Can We Continue to Draw the Line?

Chromatographic peak integration continues to be a major regulatory issue and was first discussed in this column in 2015. Is the approach to manual intervention and manual integration outlined still acceptable in the light of regulatory citations and guidance documents published since then?

Peak integration is at the heart of chromatographic analysis. Understanding how data are acquired by a chromatography data system (CDS), how peaks are integrated, and how each integration parameter operates is essential to effective analysis. The key reference on chromatographic integration was written over 20 years ago by Normal Dyson (1). Chromatographic integration in regulated laboratories was discussed in this column in 2015 (2) where a structured approach to first manual intervention (change of peak windows and processing parameters with automatic baseline placement) and then manual integration (manual repositioning baselines) was presented and discussed. The aim was to take a scientifically sound approach to integration in a regulated laboratory. Manual intervention can be applied to any analysis, but should be applied consistently to all injections in a sequence because chromatography is a comparative analytical technique. Newton and McDowall also discussed peak integration in the third part of a six-part series on data integrity, which included an outline of the order of processing of files in a sequence and the contents of an integration standard operating procedure (SOP) (3). Manual integration needs to be carefully controlled and managed because this is an area where an inspector will focus. Before we look in detail at integration, let us step back and look at the applicable regulations.

Laboratory Controls and Record Requirements

It is important to understand the regulatory requirements for laboratory controls and records as these provide a major input to any discussion. The US good manufacturing practice (GMP) regulations for laboratory controls include this requirement (4):

21 CFR 211.160(b) Laboratory controls shall include the establishment of scientifically sound and appropriate specifications, standards, sampling plans, and test procedures designed to assure that components, drug product containers, closures, in-process materials, labeling, and drug products conform to appropriate standards of identity, strength, quality, and purity.

This is a relatively simple regulation to understand: everything done in the laboratory including peak integration must be scientifically sound. A similar requirement for scientific soundness in the laboratory is found in section 11.12 of EU GMP Part 2 for active pharmaceutical ingredients (5).

As chromatography is a comparative analytical technique all injections should be integrated in the same way as much as possible. This generalization is tested near the limits of quantification and where complex mixtures are separated. Next, we have:

21 CFR 211.194(a) Laboratory records shall include complete data derived from all tests necessary to assure compliance with established specifications and standards, including examinations and assays,….. (4).

The requirement for laboratory records is also simple: complete data has been discussed in this column earlier (6) and other articles (7,8). The EU GMP regulations are not as simple to interpret; chapter 4 refers to raw data that is not defined (9). A discussion of the meaning of raw data has been presented earlier and is equivalent to complete data in the US GMP regulations (6).

Don’t Do This in Your Laboratory

How are these regulations interpreted by inspectors? Here are some examples involving scientifically unsound practices and uncontrolled integration from the FDA:

Integration of chromatograms for method STM-0076 <redacted> has been performed inconsistently. The <redacted> chromatograms exhibit tailing. Prior to March 2017 <redacted> was generally integrated as an impurity. In March 2017 a recommendation was made in STM- 0076 to <redacted> at this retention time.
The recommendation is often but not always followed and results in this area being integrated <redacted>. There is a lack of scientific justification to support if the tailing portion should be integrated. The change ... was implemented without fully evaluating the impact on previously processed data (10).

Your test methods were not capable of demonstrating the purity of your drugs... analysts reprocessed data up to 12 times, and only included the final result in the report for review by Quality Assurance. Your Deputy Manager, Quality Control stated that it is common practice to “play with parameters” to get the proper integration (11).

Failure to ensure that all test procedures are scientifically sound and appropriate to ensure that your API conform to established standards of quality and purity.
You failed to establish adequate test procedures. For example, your analyst manually integrated a HPLC test for <redacted> API despite the fact that the chromatogram lacked peak resolution. …... You lacked an approved protocol for manual integration or quality oversight of the practice (12).

