Tathagata Ray is Area Manager, Process Development at Millipore India Pvt. Ltd (India).

Tathagata Ray

The COVID-19 pandemic has given the bio/pharma industry lessons on being prepared to manufacture parenteral formulations for human use in emergency situations. Vaccines, remdesivir injection and amphotericin injection, are such recent examples. The challenge then becomes having enough flexibility in the manufacturing lines to facilitate manufacture of large volumes of unit doses of injectables as may be required with shorter changeover times, while assuring compliance and patient safety from the product contact parts used in the manufacturing line. Single-use manufacturing solutions provide the flexibility in terms of batch sizes, fill volumes, and ease of manufacturing, especially in terms of manufacturing different products in the same line with minimum line changeover requirements. This article reviews and elaborates the initial risk assessment process for extractable and leachables arising from single-use product contact manufacturing parts as per 

) chapters <665> and <1665>, which go into effect May 1, 2026 (1,2). The determined risk can then be used to build the level of extractable profiling and process equipment-related leachables (PERLs) followed by assessing the patient safety for regulatory submissions. 

Application of the Risk Evaluation Matrix as per 

<665> and <1665> for Evaluation of Leachables Risk from Single-Use Components Used in Biomanufacturing

Pharmaceutical Technology’s Trends in Manufacturing

Manufacturing systems used for biopharmaceutical drug substances or drug products can be partially or completely constructed from plastic materials. During the manufacturing process, the drug product and its components could directly come in contact with the plastics and potentially interact with one or more of the manufacturing system’s product contact components. The substances that leach from a product contact manufacturing component could become incorporated into the process stream. If these leached substances persist in the process stream and continue through the process operations, they could accumulate in the drug substance or drug product as PERLs (1,2). When present in the drug product, these PERLs can potentially alter the critical quality attributes of the drug product, such as its stability, efficacy, and safety. 

Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products

, addresses these interactions by providing a risk-based means for chemically characterizing and qualifying plastic components used to manufacture biopharmaceutical drug substances and pharmaceutical and biopharmaceutical drug products. 

Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products

, communicates the key concepts behind and provides additional information and guidance regarding the applicability and the application of 

 <665>. The authors discuss a practical approach by which 

 <1665> can be applied in a step-by-step manner for the risk assessment, using a case study involving the manufacturing process for remdesivir injection and single-use technologies (Merck).

The testing of a product contact component is driven by the risk that the component may be unsuited for its intended use based on chemical or biological effects attributed to PERLs (1). As such risks increase, the degree of required testing also increases. The risk assessment process is accomplished by a risk evaluation matrix that establishes the appropriate contributors to the dimension of risk, provides a quantifying means for the risk in each dimension, quantifies the total risk for each dimension, and links the quantified risk to an appropriate characterization strategy.

Pharmaceutical Technology’s Trends in Manufacturing May 2024 eBook

 <665>, Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products. 

 <1665>, Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products. 

Ramesh Raju Mavuleti*, rrajum@gmail.com, is head of operations, Validation Services India. Subhasis Banerjee, PhD, is principal technical application expert, Bioprocessing APAC. Tathagata Ray is consultant, Global Strategy Deployment; all at Merck Life Sciences Pvt, Ltd. K. Vasantakumar Pai, PhD, is professor and chairman, PG Department of studies & research in Industrial Chemistry, Kuvempu University. K. Sreedhara Ranganath Pai, PhD, is professor, Department of Pharmacology, Manipal Academy of Higher Education. Somasundaram Gopalakrishnan is senior consultant, Global Biopharm Center of Excellence, Merck Pte. Ltd.

*To whom all correspondence should be addressed.

When referring to this article, please cite it as Mavuleti, R. R.; Pai, K. V.; Pai, K. S. R.; et al. Application of the Risk Evaluation Matrix as per 

<665> and <1665> for Evaluation of Leachables Risk from Single-Use Components Used in Biomanufacturing. 

Articles

Determining E&L risk from single-use components can be used to build the level of extractable profiling and PERLs.

Application of the Risk Evaluation Matrix as per USP <665> and <1665> for Evaluation of Leachables Risk from Single-Use Components Used in Biomanufacturing

The adoption of single-use technologies (SUTs) in biopharmaceutical manufacturing reduces manufacturing costs, increases the manufacturing line flexibility, and improves the speed and efficacy of the line changeovers or the batch turnaround times. The closed manufacturing systems also facilitate production of a safe and regulatory-compliant product. SUTs are used throughout the biopharma manufacturing process flow, the single-use filter capsules, tubing, and connectors being the most widely used in the final fill/finish applications of the injectable preparations. The single-use bioreactor bags and buffer storage bags are used for upstream applications. This paper reviews the drivers for adoption of SUTs and how the risks from the use of these technologies can be overcome in a planned, proactive, and compliant manner. 

