Specialized and Non-Routine Projects

Our comprehensive pyrogen testing program includes specialized services designed to address complex analytical challenges, ensure data integrity over time, and systematically investigate any deviations from expected results. These non-routine projects are critical for regulatory submissions, process validation, and maintaining a state of cGMP compliance.

Low Endotoxin Recovery (LER) Studies: A Methodical Investigation of the "Masking" Phenomenon

Low Endotoxin Recovery (LER) is a well-documented phenomenon characterized by a time- and temperature-dependent loss of detectable endotoxin activity. It occurs when purified endotoxin is spiked into an undiluted product matrix, most notably in biologic formulations that contain a combination of chelating agents (e.g., citrate buffer) and non-ionic surfactants (e.g., polysorbates).This "masking" effect, where the endotoxin's structure is altered such that it is no longer recognized by Factor C-based assays, cannot be reversed by simple sample dilution.

Recognizing the potential risk that actual contamination could be similarly masked, both the FDA and EMA request LER hold-time studies as part of Biologic License Application (BLA) submissions. These studies are designed to demonstrate that the chosen endotoxin test method remains capable of detecting contamination throughout the manufacturing process and sample storage hold times.

A typical LER hold-time study is designed as follows:

• Spiking and Controls: The undiluted product is spiked with a known, low-level concentration of a standard endotoxin (Control Standard Endotoxin or Reference Standard Endotoxin, as recommended by PDA Technical Report 82). A parallel spike is prepared in LAL Reagent Water (LRW) to serve as the uninhibited control, representing 100% recovery.

• Hold Conditions: The spiked product and LRW control samples are aliquoted and held for a pre-defined period (e.g., 7 to 14 days) at temperatures that are representative of the manufacturing and storage process (e.g., room temperature and/or 2-8°C).

• Methodology: Two primary approaches can be used to manage the testing over time. In the chronological method, samples are spiked at time zero and tested at each subsequent time point (e.g., day 1, 3, 7). In the reverse spike method, aliquots are spiked at staggered intervals such that all time points can be tested simultaneously on a single analytical plate at the end of the study, which minimizes inter-assay variability.

• Acceptance Criteria: The endotoxin recovery from the product matrix is calculated relative to the LRW control at each time point. LER is generally considered to be confirmed if the recovery drops below 50% for two consecutive time points during the study.

Sample Hold-Time Studies: Validating Sample Integrity from Collection to Analysis

While procedurally similar to LER studies, a QC sample hold-time study serves a different but equally important GMP purpose. This study is not designed to investigate matrix masking effects, but rather to validate the maximum allowable time that a sample can be stored under specified conditions (e.g., refrigerated at 2-8°C) between its collection from the manufacturing line and its analysis in the laboratory, without compromising the accuracy of the test result.

The procedure involves spiking representative samples with a low level of endotoxin, storing them under the proposed conditions, and testing aliquots at various time points (e.g., 0, 24, 48, 72 hours). The time interval during which endotoxin recovery remains stable and within acceptable limits defines the validated hold time, which is then formally incorporated into the laboratory's Standard Operating Procedures (SOPs) for sample management.

Out-of-Specification (OOS) Investigations: A Structured, Phase-Based Approach

An Out-of-Specification (OOS) result is any test result that falls outside the established specifications for a product. Under cGMP regulations, any OOS result mandates a formal, thorough, and documented investigation to determine its root cause, even if the decision is made to reject the affected batch. The investigation process is structured into two distinct phases to ensure it is systematic and unbiased, and to prevent the practice of "testing into compliance."

• Phase I: Laboratory Investigation
  The immediate objective of Phase I is to determine if the OOS result was caused by a clear, assignable error within the laboratory. This is a prompt and focused inquiry that includes a comprehensive review of all aspects of the original analysis, including analyst training records, instrument calibration logs, reagent and standard preparation records, raw data, and calculations. If a clear laboratory error is identified and documented (e.g., incorrect dilution, instrument malfunction), the initial result is invalidated, and a formal re-test can be performed. If no assignable cause is found, the OOS result is considered valid, and the investigation must proceed to the next phase.

• Phase II: Full-Scale Investigation
  When no laboratory error is found, the investigation expands beyond the QC lab to encompass the entire manufacturing process. The objective is to identify the true root cause of the pyrogen contamination, assess the potential impact on other batches of the same or different products, and implement effective Corrective and Preventive Actions (CAPA). This cross-functional investigation involves a deep dive into production records, raw material testing data, water system monitoring trends, environmental monitoring data, and sampling procedures. Formal Root Cause Analysis (RCA) tools are employed to systematically identify the source of the contamination. The resulting CAPAs are implemented to address the root cause and prevent its recurrence, with subsequent effectiveness checks to ensure the actions were successful.