SUBSCRIBE

Breaking News on Contract Research, Manufacturing & Clinical Trials

Content Provided by Reading Scientific Services Ltd.

Reading Scientific Services Ltd.
Impurity isolation and sample purification

This content is provided by Reading Scientific Services Ltd.. Learn more

The opinions expressed within this Promotional Features article are those of Reading Scientific Services Ltd. and not necessarily those of Outsourcing-Pharma.com

Promotional Features gives industry stakeholders the opportunity to connect directly with you, our readers. They can contribute to the conversation on industry topics by providing content on Outsourcing-Pharma.com

For more information on Promotional Features, contact us here

Impurity isolation and sample purification

09-May-2016 Last updated on 09-May-2016 at 16:46 GMT2016-05-09T16:46:49Z

By Thibaut Monet-Dedreuil, Technical Specialist, RSSL

 

During a manufacturing process or a stability study for an Active Pharmaceutical Ingredient (API) or drug product, a sample may be found to contain impurities. These impurities might occur as degradation products, as intermediates/process impurities originating from the synthetic route, as an unwanted by-product of side-reactions or due to contamination of the original sample. Such impurities can arise at very low concentrations relative to the API, perhaps as little as 0.05% w/w, but it is important that when discovered they are isolated and identified. The ICH Q3 guidelines for Impurities in New Drug Substances (Q3A(R2)) and for Impurities in New Drug Products (Q3B(R2)) clearly state that degradation products observed during manufacturing and stability studies, conducted at the recommended storage conditions, should be identified when present at a level greater than the identification thresholds given in Tables 1 and 2.

 

Table 1: Thresholds for Degradation Products in New Drug Substances (Q3A(R2))

Maximum Daily Dose1

Reporting Threshold 2,3

Identification Threshold3

Qualification Threshold3

≤2 g

0.05%

0.10% or 1.0 mg / day   intake*

0.15% or 1.0 mg / day   intake*

>2 g

0.03%

0.05%

0.05%

1. The amount of drug substance administered per day

2. Higher reporting thresholds should be scientifically justified

3. Lower thresholds can be appropriate if the impurity is unusually toxic

*whichever is lower

 

Table 2: Thresholds for Degradation Products in New Drug Products Reporting Thresholds (Q3B(R2)

Maximum Daily Dose1

Reporting Threshold 2,3

Identification Thresholds 2,3

Qualification Thresholds 2,3

≤1 g

0.1%

-

-

>1 g

0.05%

-

-

<1 mg

-

1.0%* or 5 µg TDI*

-

1 mg -10 mg

-

0.5%* or 20 µg TDI*

-

>10 mg-2 g

-

0.2%* or 2 mg TDI*

-

>2 g

-

0.10%

-

<10 mg

-

-

1.0%* or 50 µg TDI*

10 mg - 100 mg

-

-

0.5%* or 200 µg TDI*

>100 mg - 2 g

-

-

0.2%* or 3 mg TDI*

> 2 g

-

-

0.15%

1. The amount of drug substance administered per day

2. Threshold for degradation products are either expressed as percentage of the drug substance or as total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degradation product is unusually toxic.

3. Higher threshold should be scientifically justified

*whichever is lower 

When the impurities are known impurities of the API, they can often be identified via retention time match and spectral or mass spectrometry (MS) confirmation. However, in most cases, when unknown degradation products are found, they require isolation and identification. On occasions, quantities as low as 5 mg can be sufficient for structural elucidation but it is preferable to isolate around 20 - 40 mg of impurity for full structural elucidation. 

 

Preparative LC

The technique best suited to isolating impurities is preparative liquid chromatography (LC), using low or high-pressure columns. The technique requires loading of a preparative-scale LC column with repeated doses of the sample, and collecting fractions either using known time intervals or mass-based fraction collection (using the mass of the molecule you are collecting). This allows isolation and concentration of sufficient impurity for identification purposes using highly sophisticated techniques, such as nuclear magnetic resonance (NMR) spectrometry, Fourier transform infrared spectroscopy (FT-IR) and MS,  to help identify the chemical impurity. Of course, there are some challenges with this approach. The process of isolating and concentrating the impurity is not always as straightforward as it may sound above. 