Review of SUT Adoption in Biopharma Manufacturing

 Pharmaceutical Technology’s Quality and Regulatory Sourcebook 2024

The adoption of single-use components emerged from the mid-1980s. Initially, blood bags replaced glass bottles and soon became available with a plastic tube or two as well as connectors, valves, and vials for taking biological samples. The polyvinyl chloride blood bags used for blood donations were more than just bags. Evolving from these examples are the current single-uses systems (SUS) that are gaining wider use in the biopharmaceuticals industry, especially for the production of specialized and/or small-batch biopharmaceutical drugs. These SUS are the alternatives to the systems made of relatively inflexible stainless-steel vessels and reactors, hard piping, valves, and so on. Such fixed systems must be cleaned and sterilized between batches, a relatively labor and energy-intensive operation. The adoption of aseptic connectors, tubing, mixers, storage bags, and sampling bags and usage of capsule filters in a wide range of applications in the late 1990s lead to the “plastic factory” concept in the 2000s. The technology was further expanded to the upstream and downstream unit operations in the biopharma industry, such as the bioreactors, chromatography, virus removal filtration, concentration, and purification steps. This expansion has evolved over time to the current-day concept of closed continuous processing technology, which is almost completely based on SUTs and assemblies (1,2).

Single-use or disposable bioprocessing equipment is now used for ≥85% of pre-commercial scale (i.e., preclinical and clinical) biopharma manufacturing and is increasingly being adopted for commercial product manufacturing, according to an industry survey (3). The leading reasons cited in the survey as being “very important” for the adoption of SUS include a decrease in the risk of cross-product contamination, cited by 46.2% survey respondents; eliminating cleaning requirements, cited by 41.2% of respondents; reducing time to get facility up and running, cited by 44.1% of respondents; and reduction in capital investment in facility and equipment, cited by 40.4% of respondents.

A study found that SUTs, when adopted, required 87% less water (primarily by reducing steaming in place [SIP], cleaning, and changeover between batches), 21% less labor (primarily by reducing cleaning in place [CIP] activities), 38% less space, and 29% less energy (4). According to industry sources (5), SUS lower operating costs by offering 46% water and energy reductions, a 35% more favorable carbon dioxide footprint due to lower facility emissions, and a 40% lower initial investment cost. The technology allows biopharma manufacturers to push products to market faster by increasing throughput and making scalability easier (5). The availability of this technology has made new biopharma companies adopt SUS, as it requires less upfront capital investment and enables quick advancement of development efforts towards new products (6).

1. Rathore, A. S.; Agarwal, H.; Sharma, A. K.; Pathak, M.; Muthukumar, S. Continuous Processing for Production of Biopharmaceuticals. 

 (8), 836–849. DOI: 10.1080/10826068.2014.985834.
2. Klutz, S.; Magnus, J.; Lobedann, M.; et al. Developing the Biofacility of the Future Based on Continuous Processing and SUT. 

, 120–130. DOI: 10.1016/j.jbiotec.2015.06.388
3. Langer, E. S.; Rader, R. A. Biopharmaceutical Manufacturing is Shifting to SUS. Are the Dinosaurs, the Large Stainless-Steel Facilities, Becoming Extinct? 

 online, Oct. 23, 2018.
4. Lim, J.; Cox, S.; Leveen, L.; Monge, M.; Sinclairm A. The Environmental Impact of Disposable Technologies. 

.
5. Single Use Support. Pioneering Biopharma. 

 (accessed Jan. 21, 2024).
6. Das, S. The Appeal of Single-use Tech. 

Ramesh Raju Mavuleti*, rrajum@gmail.com, is head of operations, Validation Services India; Subhasis Banerjee, PhD, is principal technical application expert, Bioprocessing APAC; and Tathagata Ray, is consultant, Global Strategy Deployment; all at Merck Life Sciences Pvt, Ltd.; K. Vasantakumar Pai, PhD , is professor and chairman, PG Department of studies & research in Industrial Chemistry, Kuvempu University; K. Sreedhara Ranganath Pai, PhD, is professor, epartment of Pharmacology, Manipal Academy of Higher Education; and Somasundaram Gopalakrishnan, is senior consultant, Global Biopharm Center of Excellence, Merck Pte. Ltd.

