How Are Analytical Techniques Used in Aseptic Processing?

Are analytical techniques successfully applied to the monitoring and control of aseptic processing and are the results generated effectively used individually or collectively employing a manufacturing management or enterprise system? The short answer, sadly, is no. Major obstacles are not all measurements are made remotely with continuous, automated data collection, with many still made periodically and not continuously with handheld instruments or read from instrument panels because sensors suitable for continuous monitor are often not commercially available. As processing equipment is purchased from different vendors, this results in piecemeal application and a lack of interconnectivity and compatible software between instruments, which is necessary for the successful introduction of a manufacturing management system.

This discussion is divided into analytical techniques associated with cleaning and disinfection; cleanroom operation; packaging component preparation; water for injection production; sterile product preparation; and aseptic filling, stoppering, and sealing. The author also looks at inspection and how the data collected are leveraged to control, mitigate risk, and optimize aseptic processing.

What Are Some Analytical Methods and Techniques?

Analytical methods may be classified as chemical, physio-mechanical, or microbiological methods. The options are that samples may be collected and conveyed to a laboratory for testing with a delay to obtaining the results or sensors may sample and generated results in the aseptic processing area. Real-time monitoring may be considered within the concept of process analytical technology (PAT), measuring critical process parameters beyond the traditional finished product testing by making in-line, data-driven decisions to adjust filling parameters or halting operations, significantly enhancing control, reducing rejection, and complying with good manufacturing practices (GMP).

What Are Common Aseptic Processing Steps and Opportunities for the Application of Analytical Methods?

The following is a list of processing steps and opportunities for in-process monitoring:

• Cleaning and disinfection steps
  • adenosine triphosphate (ATP) measurement for cleaning verification
  • process equipment clean-in-place/sterilize-in place monitoring
  • space disinfection–temperature, humidity, vapor-phase hydrogen peroxide concentration, and residuals
  • Cleanroom operating and environmental monitoring parameters
    • airflow velocity
    • laminar airflow

• Air changes per hour
  • HEPA filter integrity or leak tests
  • space pressurization
  • viable and non-viable particulate counts
  • temperature and relative mumidity
• Packaging component preparation
  • water-for-injection production
  • vial and stopper washing
  • depyrogenation tunnel temperature and belt speed
  • autoclave cycle temperature and duration
• Product preparation
  • bulk product mixing
  • bacterial endotoxin assay
  • bioburden monitoring
  • sterilizing filter integrity testing
  • product temperature
  • suspension homogeneity
• Product monitoring
  • fill weight/volume accuracy
  • filling rate
  • seal force monitoring
  • container headspace analysis
  • visual inspection for container-closure defects and visual particles.

How Are Analytical Methods Classified?

United States Pharmacopeia (USP) chapter <1058> Analytical Instrument Qualification classifies instruments as belonging to groups A, B, and C.1 Group A includes the least complex, standard laboratory instruments that are used without measuring a parameter or needing calibration like a magnetic stirrer. Group B includes instruments that may provide a measurement such a pH meter, an oven, or process tank mixer that may require only routine calibration, maintenance or a performance check. Group C includes analytical instruments with significant degree of computerization and complexity such as a Raman spectrometer, which will require full validation of both software and hardware. See Table I for a summary of representative analytical methods with their requirements, criticality, frequency of measurement and their USP <1058> validation classification when used in cleanroom operations.

Transition from Periodic to Continuous Monitoring

Periodic monitoring confirms the compliance of the operating parameter to the engineering and design specifications of an aseptic processing area, but unlike continuous monitoring, it will not detect anomalies in real time (Table I). Aso, periodic monitoring does not provide an opportunity to optimize cleanroom operation based on experience such as lower air velocity overnight, on long weekends, or during plant shutdowns for maintenance to achieve significant reduction in energy consumption and reducing airborne contamination during routine operations.

How Is State of the Industry Advancing to State-of-the-Art Analytical Technologies?

State-of-the- art technologies represent opportunities for improving aseptic processing through in-monitoring and control. The technologies include the following:

• ATP bioluminescence
• headspace analysis for vial leak detection
• biofluorescence particle counting for continuous air and bioburden monitoring
• air velocity sensors
• suspension homogeneity of adjuvant-assisted vaccines
• machine learning to implement statistical process control.

ATP Measurement

ATP measurement for cleaning verification is widely used in the food industry and may have a role in the biopharmaceutical industry. The area subject to cleaning is sampled with a swab, and the ATP is retrieved and assayed using a dairy industry standard method (14.057 Type B)2 expressed in relative light units (RLU) per 50 cm2. However, the method is insufficiently sensitive to enumerate low colony forming unit (CFU) levels.

