Reference vs Retention Samples: What is the Difference?

In pharmaceutical manufacturing, reference and retention samples allow manufacturers to verify product integrity, investigate quality concerns, and support regulatory inquiries.

Proper management of these samples helps address potential issues such as stability concerns, labeling discrepancies, and customer complaints. Regulatory authorities, including the FDA, EMA, and WHO, require adherence to strict guidelines for their collection, storage, and retention.

This article outlines the differences between reference and retention samples, storage requirements, and the key regulatory guidelines that govern their management.

What Are Reference and Retention Samples?

Reference and retention samples serve distinct yet complementary roles in Good Manufacturing Practice (GMP):

Reference Samples

A reference sample is a portion of a batch of starting material, packaging material, or finished product retained during its shelf life. Reference samples should also be retained from critical intermediate stages, particularly those subject to analytical testing and release. 

Reference samples are kept for future analytical testing, ensuring the samples meet specifications. They are significant for investigating potential quality defects and stability concerns.

Retention Samples

A retention sample is a fully packaged, market-ready unit of the finished product. It serves primarily for identification and regulatory verification. Retention samples allow authorities and manufacturers to confirm the following:

• correct labeling and packaging
• batch number and expiry date
• presentation of the final product.

Unlike reference samples, retention samples are not used for testing unless a regulatory investigation requires it.

In certain cases, a fully packaged finished product may serve as both a reference and a retention sample. However, this is only acceptable if the sample quantity is sufficient for analytical testing while also being available for visual inspections. Manufacturers must ensure regulatory compliance for both purposes, and when necessary, retain separate samples to meet each requirement.

Why are Reference and Retention Samples Important?

Reference samples include materials from various production stages to allow for future testing. Retention samples, on the other hand, consist of the final packaged product for verification purposes. While a finished product may serve both roles in certain cases, regulatory requirements must be carefully met to ensure compliance.

Reference Samples

Reference samples are retained for future analysis, allowing manufacturers to verify product quality, investigate complaints, and confirm compliance.

• Future Analytical Testing  

They verify product quality when stability, contamination, or formulation changes are in question, ensuring compliance with approved specifications for retesting.

• Complaint & Recall Investigations 

Suppose a patient or healthcare provider reports a quality issue. In that case, reference samples help determine whether the problem occurred at release time and pinpoint whether the issue originated from manufacturing, storage, or distribution.

• Regulatory Inspections 

During GMP audits, authorities such as the FDA, EMA, and WHO may request access to reference samples to confirm compliance with quality control standards.

Retention Samples

Retention samples ensure the finished product’s packaging, labeling, and presentation meet regulatory requirements.

• Identification

Confirmation that the approved specifications match the batch number, expiry date, labeling, and packaging and that the product was correctly labeled and packaged before distribution.

• Market Complaint Investigations 

Retention samples allow manufacturers to verify whether reported market complaints, such as mislabeling, damaged packaging, or batch-related issues, existed at the time of release. This helps determine if corrective actions are needed for future production or distribution practices.

• Regulatory Inspections & Batch Release Audits 

Regulatory authorities may inspect retention samples to confirm that a batch was manufactured, tested, and released correctly, ensuring compliance with GMP and GDP requirements.

• Cross-Checking Product Variants for Different Markets 

Some products are packaged differently for multiple countries. Retention samples help verify that the correct packaging components, language versions, and labeling were used and assist in detecting packaging errors or counterfeit products.

Types of Reference and Retention Samples

Reference and retention samples are categorized based on their purpose and stage of collection in the manufacturing process. Reference samples are taken from various production stages to allow for future testing, while retention samples consist of the final packaged product.

Reference Sample Types

Reference samples include materials from different stages of manufacturing to allow for future analysis:

• Starting Materials – APIs and excipients

The manufacturer must retain a reference sample of starting materials used in production, except for certain materials such as solvents, gases, and water. This allows for retrospective quality checks and helps identify potential issues related to raw material consistency.

• Packaging Components – Labels, cartons, inserts, containers, etc. 

Each packaging site must store reference samples of primary and printed packaging materials from each batch. Since packaging directly impacts patient safety and regulatory compliance, maintaining samples ensures that labeling, carton inserts, and primary packaging meet approved specifications.

