Feliza Mirasol

Dissolution testing is an important aspect of drug development, allowing drug developers to determine the stability of a drug as well as providing 

For solid oral dosage forms, dissolution testing is a requirement, necessary for detecting physical changes in an API as well as in the formulated product (1). Furthermore, including this analytical test early in the drug development lifecycle can allow drugmakers to optimize the drug’s release in a particular formulation.

With oral administration, drug absorption from a solid dosage form depends on the release of the active substance and its ability to be absorbed through the gastrointestinal (GI) tract of the patient. These two critical steps, solubilization and absorption, make 

 dissolution studies relevant predictors of a drug’s 

 dissolution tests for solid oral dosage forms (e.g., tablets, capsules) are thus used to assess lot-to-lot drug product quality, guide the development of new formulations, and ensure continuing product quality and performance after changes in formulation, manufacturing processes, manufacturing scale up, and manufacturing site (1).

A major issue in drug development is the need to enhance the level of drug in the patient and the availability of the drug to be absorbed by the patient’s body to have the intended therapeutic affect for which it was made. The challenge has always been that, without correct optimization of drug levels and solubility, a drug may not have sufficient therapeutic effect. Whereas, too high drug levels and solubility can conversely lead to adverse events or toxicity in the patient. These factors make dissolution testing crucial to the drug development process.

For most dosage forms to be therapeutically effective, APIs must be absorbed into a body’s circulatory system, normally through the GI tract as mentioned earlier, for the drug to be transported to the targeted site. The dissolution and absorption process, therefore, contribute to the drug’s bioavailability, and it is in understanding this multi-step process that a proper 

 dissolution testing method can be developed.

 conditions, including media (e.g., solvent) and hydrodynamics, can simulate 

 conditions. However, conditions that are optimal for quality control purposes may not be applicable for establishing 

 correlation (IVIVC) (relationship between 

 response). This may make it necessary to establish two dissolution tests, one to meet development objectives and another to meet regulatory demands (3).

Dissolution tests should be robust and reproducible and should have the ability to discern changes in product performance. Typically, the characteristics of a drug’s dosage form dictate what specific dissolution testing technique should be used; however, industry testing standards are given by the United States Pharmacopeia (USP) (2).

USP describes four apparatus setups for dissolution testing techniques:

Apparatus 1 comprises a vessel (can be covered), a motor, a metallic drive shaft, and a cylindrical basket.

Apparatus 2 comprises the assembly in Apparatus 1 but adds a paddle element (blade).

Apparatus 3 comprises a set of cylindrical glass vessels flat at the bottom, a set of glass reciprocating glass cylinders, inert fittings, nonsorbing, nonreacting screens, that cover the tops and bottoms of the glass cylinders, and a motor and drive assembly to move the cylinders.

Apparatus 4 comprises a reservoir and pump for dissolution medium, a flow-through cell, and water bath (2).

Dissolution testing has also historically been used for quality control, in R&D applications to detect the effects of critical variables in the manufacturing process, and for comparative 

Poorly soluble drugs are another challenge to dissolution testing, particularly when the dosage form is meant to be an immediate-release formulation. Challenges to testing the dissolution of poorly soluble drugs include developing an appropriate test method, validating the method, demonstrating that the method is appropriately discriminatory, and dealing with the potential of an 

With poorly soluble drugs, the extent of drug release tends to be low, and it is common that the dosage from cannot be fully dissolved. Additionally, the rate at which drug is released is too slow, which hinders attainment of meaningful data from dissolution testing. Some approaches to improving the dissolution of poorly soluble drugs have been studied (4).

To start, increasing the volume of the dissolution solution helps to optimize the maximum dissolvable dose. Other approaches include changing the dissolution medium to another solution/compound that increases the solubility of the compound or reducing dissolution sink requirements. In addition, nonaqueous solvents can be used if appropriate. Media that has been known to improve saturation solubility include biorelevant media, surfactants with mixed micelles, and certain nonaqueous media, in addition to water.

Dissolution data for regulatory consideration

Dissolution testing has increasingly become an important tool in regulatory decision-making over a drug, especially generic drugs. The testing can be used to waive requirements for 

 bioequivalence studies, for example, in addition to its role in determining formulation development, in monitoring manufacturing processes, and its use as a quality control test. It also plays a role in predicting the potential for dose-dumping in the case of a modified-release drug product if taken with alcoholic beverages. 

 dissolution testing can reduce the regulatory burden on FDA as well as alleviate the need for human studies that may be unnecessary for generic drug development. At the same time, the quality of the generic drug product would not be impacted (5).

