Robert Dream is managing director of HDR Company and a member of PharmTech’s Editorial Advisory Board. 

Robert Dream

 video series, Robert Dream, managing director of HDR Company and member of 

 editorial advisory board, provided an overview and definition of hold times in biopharmaceutical manufacturing. In part 2, Dream goes into further depth about the regulations involved and the risks that should be mitigated.

According to Dream, there is no single universal regulatory limit for biopharmaceutical hold times. Regulatory agencies, both in the United States and the European Union, expect pharmaceutical companies to establish, scientifically justified, validated, and documented hold times that ensure product quality, safety, and efficacy throughout the manufacturing process.

Key regulations and guidelines regarding hold times include current good manufacturing practice (GMP) regulation 21 

Pharmaceutical Inspection Co-operation Scheme

 (PIC/S) GMP guides. In addition, regulatory agencies expect hold time studies to be incorporated into process validation or documented as separate studies where appropriate.

Videos

In part 2 of his overview on hold times in biopharmaceuticals, Robert Dream details the regulations that govern hold times and how to mitigate risks that can occur during hold times.

Pharma Fundamentals: Risks and Regulations for Hold Times in Biopharmaceuticals

Pharma Fundamentals: Hold Times in Biopharmaceuticals: Risks and Regulations

Establish maximum hold time for all critical process intermediates.

Justify hold times with the stability, microbial, and process performance data.

Define and monitor storage conditions (eg, temperature, light exposure container closure, etc.).

Reassess hold times following process changes or deviations.

Maintain comprehensive documentation to support regulatory inspections and audits.

There are some differences between the US and EU regulations. FDA 21 

Parts 210 and 211 do not prescribe specific maximum hold times. Instead, manufacturers must establish written procedures and demonstrate that holding periods do not adversely affect product quality, strength, purity, or safety. Hold times justification is typically evaluated during inspection as part of process validation, stability programs, and current GMP compliance.

EU GMPs provide more explicit expectations that maximum storage periods, hold times for intermediate bulk products and materials, should be defined, documented, and supported by validation or stability data. EU inspections often expect predefined maximum hold times to be included in manufacturing documentation and validated as part of the quality system.

What Are the Associated Risks for Hold Time and How Are They Mitigated?

During hold times, materials remain vulnerable to physical, chemical, microbial, and environmental risk. The following are risks that compromise quality during hold times and how to prevent them.

Microbial contamination prevention. Use sterile closed storage, containers, maintain aseptic handling, procedures, control storage temperature, establish validated maximum hold times, perform routine environmental and microbial monitoring.

Chemical degradation prevention. Store materials at validated temperatures, maintain appropriate pH conditions, minimize exposure to oxygen and light, use suitable stabilizing buffers, conduct stability studies to define acceptable hold times.

Physical instability prevention. Avoid excessive mixing or agitation, prevent repeated freeze-thaw cycles, use validated storage conditions, monitor product appropriate and critical quality attributes.

Temperature excursion prevention. Continuously monitor storage temperatures, use calibrated temperature-controlled equipment, use an intermediate alarm system for temperature deviations, document and investigate any exposure.

Cross-contamination prevention. Use dedicated or properly cleaned equipment, follow validated cleaning procedures, segregate materials appropriately, train personnel in GMPs.

Container closure issue prevention. Use qualified compatible containers, verify container closure integrity, inspect containers before and after storage, select materials that have been validated for biopharmaceutical use.

Click the video above to learn more about validation requirements and best practices for designing hold studies, preventing contamination risks, and maintaining compliance with regulations.