Method QC/STP/I2252-04 was not followed in analysis if <redacted> tablets for <redacted> by gas chromatography. The method requires the standards be prepared with <redacted>. During the preparation of standards for sequences QC863VEN1606A, the standards were prepared with both <redacted> and <redacted>, so the same standards could be used to evaluate <redacted> tablets for <redacted> and <redacted> tablets for <redacted>. The <redacted> and <redacted> peaks coelute, potentially reducing the accuracy of the standard area count compared to the approved method (13).

Defining Manual Integration

In an earlier “Questions of Quality” column on integration (2) it was noted that there was no definition of manual integration. This column implicitly defined manual integration as manual placement of the baseline by a chromatographer. The Parenteral Drug Association’s (PDA’s) Technical Report No. 80 defines manual integration as a:

Process used by a person to modify the integration of a peak area by modifying the baseline, splitting peaks or dropping a baseline as assigned by the chromatography software to overrule the pre-established integration parameters within the chromatographic software (14).

Is this definition acceptable? It is wordy, repetitious, and could be better phrased. The use of the word overrule is contentious. As noted above, CDS software is not perfect and an application can struggle to separate overlapping peaks obvious to a trained eye. The biggest issue with this definition is that there is no mention of scientific soundness as defined in the FDA GMP regulations presented earlier (4).

A simpler, more concise, and better definition of manual integration could be “manual repositioning of peak baselines with scientific justification for their positioning”.

Implicit within this definition is the use of CDS software-otherwise you’d be drawing baselines on paper. However, this also requires that the chromatographer is trained, and ideally software technical controls should prevent manual repositioning of baselines where this is not justified by the type of the analysis. In addition, the audit trail should record the actions of the analyst optimizing the peak integration, and the system should provide a means to recall the original automated integrated chromatogram.

Should Manual Integration Be Banned?

From the citations above, would it be reasonable to ban manual integration in regulated laboratories? Let’s think this through. Experienced analysts know that chromatographic analysis can be affected by temperature, humidity, column history, as well as mobile phase preparation, so that one day’s analysis often varies slightly from the previous day’s run. To achieve consistent output and measurement, it is critical to adapt and optimize factors such as peak detection threshold or retention time windows to ensure consistent, correct, and accurate integration. But how can reviewers, approvers, quality, or outside auditors recognize the legitimate vs. egregious use of manual integration?

Banning the use of manual integration is a common response to avoid questions about data integrity. However, there are three outcomes to this crude action:

• Laboratories will have to accept poor and inconsistent integration.

• Analysts will find a workaround that permits them to integrate each chromatogram with a different set of integration parameters (typically involves performing quantification in a laboratory information management system [LIMS], or worse, a spreadsheet, without traceability back to the integration methods).

• Analysts will be forced to spend hours of their day developing complex and manipulative methods to address variations between chromatograms with a single processing method. Typically, this will require many “integration events” that could even include placing peak starts and ends at specific time points; in effect, performing manual integration to satisfy the “no manual integration” rule and deceive the reviewer.

In the wrong hands, with the wrong intent, and without a robust training and review process, altering chromatographic peak processing parameters has been misused by analysts to falsify results. How can this be managed?

The Changing Regulatory Landscape

Since the earlier “Questions of Quality” column on integration (2) there have been many publications on data integrity from regulatory authorities such as Medicines and Healthcare products Regulatory Agency (MHRA), World Health Organization (WHO), European Medicines Agency (EMA), Pharmaceutical Inspection Co‑operation Scheme (PIC/S), and Food and Drug Administration (FDA) (15–20), as well as industry bodies such as GAMP, PDA, and European Compliance Academy (ECA) (14,21,22). There is also the recent publication of the draft of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M10 on bioanalytical method validation (23) that combines the current views outlined in earlier guidance documents issued by the EMA and FDA (24,25). However, only the PDA and ICH guidance documents have specific sections on chromatographic integration (14,23).

Guidance for Integration

The PDA’s Technical Report No. 80 has a large section on chromatographic integration, and the guidance document illustrates both acceptable and unacceptable peak integration practices (14). ICH M10 section 3.3.6 outlines the current thinking for integration of chromatograms in bioanalysis (23):

• Chromatogram integration and reintegration should be described in a study plan, protocol, or SOP.

