Troubleshooting Low Potency Results in Solid Oral Dosage Forms: Is it the

Method or the Formulation?

SAMPLE PREPARATION/EXTRACTION

Carlos Lee Research Analytical, Pfizer Global Research and Development

Keywords: Accelerated Solvent Extraction, ASE, Microwave Assisted Extraction, MAE, Extraction, Sample

Preparation, Automated, Spray Dried Dispersion, Low Potency Tablet

Abstract

Low potency values are common occurrences in drug product HPLC assays. Considerable time and resources are

often committed to determine if the low potencies are method or formulation related. The need for increased

business efficiency throughout the drug development process requires analysts to develop effective strategies to

quickly resolve or eliminate the method vs formulation issue. Numerous tools are available to help rapidly

troubleshoot and resolve the method vs formulation issue. These tools, if used appropriately,

can provide rapid resolution to the method vs formulation debate and can also aid in the development of

robust/rugged analytical methods for solid oral dosage forms.

Introduction

Low potency results are relatively frequent occurrences in drug product HPLC assays. These low potency results

are driven by several factors, most of which can be divided into two main categories: formulation and method. One

of the most common formulation related factors involves entrapment of the API onto manufacturing equipment

and/or API storage containers/bags. Entrapment issues are often common in low dose formulations and are

usually addressed by coating the equipment with excipients prior to addition of API, and/or

rinsing API containers/bags with excipients to ensure quantitative transfer of the container/bag contents. Another

formulation related factor that can contribute to low potency values involves undercharging of the API during drug

product manufacture. Weighing errors

and/or miscalculation of the API activity factor are often the source of these low potency values. The latter is more

prevalent when the API is a salt, whose counterion contributes significantly to the molecular weight of the drug

substance. Segregation is another formulation

related factor that contributes to low potency results during assay of solid oral dosage forms. This involves physical

separation or de-mixing of the API from excipients in the formulation. Segregation may occur due to particle size

and/or flow differences between the API and excipients. Segregation often leads to super-potent and/or sub-potent

solid oral dosage forms and contributes to inhomogeneity of the manufactured supplies.

To fully understand the many method related factors that can contribute to low potency results, one needs to have

a clear understanding of the critical steps in the sample preparation extraction (SP&E) process. As shown in Figure

1, the first critical step in the SP&E process involves disintegration or dispersion of the solid oral dosage form to

create small granules/particles. This increase in surface area is then followed by Step 2, which involves dissolution

of the API in the diluent. Factors that influence these two critical steps will impact the rate and extent of extraction

of the API from the tablet matrix. Two such factors are dissolving solvent selection and agitation techniques. These

two factors contribute to the development of inefficient and inappropriate analytical methods for solid oral dosage

forms and should be carefully and thoroughly evaluated when troubleshooting low potency values.

In this paper, effective strategies for troubleshooting low potency results in drug product HPLC assays will be

discussed. These strategies, which include the use of non-traditional extraction techniques, can help to

significantly reduce the time it takes to troubleshoot low recovery results, allowing project timelines to be

maintained.

Non-Traditional SP&E Tools

Low potency values observed during HPLC assays often lead to the proverbial method vs. formulation debate

between analysts and formulators. The onus is often on analysts to show that the low potency results is or is not

method related. Because of the need to increase business efficiencies and because of tight project timelines,

today's analysts must use effective strategies to quickly and effectively resolve or eliminate the method vs.

formulation issue. The application of non-traditional tools can help facilitate rapid troubleshooting of low potency

results and help identify the source of these issues. Two non-traditional SP&E techniques that are very effective in

troubleshooting low potency results are 1) Pressurize Fluid Extraction (PFE)or Accelerated Solvent Extraction

(ASE); and 2) Microwave Assisted Extraction (MAE). These two techniques have received limited attention over

the last 5-10 years as SP&E techniques and little, if any; have been said about their potential as troubleshooting

tools for low potency results (1, 2, 3, 4, 5, 6, 7, 8).