Video image credits: Flags, Dmytro; White Clock and Vial, Olga; Compliance, WrightStudio; GMP, wladimir1804; Risk, Dilok; Black Clock and Syringe, Verrone; Vials and Check Mark, everythingpossible; all from stock.adobe.com

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https://www.pharmtech.com/view/pharma-fundamentals-hold-times-in-biopharmaceuticals-an-introduction

Pharma Fundamentals: Hold Times in Biopharmaceuticals


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Pharma Fundamentals: Biologics Development and PDA’s Technical Report No. 56


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Pharma Fundamentals: Hold Times in Biopharmaceuticals, Risks and Regulations

In this Pharma Fundamentals episode, Robert Dream, managing director of HDR Company and a 

 Editorial Advisory Board member, explains hold times in biopharmaceutical manufacturing. A hold time is the maximum validated period an intermediate or product can sit between processing steps without harming quality, safety, purity, potency, or stability. Pauses occur for reasons like equipment availability, quality control testing, scheduling, cleaning, or transport, and materials must remain under controlled conditions (refrigeration, freezing, controlled room temperature, nitrogen blanketing, light protection) throughout.

Dream distinguishes drug substance (the API—monoclonal antibodies, recombinant proteins) from drug product (the final formulated medicine), noting hold times apply at multiple points and that total cumulative hold time across the whole process matters, not just individual step-to-step holds. Hold times are established through validation studies testing material at set time points (e.g., 0, 12, 24, 72, 96 hours) until failure defines the ceiling; manufacturers then apply a safety margin below that maximum. Monitored parameters include temperature, sterility, bioburden, pH, protein concentration, appearance, potency, and purity.

This episode of Pharma Fundamentals provides an introduction to hold times in biopharmaceutical manufacturing and how they differ from hold times in small-molecule drug manufacturing.

Pharma Fundamentals: An Introduction to Hold Times in Biopharmaceuticals

Pharma Fundamentals: Hold Times in Biopharmaceuticals, an Introduction

Modern illustration depicting key components and processes within the biopharmaceutical industry with medical and scientific icons | Image in Slide Credit: ©KHAN VECTOR CO. -stock.adobe.com

3d rendering of Human cell or Embryonic stem cell microscope background. | Image Source in slide: ©Anusorn -stock.adobe.com

Because biologics are large, complex, living-system-derived molecules, they are more prone to degradation (unfolding, aggregation, oxidation), microbial contamination, and temperature sensitivity, requiring more extensive and biologically specific testing (potency assays, aggregation analysis, chromatography) and generally shorter, tighter hold times. Small-molecule drugs are chemically simpler, more stable, and more forgiving of storage conditions.

Scientist using tablet checking refrigerated drugs in pharmaceutical laboratory refrigerator | Image in Slide Credit: ©EDER -stock.adobe.com

Pharma Fundamentals: Hold Times in Biopharmaceuticals

Pharma Fundamentals: Biologics Quality System

https://www.pharmtech.com/view/pharma-fundamentals-biologics-quality-system

Building a Future-Ready Contamination Control Strategy with Microbiology Testing

https://www.pharmtech.com/view/building-a-future-ready-contamination-control-strategy-with-microbiology-testing

Dream also outlines the various types of hold times: process intermediate, drug substance, drug product, bulk, pre- and post-filtration (post-filtration being especially critical since contamination can't be removed after sterile filtration), buffer, media, cleaning, dirty equipment, and sample hold times—each with distinct risks and quality attributes to control.

The episode underscores that hold time validation is a rigorous, science-driven process central to ensuring biopharmaceutical product quality throughout manufacturing.

Image credits for images used in video: EDER, juliasudnitskaya, Chanintorn.v, Sashkin, Anusorn, KHAN VECTOR CO., Corona Borealis; all from stock.adobe.com

Robert Dream, managing director of HDR Company

Robert Dream is managing director of HDR Company and a 

Scientist using tablet checking refrigerated drugs in pharmaceutical laboratory refrigerator | Image Credit: ©EDER -stock.adobe.com

In the manufacturing of biological drug substances (DS) and drug products (DP), hold times play a critical role in ensuring product quality, safety, and regulatory compliance. Hold times refer to the maximum allowable duration during which intermediate materials, bulk substances, or final products can be held before proceeding to the next step in the manufacturing process. These durations must be scientifically justified and validated.

In a video episode of Pharma Fundamentals, Robert Dream provides an introduction to hold times in biopharmaceutical manufacturing, including a comparison to small-molecule drug manufacturing. 

