01 Overcoming the “Neck-Blocking” Issue in Chiral Chromatography Packing

As a domestic manufacturer specializing in the independent production of chromatography packing and columns in China, Welch has long been involved in the research and development, as well as the manufacturing, of chiral chromatography packing and columns. We have successfully addressed numerous challenging issues faced by clients in the pharmaceutical industry. Regarding chiral chromatography packing, one crucial raw material reagent stands out – linear amylose. Most domestic manufacturers and research institutions have traditionally relied on imported reagents for linear amylose. However, about five or six years ago, certain imported linear amylose reagents were suddenly taken off the shelves in the Chinese market, ceasing sales. This situation left us and many other companies utilizing linear amylose in a predicament where the key raw material for this chiral packing was no longer available. You may wonder, how significant is the role of chiral chromatography packing and columns in the separation process? How can a seemingly minor issue with linear amylose reagents become a “neck-blocking” problem? Well, let us unravel the mystery for you.

02 The Necessity of Chiral Chromatography Packing Development

For drug molecules and intermediates, when a carbon atom is bonded to four different atoms or groups through covalent bonds, two distinct spatial structures can emerge. This is akin to an object inside and outside a mirror, where the object and its reflection cannot perfectly overlap. This phenomenon is known as chirality, derived from the Greek word “cheir,” meaning hand, as these structures resemble a pair of hands, as illustrated in Figure 1.

Chirality is a fundamental attribute of the natural world, constituting one of the essential aspects of the environment upon which humanity depends for survival. Chemical processes in biological phenomena occur in highly asymmetric environments, and large biomolecules such as proteins, nucleic acids, and polysaccharides all exhibit chiral characteristics. For instance, proteins, excluding bacteria, are composed of left-handed L-amino acids, while sugars in polysaccharides and nucleic acids adopt a right-handed D-configuration. In many cases, significant differences exist in the physiological activity, toxicity, and metabolic processes of chiral compounds. Thalidomide (thalidomide, also known as Contergan) is a classic example. Widely used in the 1960s to treat adverse pregnancies in women, it was later found to cause fetal deformities. Numerous experimental results indicate that the (R)-(+)-thalidomide isomer exhibits sedative and hypnotic effects, while the (S)-(-)-thalidomide not only lacks these effects but also has teratogenic effects on the fetus.

Another example is the chiral compound ibuprofen, where the (S)-configuration is an effective nonsteroidal anti-inflammatory analgesic, while the (R)-configuration is essentially pharmacologically inactive. Due to such differences, as early as 1992, the United States Food and Drug Administration (FDA) issued regulations for the research and preparation of chiral drugs, requiring comprehensive reports on the pharmacological activity and pharmacokinetics of individual enantiomers when submitting applications for chiral drugs. The urgent demand in the market for chiral drugs and the continuous implementation of new regulations by drug regulatory authorities stimulate the research and preparation of chiral drugs. It is estimated that by 2025, the global pharmaceutical market will reach $1.7114 trillion, with approximately 60% of small molecule chemical drugs being chiral new drugs with a single enantiomer. This vast market and expansive development space have garnered significant attention for chiral separation technology, making chiral recognition and resolution a current hotspot in scientific research.

Given this, the precise separation of chiral compounds holds significant importance in pharmaceutical analysis. Currently, methods for chiral compound separation include chemical methods, enzymatic or microbial methods, and chromatographic methods. Among these, chromatography refers to the utilization of the differential interaction forces between the two enantiomers of a chiral compound and a chiral stationary phase (CSP), causing differences in their retention times within the chromatographic system, ultimately achieving chiral separation. The fundamental principle of chiral chromatographic separation is based on the “three-point interaction” theory proposed by Professor Dalgliesh in 1952. According to this theory, to achieve chiral separation between a pair of enantiomers and a chiral selector, at least three simultaneously occurring intermolecular forces are required to form complexes of non-enantiomeric isomers with different stabilities. Furthermore, at least one of the three interaction forces must involve stereochemical interactions. The necessity of the “three-point interaction” inevitably involves considering the mutual chiral recognition in three-dimensional space.