FDA’s guidance (1) on dissolution testing discusses the Biopharmaceutics Classification System (BCS), which is based on API solubility and permeability. According to the guidance, highly permeable drugs are classified as Case 1 for high solubility and Case 2 for low solubility APIs. APIs that have low permeability are classified as Case 3 for high solubility and Case 4 for low solubility. The classification serves to provide a basis for setting up 

 dissolution specifications for the test and can also provide a basis for predicting 

The development of dissolution tests for a generic drug product must take into consideration the official methods and standards described by USP, while ensuring that the testing method is robust and can be reproduced in daily operations. The method should also be transferrable between laboratories. Finally, the test method must have the capability of distinguishing any changes in solubility behavior or drug properties that may affect the end product’s performance 

If there is an absence of an available USP method appropriate for the generic drug in question, a dissolution testing method can be developed following FDA guidelines or recommendations (6). In its guidance, FDA explains three categories under which dissolution specifications for generic products should be established:

A USP drug product dissolution test is available

A USP drug product dissolution test is not available, but a dissolution test for the reference listed new drug application (NDA) drug product is publicly available

A USP drug product dissolution test is not available, and a dissolution test for the reference-listed NDA drug product is not publicly available (1).

As shown, setting dissolution specifications for generic drugs depends on whether or not an official compendial test exists for the drug product and what dissolution test was employed for the reference drug.

Even if an FDA-recommended method is not sufficient or fitting for a drugmaker’s specific API/product, then the generic drugmaker can develop its own dissolution method (5). In the end, FDA requires comprehensive dissolution testing data in the drug’s application as part of the generic drug review process.

Guidance for Industry, Dissolution Testing of Immediate Release Solid Oral Dosage Forms

 (CDER, August 1997).
2. USP, General Chapter <711>, “

,” USP29–NF24 (US Pharmacopeial Convention, Rockville, 2011).
3. Lubrizol Life Science Health, “

 Dissolution Testing for Solid Oral Dosage Forms,” 

, March 9, 2020.
4. K. Gowthamarajan and S.K. Singh, 

 17 (3) 24–32 (2010).
5. O. Anand, et al., 


Vol. 44, No. 8
August 2020
Pages: 47–48

When referring to this article, please cite it as F. Mirasol, “Defining Drug Stability with Dissolution Testing,” 

Articles

Dissolution testing is an important aspect of drug development as it provides stability parameters and helps predict drug behavior in-vivo. 

Defining Drug Stability with Dissolution Testing

Bioanalytical studies are an important aspect in biologic drug development because data from these studies are needed to define the characteristics of potential new biologics. Bioanalyses data are also an important inclusion in regulatory filings, driving the need for outsourcing partners with in-depth bioanalytical experience and experience interacting with regulatory authorities. 

 spoke with Robert Kernstock, PhD, director, Immunoassay Laboratory Services at ICON, and Neelanjan Bose, PhD, Director of Bioanalytical Chemistry at Emery Pharma, both contract research organizations (CROs), about the need for bioanalytical testing programs and regulatory strategies for potential new biologics.

 Why is it so important to conduct bioanalytical studies during the development process of a new biological therapeutic?

 Bioanalytical studies, which are designed to provide estimates for concentration of drugs and biologics in pre-clinical and clinic studies of the therapeutic molecule or their metabolites, are critical for various aspects of human clinical pharmacology, studies related to bioavailability (BA)/bioequivalence (BE) evaluation, and some nonclinical studies requiring concentration information for pharmacokinetics, toxicokinetics, or biomarkers. Bioanalytical work serves to supplement pivotal studies and aid in the decision-making process for approval, safety, and/or labeling of a drug or biologic; in short, without proper bioanalytical data, the therapeutic product would not be approved. 

 Beyond the regulatory requirements for conducting bioanalytical studies, the scientific importance of the data that these assays generate is invaluable. Bioanalytical assays provide information on certain safety aspects of the therapeutic in determining the maximum tolerated dose. Pharmacokinetic (PK) scientists use the data to determine exposure, half-life, and other pharmacological parameters, which are used to guide decisions on how often and how much of a therapeutic should be given for efficacy without undue toxicity. 

