Purification & Separation | Chromatography | HPLC

Overview of custom purification & separation technologies, chromatography, high performance liquid chromatography (HPLC), etc. for API development.

Q1. What is API purification?

Generally in the production of pharmaceutical drugs, a large part of the production costs stem from downstream processing. One important, time consuming and expensive downstream process in pharmaceutical manufacturing is active pharmaceutical ingredient (API) purification, or simply API purification.

Purification of organic compounds is an essential process as laboratory synthesized APIs, key intermediates, and other drug substances contain impurities such as organic impurities, inorganic impurities, and residual solvents.

- Organic Impurities

ICH classifies organic impurities as starting materials, by-products, key intermediates, degradation products, reagents, ligands and catalysts. These impurities usually arise during manufacturing and storage of drugs.

- Inorganic Impurities

Inorganic impurities are classified into reagents, legends, catalysts, heavy metals, residual metals, inorganic salts, and other materials (e.g. filter aids, charcoal). These impurities usually arise during the manufacturing process.

- Residual solvents

Residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products. Drug products should contain no higher levels of residual solvents than can be supported by safety data.

Separation & Purification Processes

In processes that use a series of chemical reactions for API or complex API custom synthesis, the removal of reaction by-products including colour bodies and metals is critical to produce high quality pharmaceuticals.

Separation and purification process, in chemistry, refers to the separation of a substance into its components (separation) and the removal of impurities (purification). There are a large number of applications of separation and purification technologies in fields such as medicines and manufacturing.

API separation and purification technologies are applied to remove unwanted by-products and residual products that arise during the API manufacturing and packaging processes. The preferred methods for removing residual metal catalysts are distillation, crystallisation and precipitation.

API separation and purification technologies for organic compounds are based on the nature of the compound and the impurity present in it. These processes, among others, are explored in detail in the next question.

Q2. What are the different methods of purification for synthetic APIs?

Common methods of purification for synthetic APIs, semi-synthetic APIs, complex APIs and other drug substances include crystallization, sublimation, distillation, chromatography, ultrafiltration, nanofiltration, etc. These API separation and purification processes are explored in detail below.

Methods of Purification for Synthetic APIs or Semi-Synthetic APIs:

1. Crystallization

The applications of crystallization in the pharmaceutical industry as a purification and separation technology is used for the isolation and synthesis of pure active pharmaceutical ingredients (API), semi-synthetic APIs, co-crystals, etc.

Crystallization is completed in the following steps; (1) Impure substances are dissolved in water or suitable solvents, (2) the solution is then filtered and the filtrate is heated in a water bath, (3) the hot solution is then cooled, so that the crystals (organic substances) separate out, and finally (4) the crystals are removed via filtration and the impurities are passed onto the filtrate.

Crystallization is one of the oldest API separation and purification unit operations, and has recently contributed to significant improvements in producing higher-value products with specific properties and building efficient manufacturing processes.

2. Sublimation

Sublimation and desublimation processes are applied to certain substances which tend to decompose or polymerize at temperatures above their melting point. It is a process by which a product is transferred from its solid state directly to its gaseous state by adding thermal energy, without passing through a liquid phase.

The reversal process is called de-sublimation. The most common application of sublimation and desublimation is the purification of active pharmaceutical ingredients or drug substances. Sublimation is usually used to separate volatile organic compounds from non volatile impurities, as volatile APIs or complex APIs and drug substances sublimate on heating, whereas nonvolatile impurities don't.

3. Distillation

Distillation can be operated in batch or continuous modes, in single or multiple steps, and can be run for a variety of objectives: separation, solvent swap, drying from water, purification from volatile or non-volatile impurities and crystallization. Distillation is simple, has few moving parts, is well understood, can be simulated readily, and often has a low capital cost.

Types of Distillation

- Simple distillation is a procedure by which two liquids with different boiling points can be separated. Simple distillation (the procedure outlined below) can be used effectively to separate liquids that have at least fifty degrees difference in their boiling points.

- Fractional distillation separates a complex mixture into a number of different parts, called fractions. Fractional distillation is usually used if the boiling point of liquids in a mixture are very close to each other, usually less than 40°C.

- During vacuum distillation, liquid contained in the processed material is vaporised under reduced pressure. This substance is extracted in the form of gas. It can then be re-liquefied, collected, recirculated, or processed with a scrubber or incinerator and then sent through the exhaust. Performing processes in a vacuum is faster and more energy efficient than conventional processes. At the same time, the product is not damaged due to high temperatures.

4. Chromatography

Chromatography is based on the principle that molecules in complex mixtures applied onto the surface of or into a solid, and fluid stationary phase (stable phase) separate from each other while moving with the aid of a mobile phase.

For difficult separations where tiny amounts of impurities need to be removed, purification primarily relies on chromatography systems in its various forms, with the most common being silica gel chromatography. Chromatographic systems are classified according to their stable phase and other types of chromatographic systems include alumina or reverse-phase chromatography.

