chromatographic resolution .6. useful chiral  packing materials of high-performance liquid-chromatographic resolution –cellulose triacetate and tribenzoate-and tribenzoate coated on macroporous silica-gel[J]. Chem Lett, 1984, (5): 739-742.

Author brief introduction

Xue Kunpeng
Director of R&D at Welch Materials

Master, senior engineer in material chemistry, with 15 years of scientific research and work experience in the field of chromatographic separation materials, long-term based on the design, synthesis and preparation of various chromatographic separation materials according to the physical and chemical properties of the target, especially based on the stability of chromatographic separation materials and reproducibility studies, and have unique knowledge and insights into chromatographic separation methods.

At present, he has applied for 11 Chinese invention patents in the field of chromatographic separation materials, of which 8 are authorized and 6 are the first inventors. He has published more than 30 papers in various SCI and Chinese core journals, and undertakes more than 10 pcs pf national, provincial and municipal scientific research projects in the field of high-grade chromatographic separation materials.

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