When referring to this article, please cite it as Mavuleti, R. R.; Pai, K. V.; Pai, K. S. R.; et al. Review of SUT Adoption in Biopharma Manufacturing. 

Pharmaceutical Technology Quality and Regulatory Sourcebook

The evolution of therapeutic modalities drives the adoption of single-use technologies.

The importance of liposomes as an effective drug delivery system is well accepted in the pharmaceutical industry, but their handling remains a challenge. The delicate nature of liposomes makes their separation/concentration/diafiltration particularly problematic. Even minor shear and heat can disintegrate these nanometer-sized phosphilipid entities. Principally, tangential flow filtration (TFF) is a suitable option for liposome handing (concentration and buffer exchange); however, optimization is painstaking because of the fragile nature of liposomes. We studied different methods of TFF to better understand which factors could be modified to improve liposome handling. Our investigation showed that lowering transmembrane pressure, delta pressure and process temperature, as well as performing concentration followed by direct diafiltration helps maintain liposome integrity and minimize cost.

Liposomes are minute spherical oil droplets separated from an aqueous medium by a phospholipid bilayer. The small size of liposomes enables them to pass through epidermis and act as a carrier for liquid substances, (

) which are enclosed within the liposome’s hollow internal cavity.

In today’s pharmaceutical industry, liposomes are considered to be one of the best drug delivery vehicles available because of their unique properties; a liposome can carry hydrophilic solutes inside its cavity, as well as hydrophobic chemicals in its surrounding membrane. Meanwhile, its lipid bilayer can fuse with other bilayers, such as the cell membrane, to effectively deliver an enclosed drug substance. Because of their unique nature, liposomes can deliver a considerable variety of therapeutic agents that cannot diffuse through the membrane normally, such as a solution of DNA, hydrophilic drugs, hydrophobic chemicals, drugs requiring a slow diffusion rate and cytotoxic drugs.

Liposomes are created by continued high‑shear sonication whilst a recently introduced method  extrusion  is specifically used to create human‑use liposomes.

Although liposomes may be the ideal drug delivery vehicles for many therapeutic agents, their delicate nature and integrity have created several bottlenecks in their production.


Filtration is an effective technique that can be used to separate and study the properties of liposomes. Broadly, there are two types of filtration: normal flow filtration (NFF) and tangential flow filtration (TFF). TFF is a modified filtration technique that was developed to overcome the limitations of NFF. 

In NFF, pressure acts on the perpendicular direction to the membrane filter along with product flow, resulting in irrecoverable (during process) plugging. In TFF, pressure acts both perpendicular and parallel (along with product flow) to the membrane filter; perpendicular pressure (TMP) facilitates 'filtration', whereas parallel pressure (Δ) protects the membrane from plugging (


We conducted a study to buffer exchange liposomes from one buffer (3.3% ammonium sulfate/9% sucrose solution) to another (9% sucrose solution) using a TFF cassette. The study was divided into two parts: in the first, direct diafiltration was evaluated; in the second, the liposomes were concentrated and then diafiltered to minimize the process time (because lower diafiltration volumes were required). For both parts, the integrity of liposomes was measured after the process, as was diafiltration efficiency (conductivity measurement), process time, membrane area requirement and recovery. The experimental set‑up is explained in 

In the first part of the study, the liposome solution was directly diafiltered, after which three main parameters were observed and analysed: 1) recovery of liposome in retentate (size measurement/physical integrity checking); 2) gradual decrease of salt (ammonium sulfate) concentration permeate or diafiltration efficiency (conductivity measurement); 3) process flux. 

A Millipore Pellicon 2 Mini System was installed (

 cassette (300 kD membrane) and the system was flushed with 8.2 L of water for injection (WFI). Cassette integrity was measured using the manual air diffusion method and was found to be normal. Normalized water permeability (NWP = flux of water per unit pressure) was measured to evaluate whether the cassette had been cleaned properly. Pre‑use NWP was 62.76 LMH/psi (litres/m²/hour psi), which was within the acceptable limit of 50 LMH/psi. After flushing the system/cassette, a diafiltration study was conducted on the liposome solution. The temperature was kept at ∼9 ºC to maintain liposome integrity.