Chromatography exhibits strong chiral separation capabilities, and the biological activity of chiral compounds remains unchanged after chromatographic separation. As the core of the chromatographic column used for separating chiral compounds, the development of chiral chromatography packing undoubtedly plays a crucial role in enhancing the selectivity of chiral chromatography separation and achieving the separation of chiral compounds.

Table 1: Classification of Chiral Chromatography Separation Materials

Currently, polysaccharide-derived chiral chromatography packing, particularly cellulose and linear amylose benzoate ester derivatives, is widely used. Among these, the research led by Professor Okamoto’s group at Osaka University in Japan has been particularly extensive. As early as 1984, Okamoto’s group[2,3] applied cellulose derivatives by physically coating them onto the surface of silica gel to form a polymer-coated silica gel membrane, overcoming the mechanical instability issue of microcrystalline cellulose triacetate (MCTA) as a CSP. In subsequent work, Professor Okamoto’s group synthesized a series of cellulose and linear amylose derivatives, uniformly coating them onto silica gel to produce CSP, completely solving problems such as low mechanical strength and poor stability of chiral packing. Up to now, the most widely used in the market is the “Four Kings,” a series of polysaccharide-derived chiral chromatography columns from a well-known Japanese company. The specific models include the normal phase columns AD-H, AS-H, OD-H, and OJ-H, and the reversed-phase columns AD-RH, AS-RH, OD-RH, and OJ-RH. These columns can address the separation of approximately 90% of chiral compounds in the market, demonstrating exceptional separation efficiency.

So far, the global market for chiral chromatography fillers is essentially monopolized by this Japanese chiral chromatography column company. While other conventional reverse-phase C18 chromatography columns are priced at only a few thousand Chinese yuan each, a column containing around 3 grams of chiral filler can cost over 10,000 Chinese yuan. Domestic research institutions and companies engaged in asymmetric organic synthesis incur substantial annual expenses in procuring chiral chromatography columns. Moreover, if a chiral preparative column is needed for the separation and purification to obtain a single enantiomer target product, the cost is even higher, often exceeding 100,000 Chinese yuan per column. For manufacturers of chromatography fillers, producing one kilogram of chiral chromatography filler, when assembled into columns, can often be sold at prices reaching millions of Chinese yuan, reflecting a remarkably high added value for their products.

Despite these high profits associated with chiral chromatography fillers, their lifespan is significantly shorter compared to conventional chromatography columns, especially for coated types of chiral separation materials, which are notably delicate. The substantial profitability of chiral chromatography fillers has prompted numerous chromatography companies and technological experts worldwide to challenge these technologies. However, due to the high barriers and considerable difficulty in industrializing chiral chromatography separation technology, they have been unable to disrupt the monopoly position held by this Japanese chiral chromatography filler company.

Chiral Chromatography Filler Localization – The Necessary Path to Overcoming Technological Bottlenecks

Polysaccharide-based chiral chromatography fillers are primarily developed by coating or bonding polysaccharides such as cellulose, amylose, and cyclodextrin onto porous silica microspheres. In order to achieve the separation of enantiomers, the coated or bonded cellulose and amylose must maintain a chiral structural environment, enabling a “three-point interaction” between enantiomers and chiral separation materials. Unfortunately, the chiral structure of polysaccharide derivatives, including cellulose, amylose, and cyclodextrin, is prone to damage during the coating or bonding process. As a result, the preparation of polysaccharide-based chiral chromatography fillers not only demands high-quality silica microsphere substrates but also imposes stringent requirements on the coating or bonding process. Additionally, there are high demands for the intrinsic characteristics of polysaccharide substances, such as particle size, polymerization degree, molecular weight, and functional groups of derived derivatives.

The technological barriers to the preparation of polysaccharide-based chiral chromatography fillers are exceedingly high. Currently, the Chinese market for chiral separation fillers and chromatography columns is predominantly dominated by imported brands, with very few domestic manufacturers capable of large-scale production. Despite having the world’s largest chromatography research and application teams, China’s extensive publications on chromatography technology in SCI journals have surpassed the United States since 2011, making it the global leader. However, the majority of these research papers rely on imported brands of chromatography fillers and columns.