PFE or ASE is a technique for extracting solid and semi-solid samples at elevated temperatures and pressures,

with organic and/or aqueous solvents (6-8). MAE is similar to ASE in that it facilitates extraction of samples at

elevated temperatures and pressures. However, unlike in ASE, extractions in MAE are performed in parallel and

therefore sample throughput is higher. Additionally, unlike in ASE, samples in MAE can be agitated by stirring; a

key factor when analyzing challenging solid oral dosage forms. Furthermore, the mechanism of heating in MAE

allows for faster heating of samples and therefore faster solubilization/extraction of APIs from solid oral dosage

forms (1).

Application of ASE and MAE as Low Potency Troubleshooting Tools

ASE and MAE are potentially powerful screening tools for rapid troubleshooting of low potency results. These

essentially "infinite" extraction techniques can help pinpoint the source of low potency results - Method or

Formulation related? With as little as 2 tablets and in less than 24 hours, answers to the method vs formulation

issue can be obtained. The screening procedure involves performing extraction, using ASE or MAE, on two tablets

at:

1) Ambient temperature (25°C - 40°C) and;

2) High temperature (70°C - 100°C)

The sample diluent used in the drug product test procedure, which generated the low potency result, is used as the

extraction solvent in the ASE and MAE screen. The extracted sample is diluted to volume, filtered and assayed by

the drug product test procedure. The recovery

data generated at the two extreme temperature conditions are then categorized into one of three buckets as shown

in Table 1. Insight into the source of the low potency results is captured in the extreme right column. For example,

ASE/MAE extraction that results in low recovery at low temperatures, but quantitative recovery at high

temperatures, suggest that the low potency results are method related (Bucket A). This is because quantitative

extraction of the API at elevated temperatures shows that there were no issues with the formulation - all

the drug is present, but the analytical method was unable to completely extract it from the solid oral dosage form.

The results from the ASE/MAE studies also provide additional information to the analyst to help determine a fix for

the analytical method. The quantitative

recovery obtained at elevated temperatures suggests that extraction may be kinetically driven and that longer

extraction and/or sample agitation times may facilitate complete recovery of the analyte from the tablet matrix.

Alternatively, changing the ratio of the organic and/or aqueous composition of the diluent may also help to enhance

recovery of the analyte. Table 1 further shows that low recovery at low temperatures and similarly low recovery at

elevated temperatures suggest that we have a formulation related issue (Bucket B). If all the drug was present in

the solid oral dosage form, extraction at elevated temperatures should show increased recovery over the low

temperature extraction. The fact that no change in recovery was observed when compared to the low temperature

extraction suggests that the low assay results may be formulation and not method related. The above info should

provide the analyst with sufficient data and confidence to initiate discussions with the formulator about potential

formulation related issues that could contribute to low assay results. In Table 1, Bucket C, we see that low

recovery at low temperatures but increased recovery at elevated temperatures is a bit more challenging to

decipher. The results do not clearly suggest a formulation or

method related issue. In cases like these, the analyst should assume that the low assay results are method related

and consider the application of other non-traditional techniques to shed further light on the root cause of the low

potency results. Additionally, the analyst should

thoroughly evaluate API bio-pharmaceutical data, such as solubility in the diluent and consider drug/excipient

interaction as a likely source of the low recovery. Evaluation of the structure and physiochemical properties of the

API and excipients used in the formulation should help the analyst in determining if drug/excipient interaction could

be contributing to the low potency results. Additional tools such as differential scanning calorimetry and/or solid

state NMR could also be used to predict and/or evaluate the formulation for drug/excipient interaction.