Air Velocity Sensors

Many of the earlier sensors are only suitable for periodic, manual air velocity monitoring to conform that the cleanroom is meeting design and engineering specifications (e.g., vane aerometers).3 A preferred situation would be continuous air velocity measurements in HVAC ducts, at the HEPA filter face, and above the workspace. Modern sensor types are non-invasive with wireless communication providing continuous or periodic velocity, temperature, and humidity measurements.

Advanced sensor types and technology may be summarized as follows:

• thermal anemometry: common technology, sensitive to low airflows, using hot-wire or thin-film sensors
• hot-film anemometer: offer high accuracy and stability for low velocities (e.g., down to 0.15 m/s).
• pressure-based sensors: capacitive sensors can also measure velocity by sensing air pressure.

How Is Headspace Analysis Used for Vial Leak Detection?

Many drug products filled into vials have a headspace other than ambient air. These include a headspace vacuum with lyophilized products that draw sterile water for injection from accompanying vial using a double ended needle or a syringe to reconstitute the product or a nitrogen blanket that stabilizes the product. Unless the stopper is entire, properly seated and sealed, the vial will leak with the surrounding air replacing the headspace. The headspace can be monitored online using laser-based headspace oxygen analysis to detect leaks in the container-closure system.4 The instrument is calibrated using NIST Traceable Reference Gas Standards from 0 to 25% oxygen levels. The online inspection station would be positioned after the sealing and/or before the labeling operations with the leaking vials rejected.

Seal Force Monitoring

During the vial-sealing process, the component dimensions and materials play a critical role in creating a robust and adequate seal that will satisfy container-closure integrity (CCI).5 Although these properties of stopper diameter and height are manufactured within certain tolerances, there exist lot-to-lot variabilities and aging effects. If not taken into consideration during initial design, these factors can potentially impact the residual seal force (RSF) for a container closure system. RSF of the vial, while not predictive or causal, is correlated with CCI.

Biofluorescence Particle Counting for Continuous Air and Bioburden Monitoring

A critical step in aseptic processing is bioburden monitoring to control microbial contamination of ingredients, intermediates, and bulk drug products. In contrast to the traditional laboratory-based methods, bio-fluorescent particle counters (BFPC) operate independently of growth conditions in a plate count, which require an extended incubation time, because they rely on intrinsic, real-time, fluorescence detection to enumerate microorganisms in an air, water, or product intermediates. BFPC systems count total particles and potentially biologic particles, commonly referred to as auto-fluorescent units (AFU), through the detection of Mie scatter for particle occurrence and size, and intrinsic fluorescence for biologic (i.e., AFU) assignment. Intrinsic fluorescence can be used as an indicator of the biological nature of a particle because all viable microorganisms contain fluorophores, like NAD(P)H and riboflavin, that fluoresce when excited by 405nm LASER light, the wavelength commonly used by BFPC systems.6

Applications suitable for BFPC include bioburden monitoring of column washes, purification steps, and pre-sterile filtration bioburden in the downstream processing in monoclonal antibody production, pharmaceutical-grade water, and active air monitoring in cleanrooms. Real-time monitoring allows for in-process control including anomaly detection, risk mitigation, and the ability to proceed without waiting for laboratory generated laboratory results delayed extended incubation times.

Suspension Homogeneity of Adjuvant-Assisted Vaccines

To enhance the immunological response of recipients to many vaccines, alumina-based adjuvants are added to the formulation that adsorb the antigen to the adjuvant. The particle size distribution and the crystallinity of the aluminum phosphate depend on the source of aluminum chloride hexahydrate and sodium phosphate tribasic dodecahydrate and the aluminum phosphate preparation. The homogeneity of the formulation after mixing and filling may be verified by analytical methods including infrared and Raman spectrometry (off-line), focused beam reflectance measurement, and laser diffraction (in-line).7

What Are Some Opportunities for Aseptic Processing Optimization Through Multivariant Statistical Process Control?

Data generated during continuous monitoring may be subjected to control charting to set alert and action levels and recognize adverse trends. However, univariate control charts track only a single parameter over time, which is insufficient for complex process like aseptic processing that have multiple operating parameters and environmental monitoring results.