• Any deviation from the procedures described a priori should be discussed in the Bioanalytical Report.

• The list of chromatograms that required reintegration, including any manual integrations, and the reasons for reintegration should be included in the Bioanalytical Report. Original and reintegrated chromatograms and initial and repeat integration results should be kept for future reference and submitted in the Bioanalytical Report for comparative BA/BE (bioanalytical or bioequivalence) studies.

Gone is the burdensome FDA requirement for a manager to preapprove any manual integration (25), to be replaced by a plan or SOP for controlling the integration process with the before and after chromatograms included in the study report showing the impact of the changes of manual integration.

Why Manually Integrate Peaks?

We need to consider why we need to integrate peaks manually and the reason is that there are situations where a CDS cannot integrate peaks correctly. Some examples are:

• Split peaks

• Shoulder peaks

• Tailing peaks

• Baseline noise

• Negative peaks

• Coeluting peaks

• Rising, falling, or excessively noisy baselines

• Slowly eluting peaks (where the CDS has difficulty identifying the peak end).

The reasons for the inability of the integration method may be due to:

• Poor method development and validation where the analytical procedure or the integration method is not optimized or robust

• Requirements for quantitation of very small peaks, especially new, unexpected peaks resulting from impurities or excipients

• Complex sample matrices resulting in interfering peaks that may still be present after sample preparation, for example, biological samples, contrast media

• Analysis of complex mixtures may result in a heavy manual integration workload as the CDS method is not able to integrate all peaks automatically, for example, contrast media, fermentation samples.

There must be a scientifically sound (4) justification for manual intervention and manual integration as outlined in the earlier “Questions of Quality” column (2). Remember that manual integration slows an analytical process and is inefficient (3).

The more that peak integration is automated, the faster it is, with the bonus of lower regulatory scrutiny. Otherwise the review could take longer than the actual analysis.

This places responsibility on the laboratory to develop robust analytical chromatographic procedures with reliable separations that are fit for use. A key component of this approach is that the resultant peak integration must be consistent, not the use of consistent parameters or settings to achieve that peak integration. This is a subtle but vital difference that is not always appreciated.

Quality-by-Design for Robust Methods

The regulatory world is changing for analytical procedures and this should have a positive impact on peak integration. There is an ICH final concept paper (26) for the development of ICH Q14 on analytical procedure development guidelines and a revision of ICH Q2(R1) on validation. The current problem with ICH Q2(R1) (27) is that it is focused on method validation with nothing mentioned about the most important phase of the life cycle: method development. Understanding how a method works and what are the key variables is often overlooked due to time pressures. Failure to plan is planning to fail.

A life cycle approach has been addressed by a proposed USP general chapter <1220> on analytical procedure life cycle management (28). The USP life cycle begins by defining the analytical target profile (ATP), that is, the start of the process to define and validate a design space for each analytical procedure. It ensures that critical parameters are managed and controlled, and changes within the design space are known and predictable, for example, organic modifier changes of the mobile phase. Knowing the design space of an analytical procedure should result in better peak shape and resolution and hence accurate automated peak integration. Outcomes of this analytical procedure life cycle management (APLM) or method life cycle management (MLCM) approach should be more robust methods, reproducible chromatography, scientifically consistent peak integration, and, hopefully, reduced out of specification (OOS) results.

Is the FDA Banning the Use of Inhibit Integration Events?

The use of the inhibit integration function is a hot regulatory topic now, as can be seen from this FDA regulatory citation:

1. Failure to ensure that test procedures are scientifically sound …. Our investigators observed that the software you use to conduct high performance liquid chromatography (HPLC) analyses of API for unknown impurities is configured to permit extensive use of the “inhibit integration” function without scientific justification.

For example, our investigator reviewed the integration parameters you used for HPLC identification of impurities in release testing for <redacted>. These parameters demonstrated that your software was set to inhibit peak integration at four different time periods throughout the analysis.