SEE ALSO: Why is Packaging important in GMP?

• Critical Intermediates

In-process products that undergo analytical testing before the final formulation. This provides a means to investigate quality deviations that may have occurred before the final formulation.

• Finished Product

A finished product that is kept as a representative sample during its shelf life.

Retention Sample Types

Retention samples include the finished product maintained in its market-ready form to ensure compliance with approved specifications.

• Finished Product –A fully packaged unit (e.g., blister packs, bottles, vials in cartons) that accurately represents the product patients and healthcare providers received. 

The manufacturer, importer, or batch release site must retain reference and/or retention samples for each batch of the finished product. This ensures an intact, market-ready product is available for future examination, stability testing, and regulatory audits.

Storage Duration Requirements

Storage requirements vary depending on the sample type. Regulatory guidelines specify different storage durations for finished products, starting materials, and packaging components. 

Finished Product: 

• Reference and retention samples for each batch of a finished product must be stored for at least one year beyond the product’s expiry date.
• The reference sample must be stored in its original primary packaging or a container made from the same material as the marketed product’s primary packaging. 

Starting Material:

• Samples of starting materials (excluding solvents, gases, and water used in the manufacturing process) must be retained for at least two years after the finished product is released.
• The retention period may be reduced accordingly if the material’s stability period is shorter than two years.

Packaging Materials:

• Primary and printed packaging materials should be retained for a period justified by stability concerns, regulatory requirements, or company policies. EU GMP does not mandate full shelf-life retention but advises companies to define appropriate timelines based on risk assessment.
• This ensures that packaging-related concerns, such as labeling errors or material degradation, can be investigated throughout the product’s market life.

Sample Size and Sampling Requirements

Adequate sample sizes and clearly defined sampling procedures are crucial for proper retention of samples, allowing manufacturers to conduct thorough quality assessments and demonstrate compliance during audits.

• Sufficient Quantity for Analysis

Samples should be stored in sufficient quantities to perform at least two complete and independent analytical tests, following the methods defined in the Marketing Authorization File.

• Use of Unopened Packs

Analytical testing should preferably be performed on unopened, original packs to prevent contamination, limit exposure to environmental factors, and minimize the risk of degradation. 

• Batch Representation

Reference samples must accurately represent the batch of starting material, intermediate product, or finished product from which they were taken. Ensuring samples are representative guarantees the validity of future tests and helps confirm the product’s consistent quality.

Additional Samples for Monitoring Critical Process Points

Additional samples may be collected at critical stages during the manufacturing process to monitor any potential sources of variability closely. 

Key points include:

• Beginning or end of production, where batch consistency might vary.
• Stages subject to higher mechanical or thermal stress, as these may affect the quality and stability of the product.
• In-process steps, where variations might impact final product uniformity and effectiveness.

Multiple Packaging Operations

If a batch undergoes multiple packaging operations, at least one retention sample from each distinct packaging operation must be collected. This helps identify and trace any discrepancies or inconsistencies.

If a product is packaged in different locations, companies should ensure samples are retained from each site.

Storage Conditions for Reference and Retention Samples

Proper storage of reference and retention samples is essential to maintain their stability, integrity, and reliability. Inadequate storage conditions can lead to sample degradation, making them unsuitable for quality investigations or regulatory inspections.

Regulatory bodies require manufacturers to store these samples under controlled conditions in dedicated, monitored storage areas that protect them from temperature fluctuations, humidity, light exposure, and contamination.

• Temperature Control: Samples must be stored according to their stability requirements.
• Humidity Control: Relative humidity must be monitored, with desiccants used for sensitive samples.
• Light Protection: Light-sensitive products must be stored in opaque containers.
• Security & Access Control: Storage areas must be restricted to authorized personnel, with logs maintained for sample retrieval.

Storage Areas & Equipment

To prevent mix-ups, contamination, and unauthorized access, reference and retention samples must be stored in controlled areas. These storage spaces should be marked, well-organized, and separate from production, testing, or raw material storage.