- High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample complex mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). High Performance Liquid Chromatography (HPLC) is faster, has more surface area for interactions, and better impurity detection qualities as compared to conventional chromatographic systems.

5. Filtration

In filtration, a porous material or polymer membrane is used to separate particles of different sizes. If the pore sizes are highly uniform, separation can be fairly sensitive to the size of the particles, but the method is most commonly used to affect gross separations, as of liquids from suspended crystals or other solids.

- Ultrafiltration

Ultrafiltration uses a permeable polymer membrane to separate macromolecules based on the pore size (5–20nm) and their molecular weight cut-off (1000–100,000Da) at low transmembrane pressures.

- Nanofiltration

Like ultrafiltration, nanofiltration is based on size exclusion or particle capture and utilizes polymer membranes to separate particles. Nanofiltration (NF) rejects even smaller molecules than ultrafiltration, and has been used for water softening and removal of dissolved solids and viruses. The separation of molecules present in organic solvents by nanofiltration has great potential in industries ranging from refining to fine chemical and pharmaceutical custom synthesis.

Q3. What is chromatography and what are the different types?

Chromatography

Chromatography may be defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases. Chromatography is based on the differences in the rate at which components of a mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called mobile phase).

Chromatography of many kinds is widely used throughout the pharmaceutical industry. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (purification). Analytical chromatography, on the other hand, is done with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture.

The early stages of pharmaceutical drug development typically require chromatographic techniques that provide very high resolution. This is essential for early development as a relatively large number of process-related impurities, synthetic intermediates, and degradation products must be separated to characterize starting materials and products of chemical synthesis.

Some Types of Chromatography

1. Normal Phase Chromatography: Normal-phase chromatography (NPC) is a chromatographic type that uses a polar stationary phase and a nonpolar mobile phase for the separation of polar compounds.

2. Reverse Phase Chromatography: Reversed-phase chromatography (RPC) is a liquid chromatography technique that involves the separation of molecules on the basis of hydrophobic interactions between the solute molecules in the mobile phase and the ligands attached to the stationary phase.

3. Flash Chromatography: Flash chromatography is a process in which a vertical glass (column) is placed, and that column is packed with the solid stationary phase from which the mobile phase (liquid form) is passed through it by gravity or some external pressure.

Liquid Chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid, where sample ions or molecules are dissolved. It is carried out either in a column or a plane. Due to the differences in ion-exchange, adsorption, partitioning, or size, different solutes will interact with the stationary phase to different degrees, and therefore the separation of the compounds can be achieved.

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography, commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.

Column chromatography is one of the most common methods of protein purification. Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) are separated from each other while moving with the aid of a mobile phase (liquid). HPLC is an adaptation of column chromatography.

In HPLC, column chromatography is performed at a higher flow rate, and therefore higher pressure, than classical column chromatography. This enables the use of smaller stationary phase beads with a greater surface area to volume ratio, which greatly increases the interaction of the stationary phase and components in the mobile phase making separation more efficient and thorough.

Types of HPLC:

- Based on the mode of separation

Normal phase chromatography

Reverse phase chromatography

Partition chromatography

Displacement chromatography

- Based on principle of separation

Adsorption chromatography

Ion exchange chromatography

Size exclusion chromatography

Affinity chromatography

Chiral phase chromatography

Ion pair chromatography

- Based on elution technique

Isocratic separation

Gradient separation

- Based on the scale of operation

Analytical HPLC

Preparative HPLC

Q4. What are the process challenges associated with API separation and purification?

Some API Purification & Separation Challenges:

1. Purification Leading to Low Yields

Organic compounds are never too pure. When isolated from natural sources or created through organic reactions, they always contain other compounds. Purification is a critical step in drug manufacturing, which helps to eliminate unwanted materials that can be hazardous or compromise drug efficacy.

Purification of active pharmaceutical ingredients (purification of APIs) or compounds may at times lead to reduced quantities of final products. Having proper process controls and the support of an experienced scientific team can minimize such yield related challenges.

2. Identifying Impurities

Estimating the profile of organic impurities during API purification or purification of active pharmaceutical ingredients (purification of APIs) is a very challenging task. Various API purification and separation technologies have to be applied to do so. The latest challenge in drug-impurity proï¬ling is the determination of trace-levels of genotoxic and carcinogenic impurities.

3. Filtration Challenges

- Traditional Filtration Challenges

Traditionally, specialised filter housings such as those produced with an internal coating or made from exotic nickel alloys have been recommended for API manufacturing. However, neither of these options is ideal. Coatings are prone to mechanical damage and nickel alloys have limited resistance to corrosion by certain acids and some other chemicals. Moving to filter housings equipped to deal with difficult applications can solve such problems.