The specific choice of preparative LC method will rely on what is already known about the chemical structure of the drug molecule: its known or anticipated impurities, stability and solubility data, earlier used chromatographic methods and much, much more. This pre-knowledge will help determine what sort of chromatography might be best suited for the isolation. 

It is important to optimise the chromatography at an analytical scale before scale-up and increasing the flow rate for preparative LC. This can avoid wasting large quantities of expensive solvents, where required. When scaling up the chromatography from analytical to preparative LC, an increase in both the sample concentration and the flow-rate of the mobile phase is required. A sensible approach is to increase the sample concentration before increasing the mobile phase flow rate, and to optimise the selectivity and resolution between the peak of interest and other peaks at analytical scale. However, keep in mind that the sample loading capacity of any column must not be exceeded when performing these optimisation experiments. This allows you to scale up a method that is already optimised before using large quantities of solvent on a Prep LC. 

Clearly, the aim of this work is to enable sufficient resolution to minimise issues from co-eluting compounds and obtain as pure an impurity as possible. Hence, wherever possible, develop an LC method that does not use additives in the mobile phase. If this is not possible, the additive used must be easily removable. The mobile phase must also be able to solubilise the sample at the higher concentrations used for preparative LC, and be suitable for the NMR, MS or FT-IR analysis that will be used after isolation. Finally, the mobile phase will ideally be of low viscosity, to stop high back pressure building up in the system.

After performing the LC separation and fraction collection, the enriched or purified impurity has to be recovered from the mobile phase without degradation. This is not always straightforward as the impurity may be unstable under the conditions of isolation.

 

Performing the isolation  

It is good practise to investigate both the pooled fraction and the recovered material, so that should there have been any degradation of the unknown impurity during the recovery process, it can be detected. Thereafter, the recovered material can be subjected to the next LC separation or, following the final purification step, submitted to analytical investigation. Once again, the quality of solvents used for these separations is critical, especially if evaporation is to be applied for recovery. Impurities from the solvent could contaminate the isolated impurity, and present difficulties in the elucidation of its structure. The use of highly pure HPLC-grade solvents at the stage of final purification can help to alleviate the risk of such contamination. If solvent recycling is used, care must be taken to ensure collected fractions are not contaminated with co-eluting compounds. It is also important to ensure that the mobile phase solvent is volatile if evaporation is being used as the final isolation step. Water can often be successfully removed by salting out and evaporating the organic layer only, assuming the compound of interest is preferentially soluble in the organic media. This can dramatically reduce the evaporation time. 

After the impurity isolation is complete, it is possible to collect milligram quantities of material, which should be enough for the identification techniques mentioned above. It is also important to confirm the purity of the isolated material using high-resolution analytical columns (and more than one separation technique may be required) to confirm the purity of the isolated material.

 

Alternative use 

Preparative HPLC is also suitable for isolating reference standard-quality material. Again, this is performed by loading the sample onto the column and collecting fractions within a narrow band of the eluted sample peak, where the target molecule should be at its purest. Depending on the sample and size of column used, a few grams of reference standard can be produced in this manner. The identification techniques of NMR, FT-IR and MS can be used to aid in the characterisation of this material (along with other techniques such as Karl Fischer, HPLC, heavy metals, sulphated ash) and subsequently established as a Primary Reference Standard. Where biopharmaceutical molecules are purified, alternative techniques such as amino acid analysis, peptide mapping (using LC-MS), gel electrophoresis etc. may be used to confirm purity and identity. 

The approach is appropriate for small and large molecules, depending on the nature of the columns used, meaning it has applications for both traditional pharmaceutical and biopharmaceutical customers.

 

Conclusion 

Impurity isolation, identification and sample purification are essential aspects of pharmaceutical development, and these services are an important part of the support that RSSL can offer pharmaceutical customers, for both existing compounds and new Chemical/Biological Entities.

 

 

This article is written by/on behalf of Reading Scientific Services Ltd. and not by the Outsourcing-Pharma.com editorial team.

Get in touch with Reading Scientific Services Ltd.

Follow Reading Scientific Services Ltd.

Twitter
LinkedIn