Example Case Studies Employing ASE/MAE Screens

Case studies highlighting the application of ASE/MAE as screening tools to troubleshoot low potency results are

shown in Tables 2 and 3. In Table 2, Compound A, a Pfizer propriety 25% Spray Dried Dispersion (SDD) 5 mgA

tablet formulation, showed unexpected low potency when evaluated with the manual extraction method. The ASE

screen was applied to determine if the low potency result was formulation or method related. Extraction at low

temperatures provided low recovery of the API from the tablet matrix, while extraction at elevated temperatures

provided essentially quantitative recovery of the API. The above results suggested that the low potency results

obtained with the manual method could not be formulation related, as all the API was obviously present within the

tablet matrix (Bucket A). The results were more likely method related, with kinetics possibly playing a significant

role. As shown in Table 2, increasing the shake and sonication times from 1 hour and 30 minutes,

respectively, to 2 hours and 60 minutes, respectively, afforded quantitative recovery of the API from the tablet

matrix. Additionally, changing the sample preparation procedure by using water first, followed by the addition of

95/5 ACN/water, allowed for rapid and quantitative extraction of the API from the tablet matrix. The initial use of

water allowed for rapid disintegration of the tablet, which contained

approximately 3% of the super disintegrant, sodium starch glycolate (Explotab). Another example which highlights

the successful application of ASE/MAE screen to troubleshoot low potency results is shown in Table 3. Compound

B, another Pfizer propriety compound, provided low recovery when the manual method was applied. Tablets were

then evaluated with ASE at low temperature and high temperature. Results from the ASE studies suggested that

the low potency results were formulation rather than method related (Bucket B). This is because extraction at

elevated temperatures resulted in no change in percent recovery when compared to extraction at lower

temperatures. If additional drug was present in the formulation, an increase in recovery at elevated temperatures

would be expected. With the above data in hand, the analyst was able to convince the formulator that the low

assay results were formulation driven. Subsequent evaluation of samples from the blender and mill showed

that drug was adhering to walls of the blender and mill wall, confirming that the low assay results were formulation

driven. The issue was remedied by first adding excipients to coat the surfaces of the manufacturing equipment.

Assay of the reformulated tablets resulted in quantitative recovery of the API from the matrix, confirming that the

analytical method was suitable for its intended purpose.

As the above examples indicate, ASE and MAE have great potential to be suitable screening tools for

troubleshooting low potency results. ASE and MAE can provide insight as to the source of the low potency results -

leading to rapid resolution of the method vs formulation

debate, with minimum resource burn. Additionally, if applied early in the method development phase, ASE and

MAE can facilitate the development of robust and rugged sample

preparation/extraction procedures for solid oral dosage forms. This is because ASE/MAE can be used as

screening tools to determine if the initially selected extraction solvent is suitable for extracting the API from the

tablet matrix. This kind of information can lead to rapid method development and validation and early confidence

as to the robustness and ruggedness of the analytical method. The procedure for using ASE/MAE as a method

development screening tool is analogous to the ASE/MAE troubleshooting screen procedure described earlier. The

difference is that an additional bucket (Bucket D) is added to Table 1 (Table 4). If extraction on two tablets provide

quantitative recovery at both temperature extremes, the solvent used is suitable for extracting the API from the

tablet matrix. The ASE/MAE method development screen was used in the development of an analytical

method for Compound C, a 5 mgA immediate release tablet formulation. The dissolving solvent used in the API

assay/purity method was evaluated to determine if it was a suitable solvent for extracting the API from the tablet

matrix. ASE/MAE extractions were performed

on two Compound C tablets at low and elevated temperatures. The samples were then diluted to nominal

concentration, filtered and assayed with the API HPLC method. As shown in Table 5, quantitative recovery was

obtained at both temperature extremes (Bucket D), suggesting that the solvent is suitable for extracting the API

from the tablet matrix. With the above information, the analyst was able to develop and validate a suitable HPLC

method in less than 1 week, with minimal human and sample resources.

Conclusions

Low potency results are relatively frequent occurrences in drug product HPLC assays. These low potency results

are either method or formulation related. The current drug development climate requires analysts to quickly

troubleshoot and identify the source of the low potency values, so that clinical supplies could be reformulated or

methods redeveloped to allow for timely release to support the clinical studies. Efficient and effective

troubleshooting requires that the analysts apply appropriate strategies, including the use of non-traditional sample

preparation/extraction screening tools such as ASE and MAE.

Acknowledgements

The author would like to thank Karen Alsante for her review of this manuscript and for her constructive feedback.

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