Hold time is a central but often underappreciated concept in biopharmaceutical manufacturing. It sits at the intersection of process engineering, product stability, microbiology, and regulatory compliance. This paper takes a structured explanation covering definition, scientific basis, regulatory expectations, validation approaches, and real-world examples.

In biopharmaceutical manufacturing, hold time (or process hold time) refers to the maximum allowable time that materials (e.g., intermediates, bulk drug substance, buffers, or equipment) can be stored under defined conditions without compromising quality.

Regulatory bodies, such as the European Medicines Agency (EMA) and the World Health Organization (WHO) define process hold times as the period during which materials “may be held under specified conditions and remain within defined specifications.” Hold time study is defined as a scientific evaluation determining how long a material can be held, while maintaining identity, purity, safety, and potency, specifically as follows:

Hold time refers to the time during which a product, intermediate, or raw material is stored under specified conditions before it is processed to the next manufacturing stage.

Total hold time is the entire duration a material (e.g., an intermediate or bulk substance) is held from the point of its manufacture to its final use or packaging, including all storage and in-process steps.

In-process hold time specifically refers to the duration between processing steps, for example, the time between filtration and filling, or between chromatography and formulation. It is a subset of the total hold time and is particularly important in biologicals, where the stability of intermediates can be time- and temperature-sensitive.

Biopharmaceutical products—including monoclonal antibodies (mAbs), vaccines, and advanced cell and gene therapies (CGTs)—are inherently complex molecules whose structural integrity is closely tied to their safety and efficacy. Unlike small-molecule drugs, these biologics are highly sensitive to environmental and processing conditions, making them particularly vulnerable to time-dependent degradation during manufacturing hold steps. Prolonged or poorly controlled holds can lead to chemical changes such as oxidation, deamidation, and aggregation, as well as physical instability including protein unfolding and precipitation. In addition, aqueous processing environments increase the risk of microbial contamination, while inconsistencies in hold conditions can introduce process variability that affects yield and downstream performance. For these reasons, establishing and validating appropriate hold times is a critical component of robust and compliant biologics manufacturing processes.

Biopharmaceuticals (e.g., mAbs, vaccines, CGTs) are structurally complex and inherently unstable, making them highly sensitive to time-dependent degradation.

During manufacturing hold steps, biopharmaceutical materials are exposed to a range of risks that can compromise their quality and performance. Chemical degradation pathways—such as oxidation, deamidation, and aggregation—can gradually alter molecular structure, while physical instability may lead to protein unfolding or precipitation, reducing biological activity. At the same time, the predominantly aqueous environments used in processing heighten the risk of microbial contamination if conditions are not carefully controlled. Variability introduced during these holds can further propagate through the process, affecting overall yield and the consistency of downstream operations. Taken together, these considerations underscore the importance of establishing and validating appropriate hold times as a fundamental requirement for ensuring the reliability and robustness of commercial biologics manufacturing, such as the following:

Impact on yield and downstream performance

Because of this, validated hold times are considered essential for commercial manufacturing of biologics.

Where Hold Times Occur in a Typical Bioprocess

Hold times are an inherent part of biopharmaceutical manufacturing and can arise at nearly every stage of the process, from upstream production to final distribution. Following cell culture, harvested material may await clarification, while downstream purification introduces additional holds, such as post-Protein A chromatography pools, viral inactivation steps, and ultrafiltration/diafiltration retentate storage. In drug product manufacturing, formulated bulk solutions may be held prior to filling, and filled vials may remain in storage before packaging. Even during logistics, delays can occur before materials enter the controlled cold chain. Each of these intervals represents a potential point of risk to product quality, and as such, regulatory authorities expect all hold points to be clearly identified, scientifically justified, and rigorously validated within the overall manufacturing strategy.

Hold times can occur at nearly every stage, as follows:

Cell culture harvest waiting for clarification

Ultrafiltration (UF)/diafiltration (DF) retentate storage

Regulators expect all these hold points to be identified, justified, and validated.