We then performed step diafiltration. Briefly, 400 mL of 9% sucrose solution (no conductivity) was added to 400 mL of liposome in 3.3% ammonium sulfate/9% sucrose solution (conductivity ∼20 mS/cm). By maintaining a TMP of 0.2 bar and ΔP of 0.4 bar, ∼400 mL of permeate was passed though the membrane and the retentate volume was brought back to 400 mL. TMP and ΔP were kept intentionally low, anticipating the possible physical stress on the liposomes. The above step was repeated seven times until conductivity of ∼20 mS/cm fell to ∼0.1 mS/cm. Details schematically are presented in 

In the previous step, 400 mL of 9% sucrose was added to 400 mL of liposome solution. It was brought back to 300 mL after removing 500 mL in permeate. Throughout the process, TMP was maintained at 0.2 bar. The retentate was subjected to 100 mL (50 mLx2) of 9% sucrose wash to make the final volume of 400 mL. The permeate was checked for conductivity during each step. 

Process parameters observed in direct diafiltration of liposome are presented in 

The permeate was clear without any liposome passage and no significant change in liposome size was observed throughout the TFF process. There was no flux drop with the maintenance of constant TMP throughout the process; average flux was 32.6 LMH at 0.2 bar TMP. Initial conductivity was 20 mS/cm, which gradually came down to 0.09 mS/cm (as observed in the permeate of the eighth diafiltration volume). Mass balance and brief results are presented in 

After the process, 5 L of WFI was recirculated for 5 min and then drained off  recirculation was performed twice. The system was sanitized with 0.3N NaOH solution, which was recirculated for 10 min and then drained off. The cassette was again washed with 5 L of WFI. Post-use NWP (58.70 LMH/psi) was measured to check cleaning efficiency, which was acceptable.

Second step: intrinsic concentration followed by diafiltration


The main objective of the second part of the study was to reduce the number of diafiltration steps (by concentrating the solution first), thus reducing process time. To achieve this, the liposome solution was concentrated and then diafiltered. This was followed by observation and analysis of the three key parameters observed in the previous study. 

A Millipore Pellicon Mini System, which was installed with a Pellicon mini 0.1 m

 cassette (300 kD membrane), was used. Installation and equilibration conditions were as previously explained. Pre-use NWP was 58.70 LMH/psi, which was within the acceptable limit. After flushing the system/cassette, a concentration and diafiltration study was conducted on liposomes in 3.3% ammonium sulfate/9% sucrose solution. The temperature was kept at ∼10 °C to maintain liposome integrity.

Step diafiltration was then performed. Briefly, 400 mL of 9% sucrose solution was added with 400 mL of liposome in 3.3% ammonium sulfate/9% sucrose solution. By maintaining a TMP of 0.25 bar, ∼600 mL of permeate was passed through the membrane, and the retentate volume was brought back to 200 mL. Again, 600 mL of 9% sucrose solution was added to increase the volume to 800 mL. Details schematically are presented in 

In the last step, 300 mL of 9% sucrose was added with 200 mL of liposome solution; this was reduced to 175 mL after removing 325 mL in the permeate. Throughout the process, TMP was maintained at 0.25 bar. The retentate was subjected to 225 mL of 9% sucrose wash to give a final volume of 400 mL. In each step, the permeate was checked for conductivity. Process parameters observed in direct diafiltration of liposome are presented in 

. Permeate was clear without any liposome passage.

The permeate was clear without any liposome passage, and no significant change in liposome size was observed throughout the TFF process. There was a flux drop with the maintenance of constant TMP throughout the process; average flux was 18.6 LMH at 0.25 bar/g TMP. Initial permeate conductivity was 21.9 mS/cm, which gradually came down to 0.1 mS/cm (as observed in the permeate of the fifth diafiltration volume). Mass balance and brief results are presented in Table 4 and Table 5.

), we concluded that, for better handling of liposomes during diafiltration by TFF, the following factors should be considered:

Lower TMP and ΔP help to maintain liposome integrity. 

Less process temperature (preferably 810 °C) is favourable for maintaining the integrity of the liposomes. 

Direct diafiltration could be a better option than concentration and diafiltration, if exchange buffer/media is not expensive (flux decreases sharply with the inclusion of a concentration step). Moreover, liposome recovery is better in direct diafiltration rather than concentration and diafiltration. 

Concentration followed by diafiltration may be considered, if exotic buffer/media is used to minimize cost. 

(14), 1532–55 (2006).Protein Concentration and Diafiltration by Tangential Flow Filtration — An Overview, Millipore Technical Publications, Lit. No. TB032. 
2. R.P. Lenk 

 Method for size separation of particles, US Patent 5948441 (1999). 
3. H. Lautenschläger, “Liposomes,” in A.O. Barel, M. Paye and H.I. Maibach, Eds, 

The importance of liposomes as an effective drug delivery system is well accepted in the pharmaceutical industry, but their handling remains a challenge.