- Continuous API Filtration and Purification Challenges

Continuous custom synthesis and formulation are becoming established in the pharmaceutical industry, however connecting the two through continuous particle formation and isolation via filtration, washing and drying poses significant challenges.

4. Industrial Crystallization Challenges

One of the biggest challenges in the field of industrial crystallization is to match the changing requirements of controlling the crystal morphology, size distribution and polymorphism (in terms of product quality e.g., purity, filterability, flowability and reactivity) of drug substances.

Both, application-oriented and theoretical multi-disciplinary approaches have to be applied to solve the emerging issues that spread over fundamental aspects to commercial applications.

5. Chromatography Challenges

The main problem with chromatographic purification is its price, therefore chromatography systems can become quite expensive. Furthermore, there are scale-up challenges associated with High Pressure Liquid Chromatography (HPLC).

6. Distillation Column Challenges

Distillation columns are the primary equipment used in the industry to carry out the separation of organic chemicals. Distillation columns are known for their propensity to consume large amounts of energy, among other challenges. The main bottlenecks that hinder the separation process are: Fouling, mechanical issues, internal pressure reductions, and foaming.

- Other challenges associated with API separation and purification processes include the need to fast track APIs to market, remove residual metal catalysts, manage costs, etc.

Q5. How has the trend towards single-use technologies impacted API filtration and purification processes?

Single-use purification and isolation technologies include tubing, capsule filters, ion exchange membrane chromatography devices, mixers, bioreactors, product holding sterile bags in place of stainless steel vessels (sterile fluid containment bags), connection devices and sampling receptacles.

While single use technologies had limited applications a few years ago, these days, disposable components are becoming a more common feature of facility design from upstream to downstream processing. Single-use components reduce the risk of contamination, and can be less expensive, easier to use and provide more flexibility than traditional integrated purification and isolation technologies.

Today, these single-use purification and isolation technologies have been adopted across the upstream manufacturing process, downstream purification process, and through the fill-finish of entire classes of biologic drugs. Single-use technologies supply a number of values to any mode of bioprocessing, and can provide specific and enabling features to continuous bioprocessing implementations.

Many disposable chromatography columns meet cGMP and industry standards, and also offer reproducibility, scalability, speed, ease-of-use, and operational safety. Recent advances have made recycling and disposable chromatography systems possible at reasonable costs.

Single-use chromatography systems are designed for pilot scale, clinical production batches, and commercial production. As demand for single-use columns is rising, there is also an increasing interest towards a specialized system for single-use chromatography.

Single-use systems in general provide significant advantage over traditional stainless-steel systems for downstream processing. Some examples are API filtration and purification systems, buffer preparation systems, and sampling/dispensing systems. Key advantages of using single-use systems are speed of implementation, which is crucial for high-impact new product launches, and increased operational efficiency related to a reduction in the resources required for cleaning and setup.

Single-use technologies impact downstream processing methods, such as API filtration and purification, in the following ways:

- More flexibility

- Less up-front capital investment

- No batch-to-batch cross contamination

- No reuse cleaning validation

- Less work on scale up

- Accelerated time to market

Q6. Which are some of the leading CMOs offering API purification services?

There are numerous CMOs and CDMOs offering custom purification and separation services for APIs, complex APIs, intermediates, and other drug substances. Some of the leading contract service providers offering API separation and purification are listed below.

SANOFI Active Ingredient Solutions

Sanofi Active Ingredient Solutions cover both APIs sales and contract development and manufacturing. They provide expertise in quality, regulatory and supply and have dedicated commercial teams for customers around the globe.

SAIS is a therapeutic protein extraction and isolation service expert for custom purification & analysis of sophisticated proteins from natural origin raw materials. Their services include API protein extraction & isolation, and their biotechnological application capabilities include the production, extraction and custom purification of APIs & therapeutic proteins.

AMRI

Albany Molecular Research Inc. (AMRI) is a global contract research and manufacturing organization that has been working with the life sciences industry to improve patient outcomes and the quality of life for more than two decades.

AMRI's expert scientists routinely isolate materials of interest, including controlled substances and potent compounds, through classic and chromatography systems. Furthermore, AMRI’s inventory of chromatography systems for preparative applications (chiral and achiral) provides extensive screening options as well as quicker turnaround on processes.

Clearsynth Labs Ltd.

Clearsynth is committed to research in material science and provides high quality certified reference materials. Clearsynth services are applied at all stages of development. Clearsynth considers all issues related to final isolations and methods. It also offers custom purification and extraction services including solid phase extraction technologies.

Diverchim S.A.

Diverchim, an expert in organic chemistry, combines the services of a CRO for drug discovery and process development together with the services of a CMO to manufacture APIs and c-GMP excipients within a regulatory approved environment for 10’s g up to 10's kg.

Thanks to its innovative techniques (HPLC, SFC or combined preparative HPLC/SFC), Diverchim offers custom purification & isolation technologies to its customers and characterizes process impurities as well as reference compounds.