LC-UV-Based Synthetic Peptide Impurity Tracking and Reporting with Compliant-Ready Empower 3 Software

In this work, a UPLC-UV-based method is used for determining product purity using Empower 3 Chromatography Data Software (CDS).

This work demonstrates how Empower 3 Software can be easily employed for synthetic peptide impurity tracking in a compliant-ready workflow. Eledoisin was used as a model peptide to demonstrate how acceptance criteria can be used to assess reference sample material and a sample solution. Sample material was identified as having an impurity above the maximum allowed value for individual impurities to demonstrate failure to meet acceptance criteria. Empower reporting can then be used to retrieve and report data. In summary, this work demonstrates how Empower 3 Software offers integrated functionality for processing and reporting synthetic peptide data, thus enabling the assessment of product quality in an efficient, accurate, and compliant-ready manner.

Benefits

• Integrated tracking and reporting of synthetic peptide impurities using compliant-ready Empower 3 Software
• Customizable acceptance criteria thresholds based on ICH guidelines and USP standards
• Integrated reporting of multiple results to readily assess method acceptance criteria

The number of peptides in pre-clinical and clinical trials has seen steady growth over the past decade. Where development was previously hindered by factors such as efficient clearance rates and short half-lives, advances in formulation and alternative delivery systems have contributed to a resurgence.^1,2 Peptides make up a unique class of pharmaceuticals not readily classified as small molecules or biologics. This becomes especially important in a regulatory framework. The United States Food and Drug Agency (FDA) has revised the definition of a biological product to include a protein (except any chemically synthesized peptide), where the term protein refers to a “defined sequence that is greater than 40 amino acids in size.”³ A chemically synthesized polypeptide must be made completely by chemical synthesis and be less than 100 amino acids in size.³ Effective March 2020, a protein will require submission of an application for a biological product. With this in mind, pursuing a synthetic peptide manufacturing strategy over recombinant strategies may become more appealing in an effort to reduce costs and deliver products to market more quickly.

Analytical characterization and quality control of synthetic peptide products falls into one of four test categories: identification, assay, impurities, and specific tests. Impurities can result from the manufacturing process or from degradation during manufacturing or storage, and are typically determined by HPLC.^4-6 Treatment of the HPLC data can suffer from a variety of user-induced pitfalls. Peak area integration, for example, can often be subjective without defined processing methods in place. Also, it is not uncommon to export results to external software for processing, which can introduce transcription errors or incorrect calculations, but can also create an added burden to maintain compliance. Informatics suites that support impurity profiling workflows and offer integrated data analysis options are highly desirable, both for eliminating user error and ensuring compliance.

In this work, a UPLC-UV-based method is used for determining product purity in accordance with the International Council for Harmonisation (ICH) and United States Pharmacopeia (USP) functionalities built into Empower 3 Chromatography Data Software (CDS). The peptide used for this study was eledoisin, which is a biologically active peptide that acts as a vasodilator, and is thus used as a clinically relevant model system. Through establishing system suitability and impurity limits within the software, standards or samples not meeting acceptance criteria can be flagged and reported. By developing processing and reporting methods, future  data can be handled in a relatively automated fashion.

The synthetic peptide eledoisin (pE-PSKDAFIGLM-amide) was purchased from New England Peptide Inc. (Gardner, MA) at ≥95% purity by HPLC percent area. A stock solution of 2 mg/mL eledoisin in water was further diluted to a working concentration of 0.4 mg/mL. A Waters ACQUITY UPLC Peptide CSH C₁₈ 130 Å, 1.7 µm Column was selected for this study based on the high peak capacity separations it can provide for peptides in mobile phases with formic acid ion-pairing^.7

Verifying assay and instrument performance using Empower 3 system suitability option

Empower 3 Software can be readily tailored to meet user-defined needs. By deploying the system suitability application, the user can verify that both instrument and method requirements are met. To verify performance, a system suitability standard can be used to assess criteria aligned with the FDA.⁸ In practice, this standard and the associated criteria would undergo careful examination to determine what parameters are appropriate. For our study, more general criteria will be put into place for demonstrative purposes. We recognize the importance of assuring data quality, but we will also assume successful instrument qualification and method validation.⁹ Because the manufacturing process used to produce synthetic peptides allows for some variation in the final product, a specifically prepared system suitability standard is used. For this study, the Waters MassPREP Peptide Mixture is used as the system suitability standard, with enolase T35 being used to determine suitability.