• Dedicated Sample Storage Rooms 

Reference and retention samples must be stored in restricted-access storage rooms with strict access controls, ensuring only authorized personnel can handle them. To prevent mix-ups, these rooms must be separate from raw materials, in-process materials, and routine testing samples. Storage areas should be clearly designated, systematically organized, and securely maintained, facilitating efficient inventory management and quick retrieval for investigations or audits.

• Specialized Refrigeration & Freezers 

Validated refrigerators or freezers with continuous monitoring must be used for temperature-sensitive samples. Backup power systems and alarm mechanisms should also be in place to prevent temperature excursions that could compromise sample integrity. Routine maintenance and calibration of these units help ensure they function correctly.

• Secured Cabinets  

Some samples must be stored in secure, controlled-access cabinets, ensuring only authorized personnel can retrieve them. These cabinets should be clearly labeled, positioned in low-traffic areas, and regularly inspected to prevent unauthorized handling or accidental damage. Inventory logs or digital tracking systems should document any access or movement of samples.

Environmental Conditions

GMP-compliant storage rooms must be designed to protect samples from environmental risks. The following key conditions must be strictly controlled, monitored, and documented:

• Temperature Control – Samples must be stored according to their stability requirements:
  • Ambient storage (15–25°C) – Suitable for most solid oral dosage forms (e.g., tablets, capsules).
  • Refrigerated storage (2–8°C) – Required for temperature-sensitive pharmaceuticals, including biologics and vaccines.
  • Frozen storage (-20°C or lower) – Necessary for biologics, cell-based therapies, and deep-freeze products.
• Humidity Control — Humidity-sensitive products must be stored in areas where relative humidity is monitored and controlled. Certain dosage forms may require desiccants or humidity-controlled rooms.
• Protection from Light — Light-sensitive products should be stored in opaque containers or dark storage to prevent degradation from UV or fluorescent light exposure.

Monitoring & Control

To preserve sample integrity, continuous monitoring, alarm systems, and periodic inspections are required:

• Automated Temperature & Humidity Monitoring – Digital sensors or data loggers must continuously track and record conditions. Any deviation must trigger an immediate alert for corrective action.
• Alarm & Notification Systems—Automated alarms should notify responsible personnel of temperature excursions, equipment failures, or power outages.
• Routine Inspections – Regular physical inspections should check for signs of deterioration, packaging damage, or improper storage conditions.
• Access Control & Security – Storage areas must have restricted access, with entry limited to authorized personnel. A logbook or electronic tracking system should record sample handling, retrievals, and transfers.

Marketing Authorization Compliance

Storage conditions must adhere to the specifications outlined in the product’s Marketing Authorization to ensure that reference samples accurately reflect the quality of the product throughout its shelf life. Any deviation from approved storage conditions could compromise the validity of future testing results.

Regulatory Guidelines & Compliance Requirements

Pharmaceutical manufacturers must adhere to strict regulatory guidelines regarding reference and retention samples. While EU GMP, FDA, and WHO guidelines share common principles for storing reference and retention samples, they differ in specific requirements, such as retention periods, storage conditions, and regulatory authority. Below is a comparison:

EU GMP (Annex 19)

EU GMP guidelines are mandatory for pharmaceutical manufacturers in the European Union, focusing on traceability, stability, and regulatory inspections. They require:

• Finished product samples are to be retained for at least one year after expiry.
• Starting material samples are to be stored for two years after release unless stability data supports a shorter period.
• Storage conditions to follow the Marketing Authorization to maintain sample integrity.
• Comprehensive documentation for full traceability and audit readiness.

FDA (21 CFR 600.13 & 211.170)

FDA regulations apply to manufacturers selling in the United States, emphasizing post-market surveillance, recalls, and compliance audits. Key requirements include:

• Retention of samples for one year beyond expiry or three years after distribution, whichever is longer.
• Special storage conditions for biologics require strict temperature control and continuous monitoring to prevent degradation.
• Complete records to support regulatory inquiries, stability testing, and recalls.

WHO Guidelines

WHO provides global recommendations to harmonize reference and retention sample practices, particularly in developing regions. While not legally binding, these guidelines emphasize:

• Sufficient retention periods to allow for future investigations.
• Regular stability monitoring to ensure sample integrity.
• Comprehensive documentation to support audits and quality investigations.
• Flexibility to align with local regulatory requirements while promoting international best practices.

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