This prevalent reliance on imports is mainly due to the high technological barriers, lengthy industrialization cycles, and stringent requirements for the stability and reproducibility of the chromatography filler materials. Chromatography technology is primarily used for precise qualitative, quantitative, or purification of certain high-value target substances. A practical challenge arises as the value of customer samples often far exceeds the value of a single chromatography column. Moreover, any deviation could lead to extensive Out-of-Specification (OOS) investigations, consuming time and resources. Consequently, customers are reluctant to easily switch brands of chromatography fillers and columns.

Starch is a widely distributed polysaccharide composed of D-(+)-glucose units. Its structure is more complex than cellulose, as illustrated in Figure 3. Starch consists of approximately 20% amylose, which is a linear polymer, and 80% amylopectin, which is a branched polymer connected by C1-C6 linkages. Amylose is a linear polymer, while amylopectin exhibits a branched structure due to the C1-C6 connections.

Figure 3: Chemical Structure of Starch

Welch addressed the formidable challenge of obtaining straight-chain starch as a raw material, a bottleneck in chiral separation, by focusing on the specific needs of chiral separation during our research and development efforts. Initially, we constantly questioned our capability to solve this problem. With numerous chromatography colleagues globally unable to find suitable straight-chain starch materials for chiral fillers, why should Welch succeed? Despite facing many setbacks over the years and collaborating with renowned domestic research institutions and universities, all attempts to overcome this challenge with external support ended in failure.

Ultimately, with no viable alternatives, we had to embark on a self-driven exploration to solve this problem. Despite our team’s limited familiarity with knowledge in the field of food chemistry, and lacking profound understanding of straight-chain starch, our R&D team adopted a “slow and steady wins the race” mentality. Inspired by the craftsmanship spirit of developed countries like Germany and Japan, we experimented with various trial-and-error approaches. After years of continuous effort, we successfully developed straight-chain starch suitable for chiral material production, as depicted in Figure 4. To be honest, the moment we witnessed the near-perfect results, the entire R&D team was astonished, experiencing an intense and sudden sense of “happiness coming too unexpectedly.”

This experience profoundly taught us that even in the niche field of chromatography, where developed countries like Europe, America, and Japan excel, we can achieve the same level of success. Even if we may be a bit slower or less sophisticated, as long as we persist unwaveringly and never give up on climbing the peak of chromatography separation material research, there is nothing impossible to achieve. Overcoming this bottleneck challenge made our entire team realize that excelling in seemingly trivial and ordinary tasks, and genuinely solving commonplace problems, is the essence of true innovation!

Image 4: Sample image of the core raw material, linear amylose, developed by Welch for chiral fillers.

The ability of Welch Materials to overcome the production challenges of chiral chromatography fillers based on linear amylose clearly demonstrates the importance of perseverance and unwavering commitment. Even when the goal seems distant, as long as we embody the spirit of the Foolish Old Man moving mountains, there is the possibility of achievement. Welch’s R&D team, driven by this persistent spirit, successfully tackled the bottleneck issue in the industrial preparation of the crucial raw material, linear amylose, for chiral fillers. They completely resolved the problem of self-supply of linear amylose, addressing issues such as uneven coating processes and unstable column packing, ultimately producing high-performance linear amylose chiral chromatography fillers and columns. Currently, we can not only provide high-performance linear amylose chiral chromatography fillers but also offer a complete solution for customers with chiral purification needs. We also have the production capacity for chiral fillers ranging from milligrams to kilograms or even hundreds of kilograms. Figure 5 depicts four coating-type chiral separation materials developed by Welch based on cellulose and linear amylose, tested against the quality standards of a Japanese import company. Their separation performance is essentially on par with that of the Japanese company, with separation factors reaching 18-20 (using trans-Stilbene oxide as an example). The chromatograms illustrating their separation effects are shown in Figure 6. Figure 7 demonstrates the separation results of Welch’s chiral chromatography column applied to the separation of some chiral compounds, clearly exhibiting outstanding separation effects, all achieving baseline separation.

Cellulose derivative type:

Spherical silica gel surface coated with cellulose derivative.

Linear Amylose Derivative Type:

Spherical silica gel surface coated with linear amylose derivative.

Image 5: Four types of coated chromatography columns developed by Welch

Image 6: Standard test chromatogram of Moonlight’s linear amylose chiral chromatography column

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

Image 7: Separation effects of Moonlight’s chiral chromatography column on some chiral racemates.

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