A sample set was created with five injections of the system suitability sample at the beginning of the run. The chromatogram for the system suitability standard is shown in Figure 1. To calculate suitability results, this feature must be enabled within the processing method under the Suitability tab (Figure 2A). Suitability results can be calculated according to the United States, European, or Japanese Pharmacopeia. For our example, results are calculated according to the USP. Calculating signal to noise (s/n) in this case is not enabled, as we will select different acceptance criteria parameters. Should the user wish to use s/n as a suitability parameter, detector noise and drift must be enabled in the Noise and Drift tab. Instead, USP Tailing is used with an upper limit set to 2.0, which is selected in the Limits tab (Figure 2B). If peak tailing meets this criterion, the USP Tailing field in the Review window appears in unmodified text (Figure 2C). Should peak tailing exceed this value, it will be flagged in the Review window and appear in red text. 

Capacity factor, resolution, and theoretical plate number are also often used to establish acceptance criteria, but in this case, the default guidelines are less pertinent to our UPLC method.¹⁰ A second acceptance parameter will require peak area of the five injections RSD ≤1%, which is in accordance with USP <621>.¹¹ The mean, standard deviation, and RSD can be added as summary calculations and displayed through Empower reporting. By using a component summary, minimum and maximum limits for each of these fields can be incorporated. A final report will be generated after establishing impurity limits for reference standard and sample assessment.

Setting maximum allowed values for impurity screening in a reference standard solution and sample solution using Empower 3 impurity tab

Because chemical manufacture of synthetic peptides does not always produce the same impurities from batch to batch, impurities cannot always be easily identified based on relative retention time alone. For this reason, the following acceptance criteria for standard and sample analysis will be used:

Any individual impurity: Not more than (NMT) 1.5%

Total impurities: NMT 5.0%

In practice, more tightly defined acceptance criteria may be used, but these limits are based on the purity of the eledoisin sample. It is also possible to qualify impurities to loosen criteria in a case where a known impurity has been characterized and is known to not be harmful.

Criteria can be built into the processing method through the Impurity tab (Figure 3A). The Impurity Response should be set to % Area with eledoisin selected as the Main Component. The Maximum Allowed Values are those shown above. 

A chromatogram of the eledoisin reference standard is shown in Figure 1B, which is also representative of what a chromatogram of the eledoisin sample would look like. Figure 3B in the Review Window identifies nine impurities that will be reported in the final Empower report. From the view of this chromatogram, Impurity 1 has 0.03% area and is identified as being Below Reporting Threshold in the ICH Threshold field and is not included in Total Impurities. Impurity 3 is below the individual impurity limit for the reference standard, but the sample exceeds 1.5%. Because this value exceeds the maximum allowed value, it is highlighted in red text. Total Impurities is reported as 4.2, which meets the acceptance criteria.

Reporting of system suitability, reference standard solution and sample solution peak results using Empower 3 software

Empower 3 reporting can be customized to display data based on a user’s needs. For this example, system suitability results and individual peak tables for the Reference Standard and Sample will be reported to readily highlight any criteria that do not meet specification. Final reporting can be seen in Figure 4. System suitability results show that %RSD for peak area of the five injections is 0.6%, which meets the 1.0% criteria requirement. The acceptance criterion for USP Tailing was set at NMT 2.0, and for each of the five injections peak tailing was 1.8. Tables of peak results can be seen for both the reference standard solution and the sample solution. The reference standard meets criteria for both the total number of impurities as well as the limit of any individual impurity. Impurity 3 in the sample solution exceeds the individual impurity limit and is flagged in red text. Empower 3 Software allows data to be reported in a clear and efficient fashion so that any precautionary actions necessary can be carried out in a more timely manner.

This work demonstrates how Empower 3 Software can be easily employed for synthetic peptide impurity tracking in a compliant-ready workflow. Eledoisin was used as a model peptide to demonstrate how acceptance criteria can be used to assess reference sample material and a sample solution. Sample material was identified as having an impurity above the maximum allowed value for individual impurities to demonstrate failure to meet acceptance criteria. Empower reporting can then be used to retrieve and report data. In summary, this work demonstrates how Empower 3 Software offers integrated functionality for processing and reporting synthetic peptide data, thus enabling the assessment of product quality in an efficient, accurate, and compliant-ready manner.

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11. USP, General Chapter <621> Chromatography, USP39-NF34. Official from December 1, 2016.