Inhibiting integration at various points during release testing for commercial batches is not scientifically justified. It can mask identification and quantitation of impurities in your API, which may result in releasing API that do not conform to specifications (29).

This citation is based on 21 CFR 211.160(b) (4), which was presented earlier in this column, and the key questions to ask are if, where, and when can integrate inhibit be used? It has been said in some audits and inspections that this function cannot be used. This is an untenable situation and there is no explicit or implicit statement in the GXP regulations for this attitude. However, it comes back to scientific soundness and a laboratory must be able to justify the use of the function. Let us consider the following scenario:

• There is a baseline perturbation with a large negative peak after an injection. A peak of interest elutes shortly after the negative peak. The use of integrate inhibit is fully justified from the start of the injection until the baseline has returned to normal and before elution of the peak of interest. Otherwise there is a large probability that baseline placement of the analyte could be adversely influenced by the negative peak.

• Similar scenarios occur when extraneous peaks are washed from the column, or baseline perturbations from mobile phase changes during a gradient elution or wash at the end of a chromatogram.

What is more problematic is the use of integrate inhibit in the middle of a run as cited above (29). If system, blank, or other non‑sample peaks occur in the middle of a chromatogram, traditionally those peaks were not integrated. Because of suspicions that the excluded peaks might be real impurities, excluding these system peaks needs to be carefully documented and justified in the method development and validation reports, otherwise they should be integrated and marked clearly as system peaks.

System Evaluation Injections

Trial injections using actual samples feature in many warning letters (30) and question 13 of the FDA data integrity guidance (20) states that:

FDA prohibits sampling and testing with the goal of achieving a specific result or to overcome an unacceptable result (e.g., testing different samples until the desired passing result is obtained).

This is correct and should never be acceptable in a GXP laboratory SOP.

However, consider the following situation: you are analyzing low volume samples from a nonclinical study. There is a total volume of 20 μL plasma sample that is extracted and there is only enough for a single injection from each sample. Ask yourself the question; are you going to commit an analytical run of samples without checking that the system is ready? The cost of repeating the study is a high six figure sum if a run does not work. Therefore, from a practical perspective, we need a way of checking that a system is ready for analysis, but one that does not involve testing into compliance with samples. Ah, somebody says use system suitability test (SST) injections. The problem is that you may need several replicates to determine if the system is ready, and SST injections should never be started until you are confident that the system is equilibrated.

We propose the following approach for system evaluation, readiness injections, or equilibration checks:

• The ability to use system evaluation injections must be documented in an applicable SOP or analytical procedure.

• The minimum column equilibration time needs to be documented in the method to avoid excessive system readiness injections.

• Only system evaluation injections prepared from a suitable reference standard can be used to evaluate if the chromatographic system is ready. Records of the solution preparation must be available. Ideally, a test mixture that mimics the separation characteristics, but is easily distinguishable from real samples should be used.

• Should the maximum number of system evaluation injections that can be made be documented in the procedure before a problem with the chromatographic system needs to be investigated? If the cause is thought be an equilibration issue, waiting and injecting again should be sufficient. If the system continues to not behave, then an investigation is needed; the cause should be found, remediated, and documented in the instrument logbook before checking the system evaluation again. If the problem requires maintenance to resolve it, for example, pump seal replacement, then requalification of the pump should be conducted and documented before beginning the analysis.

• Using sample preparations as equilibration injections or “system readiness” checks must be clearly prohibited. The FDA guidance offers suggestions about the use of a well-characterized secondary standard for such a purpose.

• System evaluation injections are part of the complete data for the analytical run and must be included in the instrument logbook entries along with any investigation and remediation work on the instrument. A common practice is to store the data from these tests in a separate folder or location to the real analyses. This practice needs careful management and documentation as it becomes difficult to connect those injections to the official laboratory work. Ideally, all work including system evaluation injections should be stored in the same location.