The scientific basis of hold time in biopharmaceutical manufacturing lies in the interplay between time-dependent degradation kinetics and the surrounding environmental conditions. As biologic products are held between process steps, their stability is influenced by key variables such as temperature—often the most critical factor—along with pH, light exposure, oxygen levels, and the nature of the container closure system. These conditions can directly impact critical quality attributes (CQAs), including aggregation and fragmentation profiles, the formation of charge variants, and overall product potency. Additionally, extended holds may increase the risk of bioburden and endotoxin accumulation. A thorough understanding of these factors is essential for defining scientifically justified hold times that ensure product integrity and consistency throughout manufacturing.

Hold time is governed by the following time-dependent degradation kinetics and environmental factors:

During hold studies, samples are analyzed at multiple time points for these attributes.

Biopharmaceuticals are sensitive to environmental conditions and may degrade or lose potency if held too long. Therefore, hold times must be validated and supported by stability data. Regulatory authorities such as the FDA and EMA require manufacturers to establish, justify, and verify hold times through formal studies, as an example 

 depicts some known knowledge summary for drug substance manufacturing.

Table: Hold Times in Biologics Manufacturing

FDA’s Guidance for Industry–Process Validation: General Principles and Practices

 hold times must be validated during process qualification to demonstrate that materials held at specific stages retain their quality attributes.The document states, “Any time a manufacturing process is paused, the conditions and time must be justified by data demonstrating that product quality is not adversely affected.”

 Volume 4 (EU Guidelines to Good Manufacturing Practice) states, “The maximum hold time for any in-process material should be defined based on knowledge of the product and supported by stability data.”

 specifies that time limits for storage of starting materials, intermediate and bulk products should be based on data that demonstrate no adverse impact on quality.

International Council for Harmonisation (ICH) guidelines. 

, states that hold time studies should be performed when necessary to verify that intermediate and bulk products meet acceptance criteria throughout storage.

 states, “Hold times should be established based on a combination of development data and GMP experience, ensuring that product quality attributes are maintained.”

ICH Q1A(R2) (Stability Testing), focuses on final products; however, it implies that intermediate hold times must not adversely affect the final product's stability.

The objective of hold time validation is to establish maximum allowable hold time with scientific evidence.

A typical approach to study design is as follows:

Use production-scale or representative batches

Sample at multiple time points (e.g., 0, 6, 12, 24, 48 hours, etc.)

Analyze CQAs and microbiological parameters.

Validation demonstrates that no significant degradation occurs and product remains within specifications. Hold time validation involves the following:

Designing studies under worst-case conditions (e.g., highest temperature within range, longest duration).

Testing critical quality attributes (CQAs) such as potency, purity, appearance, pH, and microbial load at multiple time points.

Establishing limits that ensure the product remains within specifications over the defined hold period.

Validation studies typically assess the following:

Chemical stability (e.g., pH, potency, degradation products).

Microbiological control (e.g., bioburden testing).

Physical characteristics (e.g., viscosity, color, turbidity).

For example, a protein solution held between UF/DF and sterile filtration might be validated for up to 24 hours at 2–8 °C, with testing on aggregation and potency confirming stability.

When establishing hold times and storage conditions, several key considerations ensure robustness and product quality. Worst-case conditions should be deliberately evaluated, including the longest anticipated hold time, the highest temperature the material may encounter, and the most sensitive material fraction, as these represent the greatest risk to stability. Equally important is a clear definition of time zero (T₀), which must be unambiguous—such as marking it at the end of collection or the start of pooling—to ensure consistency across studies. Scale also plays a critical role, because lab-scale results may not directly translate to manufacturing scale due to differences in equipment, volumes, and environmental exposure. A well-designed sampling strategy is necessary to capture both the earliest and latest material exposure, providing a complete picture of potential variability over time. Finally, a risk-based approach should guide validation efforts, recognizing that not all hold points carry the same level of risk; tools such as failure mode effects analysis (FMEA),

 combined with process knowledge and historical data, help prioritize focus where it matters most.