Multivariate statistical process control (MSPC) is defined as the application of multivariate statistical techniques to analyze complex process data with potentially correlated variables.8 For example, a publication on monitoring surgical suites demonstrated a moderate correlation (around 0.7) between non-viable particle counts, biofluorescent particle counts and colony-forming units.9 Other parameters are likely to be proven to be correlated. MSPC in combination with automated data collection and analysis may be used to generate control charts based on a multivariate (chemometric) model. These charts can then be used to control and improve manufacturing processes including environmental control in aseptic processing areas, sound alarms when anomalies are detected, and report summaries on a dashboard.

Other publications point out the application of MSPC to downstream processing of monoclonal antibodies, which is a stepwise, continuous operation where the process is advanced without data at risk.10

What Are the US Regulatory and Compendial Expectations?

Regulatory agencies expect that analytical methods be qualified for their intended use. Specifically, the current good manufacturing practice (cGMP) regulations [21 Code of Federal Regulations 211.194(a)] require that test methods,11 which are used for assessing compliance of pharmaceutical articles with established specifications, must meet proper standards of accuracy and reliability. Methods not recognized in the pharmacopeias or in standard methods as official test methods must be subject to method validation accordance with USP <1225> Validation of Compendial Procedures12 for chemical tests or for alternative methods to compendial microbial tests USP <1223> Validation of Alternative Microbiological Methods.13 Whether laboratory and processing measurement equipment requires full validation or merely qualification usually met through calibration is less clear-cut. The USP <1058> classification will provide useful guidance.

Other major regulatory requirements are maintaining a high level of data integrity and the full investigation and correction of GMP deviations and product failures.

Conclusion

With the transition from small-molecule pharmaceuticals to high value, large-molecule biological products, the cost of product rejection has grown enormously. A greater level of in-process monitoring and control with analytical results collected continuously, analyzed statistically, and communicated to the operators and their supervisors in the aseptic processing area is critical to improve quality and reduce costs. In-process measurements must be collected via sensors, preferably with wireless communication, without human intervention that are the major source of microbial contamination. The overall goal of analytical technologies is to build a manufacturing management (enterprise) system, based on in-process monitoring data, to prevent product failure, and optimize the multiple inputs into aseptic processing. Perhaps in the future in-process monitoring may begin to replace finished product testing for product release.

References

1. USP <1058> Analytical Instrument Qualification. USP-NF 2025.
2. Standard Methods for the Examination of Dairy Products 14.057 ATP Bioluminescence -3ATP- Specific Luciferin/Luciferase Reaction J. L. Kornacki, E. T. Ryser and C. M. Mangione (editors) APHA/Academic Press 18th Edition 2024
3. Whyte, W. Chapter 10 Measurement of air quantities and pressure differences. Cleanroom Technology–Fundamentals of Design, Testing and Operation; 2001:123-130
4. Caudill AA, Victor K, et al. A container closure integrity test method for vials stored at cryogenic conditions using headspace oxygen analysis. PDA J. Pharm. Sci. & Technol. 2024;78 (6): 707-729.
5. Cabrita, DL, Currier G, et al. Development of probabilistic model to predict residual seal force of crimped vial seals. PDA J. Pharm. Sci. & Technol. December 2025, pdajpst.2025-000041.1; doi: 10.5731/pdajpst.2025-000041.1
6. Martindale C, Dreyer C, et al. Considerations for the validation of non-CFU based bio-fluorescent particle counting technologies. PDA J. Pharm. Sci. & Technol. 2025; 79 (6) 694-706
7. Mei C, Deshmuka S. et al., Aluminum phosphate vaccine adjuvant: analysis of composition and size using off-line and in-line tools. Comp. & Struct. Biotech. J. 2019; 17: 1184-1194
8. Manzano T. and Whitford W. AI applications for multivariate control in drug manufacturing. In A Handbook of Artificial Intelligence in Drug Delivery. A. Philip, A. Shahiwala, M. Rashid and M. Faiyazuddin (editors) Academic Press 2023; 55-82
9. Larsson L-L, Nordennadler J. et al. Correlation between a real-time bioparticle detection device and a traditional microbiological active air sampler monitoring air quality in an operating room during elective arthroplasty surgery: a prospective feasibility study. Act. Orthopaed. 2025; 96: 176-181
10. Undey C, Ertunc S, et al. Applied advanced process analytics in biopharmaceutical manufacturing: challenges and prospects in real-time monitoring and control. J. Process 2010; 20(9): 1009-1018
11. 21 US Code of Federal Regulations 211.194(a) Laboratory records
12. USP <1225> Validation of Compendial Procedures. USP-NF 2025.
13. USP <1223> Validation of Alternative Microbiological Methods. USP-NF 2025.

About the author

Tony Cundell, PhD is the principal consultant at Microbiological Consulting, LLC.