Five Rules of Integration

An integration SOP was discussed earlier (3,25) to help understand what should be in it and the associated training. There are five rules to consider:

• Rule 1: The main function of a CDS is not to correct your poor chromatography.
  This places greater emphasis on the development of robust chromatographic procedures so that the factors involved in the separation are known and controlled adequately. Whenever possible, separations should be developed such that automatic integration is the norm not the exception. Management need to understand that adequate time must be given to method development and validation. This is especially true for pharmacopoeial methods that never work as written.

• Rule 2: Never use default integration parameters, always configure specific integration for each method.
  Without exception, peak integration and result processing must be defined and validated for each method so that all peak windows and names are defined and if necessary any system peaks are identified. Using a default or generic method results in excessive need for manual integration to name and calculate peaks.

• Rule 3: Always use automatic integration as a first option and control manual integration practices.
  Remember that the use of manual integration is a regulatory concern and use needs to be scientifically sound. Also be aware that, as discussed earlier, manual integration slows down a process, so see Rule 1 to get the right method depending on the sample matrix and peaks of interest.

• Rule 4: Understand how the CDS works and how the numbers are generated.
  This requires basic training in the principles of peak integration and how a CDS works. The problem is that with mergers, acquisitions, and encouraging experienced analysts to retire and employ younger workers, skills are being eroded and a CDS can be looked at as a black box that always gives the right answers.

• Rule 5: Use your brain-think.
  This rule is sometimes difficult to follow but follows on from Rule 4. You can have what appears to be a perfect separation and peak integration, but look at peak start and end placement-do they look right? Use the zoom and overlay functions of the CDS to see if standards and samples have the right peak shape. The analyst is responsible for executing applicable procedures correctly, which includes correct peak integration. The reviewer, however, also has a role to ensure that all integration (whether automated, optimized, or manually placed) follows the method guidance for placing baselines as the SOP describes, especially when the representative area for unresolved peaks are being estimated. Significant peak area manipulation should be easily noticed by an experienced reviewer.

Quo Vadis Peak Integration?

If you think that peak integration is a regulatory issue now, what will it be like in the future? The May 2019 supplement to LCGC Europe gives an interesting glimpse via an article from Wahab et al. (31), who discuss advanced signal processing techniques that could be used in chromatographic integration. The techniques listed are:

• Deconvolution of extracolumn effects by Fourier transformation for removing band broadening

• Peak area extraction by iterative curve fitting for partial overlapping peaks in a chromatogram

• Model-free approaches for peak information extraction is another approach for extracting peak areas from overlapping peaks in complex matrices

• Direct resolution by power law increases resolution by reducing peak width and trailing

• Direct resolution enhancement by even derivative peak sharpening also increases resolution by reducing peak width.

It is beyond the scope of this column to present and discuss what is already in this paper (31), but if any of these techniques are integrated into a chromatography data system, then their use needs to be justified scientifically. This means from development through validation to use of a method.

If regulators are worried by peak integration now, they could be paranoid in the future!

Summary

At the start of this column we asked the question: is the approach to manual intervention and manual integration still acceptable in the light of regulatory citations and guidance documents published since 2015? Yes is the answer, but the integration parameters used in each method need to be scientifically sound and justified on a method by method basis. This means that more attention to detail must be made when developing each method, understanding and controlling the factors that influence chromatographic separation and peak shape. Good peak integration requires good chromatography. The bottom line is-are you in control of the analytical procedure and peak integration?

References

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1. R.D. McDowall, LCGC Europe27(9), 486–492 (2014).

1. M.F. Wahab, G. Hellinghausen, and D.W. Armstrong, Recent Developments in HPLC and UHPLC, LCGC Europe Supplement32(s5), 22–28 (2019).

Heather Longden is a Senior Marketing Manager, Pharmaceutical Regulatory Compliance at Waters Corporation in Milford, Massachusetts, USA.

“Questions of Quality” editor Bob McDowall is Director of R.D. McDowall Limited, Bromley, Kent, UK. He is also a member of LCGC Europe’s editorial advisory board. Direct correspondence about this column to the editor‑in‑chief, Alasdair Matheson, amatheson@mmhgroup.com