Hold Times in Pharmaceutical Manufacturing

The main objectives of establishing hold times in pharmaceutical manufacturing are to ensure consistency and reproducibility in product quality, to prevent microbial contamination or chemical degradation, and to comply with regulatory expectations.Hold time is not a single concept but rather spans multiple stages of manufacturing, each with distinct considerations.

Intermediate hold time refers to the period between unit operations, such as after chromatography and before filtration, for example when a protein eluate is held overnight prior to pooling.

Bulk hold time applies to the storage of the drug substance before formulation or fill-finish, often under controlled conditions such as 2–8 °C to maintain stability.

In-process hold time captures delays within active processing steps, such as pauses between filtration and chromatography, where material may be temporarily idle but still exposed to process conditions.

Clean hold time, in contrast, relates to equipment rather than product, defining how long cleaned equipment can remain unused before it must be re-cleaned, typically measured from the end of cleaning to the next use.

Buffer or media hold time addresses the stability of prepared solutions prior to use in manufacturing.

Broadly, these categories can also be grouped as intermediate hold time (between steps like granulation and compression), bulk product hold time (storage before packaging), and solution or suspension hold time (when materials such as APIs in solvent are held before further processing), highlighting the need to evaluate stability and risk at each stage.

Designing a hold time study requires a structured approach to ensure materials remain stable and within specification throughout storage or processing delays. Key considerations include sampling at predetermined intervals—such as 0, 24, 48, and 72 hours or longer depending on process needs—to capture any time-dependent changes.

Storage conditions should closely mimic actual manufacturing environments or reflect worst-case scenarios, including factors like temperature and humidity. Container compatibility is also critical, as the selected containers must not interact with or compromise the material’s stability.

In addition, appropriate testing parameters must be defined, encompassing physical, chemical, and microbiological attributes relevant to the specific stage of the product. Ultimately, the study must demonstrate that the material consistently meets established specifications over the designated hold time. For microbiological evaluation, particular emphasis is placed on both sterile and non-sterile products, with testing aligned to pharmacopeial standards such as 

The study must demonstrate that the material remains within specification throughout the designated hold time. For microbiological testing, particular attention is given to sterile and non-sterile products (USP <61>, <62>, <71>).

For example, in oral solid dosage forms, the granulation may be held before tableting. A study might evaluate moisture content, particle size, and potency over several days. For sterile products, bioburden levels and endotoxins must remain within limits during hold times before sterilization or filling.

 found that failure to validate hold times was a common reason for regulatory observations, particularly in aseptic manufacturing. Therefore, documenting hold time data and incorporating them into the batch record is essential.

In biologic manufacturing, aseptic processing solution (APS) or similarly termed intermediate solutions are often held for defined periods between processing steps. APS holding time refers to the duration for which a sterile or aseptic product, solution, or intermediate can be held under controlled conditions before the next processing step while maintaining its quality, safety, and efficacy.

APS holding time is crucial due to the sensitivity of biologic products, which often include proteins, mAbs, or vaccines that are prone to degradation, aggregation, or microbial contamination. The time between production steps must be validated to ensure product integrity is not compromised.

The following are key reasons for controlling holding time:

: Proteins may degrade or lose activity if held too long.

: Aseptic conditions must be preserved to avoid contamination.

: Variability in holding time can introduce inconsistencies in yield or efficacy.

Articles

Hold times define maximum allowable storage durations for intermediates, bulk drug substance, buffers, and even cleaned equipment under specified conditions without compromising identity, purity, potency, or microbiological control. In biologics manufacturing, time- and environment-driven degradation (oxidation, deamidation, aggregation), physical instability, and bioburden/endotoxin risks make hold control a critical process element. Regulators expect all hold points to be identified, justified with stability data, and validated under worst-case conditions. Robust study design, documentation, and lifecycle change control mitigate inspection risk.

The scientific basis, regulatory expectations, validation approaches of hold times in biopharmaceuticals are presented along with real-world examples.