	Bioanalytical assays extend beyond simply measuring drug concentrations over time but are also used to assess drug efficacy by way of pharmacodynamic (PD) endpoints (i.e., biomarkers). Biomarker assay results may provide early indicators of efficacy, or even safety issues. They can also be used to stratify patients to predict responders or non-responders.  Another key bioanalytical assay for biotherapeutics is the assessment of immunogenicity (both wanted and unwanted). For vaccine development, a positive immunogenicity result may be considered to be potential evidence that the vaccine is working as intended. Unwanted immunogenicity is much more complicated as the impact of anti-drug antibodies may influence the pharmacokinetics, efficacy, and safety, and has to be looked at down to the individual patient level.

 What type of data/information in particular do global regulatory authorities require from bioanalytical studies?

 For any regulated bioanalytical study to occur, a validated method is required. FDA has issued guidance documents detailing the scope of bioanalytical method validation required for PK/PD endpoints, but it is also important to consider other regional guidance documents (e.g., European Medicines Agency, Pharmaceuticals and Medical Devices Agency [Japan], Agência Nacional de Vigilância Sanitária [Anvisa] [Brazil], etc.) when conducting method validations. The general assessments for method validation consist of accuracy, precision, selectivity/specificity, linearity, robustness, and stability.  

	Depending on the type of assay, certain parameters may be added or removed to meet the assays’ context-of-use, and the analytical acceptance criteria may also vary. For immunogenicity assays, a statistical report or summary is required to justify your cut point(s). A well-described validation plan detailing the experiments and a priori acceptance criteria should be written and approved resulting in a bioanalytical report summarizing the experiments in tables, descriptions of deviations, and any pertinent conclusions. A quality statement from a quality assurance unit is typically included in the report for any regulated work. 

	Once the methods are validated, the sample analysis commences that follows bioanalytical plans and/or standard operating procedures (SOPS). The reported data typically contain information on subject (or animal) number, dose/treatment group, time point, and analytical result. A listing of assay performance characteristics, which include tables of assay control results, run summaries, and calibration curve results, are typically provided. Additional information, such as incurred sample reanalysis results and sample condition (e.g., hemolyzed) may also be included.

 On a broader perspective, FDA requires PK, toxicokinetic, or biomarker concentration evaluation through bioanalytical studies. It is critical that the data [are] generated via phase-appropriately validated methods (i.e., the methods are ‘fit-for-purpose’) and in many cases adhere to 

 58), Good Laboratory Practice for Nonclinical Laboratory Studies (1). These involve much experimentation, data curation and storage, quality review, personnel training, and generation of SOPs-all related documentation should be available for review by FDA, along with the bioanalytical report. 

	On a global scale, requirements and expectations around regulated bioanalysis generally follow the same thread as FDA, but with specific regional differences. Most jurisdictions have independent bioanalytical method validation guidance. 

 What types of approaches or strategies are best to plan out early on in the drug development process? 

 Bioanalytical studies are challenging to design and plan properly at the onset of the drug/biologic development process, as they involve samples from multiple pre-clinical species, tissue types, and human-derived samples with a diverse (and in most cases unknown) genetic and metabolic makeup. The bioanalytical methods need to be robust enough to work with the variability that comes with such a diverse set of samples. 

	It is thus important to anticipate these challenges early on while in the R&D phase, and develop sample preparation protocol(s) and method(s) that can work with such diverse types of samples, varying sample amounts, and be able to account for less-than-ideal sample handling during shipment and storage. It is also preferable to start the method validation process early that ensures that the data are reliable. While FDA guidance suggest that the level of validation should be appropriate for the intended purpose of the study, it is often helpful and cost-effective in the longer term to expand validation a bit beyond that so as to get better prepared for the later development process.  

 Early in clinical development, it is important to understand the context-of-use for your bioanalytical assays, what type of therapeutic you have, and how your clinical studies are going to evolve. For instance, if you think your lowest effective concentration of your therapeutic is 500 µg/mL (trough levels), then developing an immunogenicity assay that is tolerant to high levels of therapeutic would be a critical consideration early in development. Whether or not your Phase I study is going to be in patients or healthy volunteers is another important consideration. 

	Similarly, the disease-state biomarker assays may require different sensitivities than if it was in a normal population. Understanding the sensitivity requirements for your PK assay is also important. Intravenous administration of the therapeutic may require a less sensitive assay in your serum samples, whereas an ocular injection of the therapeutic will require a very sensitive serum PK assay to measure circulating drug levels. When conducting a preclinical toxicology study, the PK assay may not need a very low limit of quantitation since the therapeutic will be dosed at high(er) levels, and the immunogenicity assay may not have a confirmatory tier as an immune response is expected from a foreign protein. Perhaps you have a novel cell therapy, and a flow cytometer is used to collect ‘cellular kinetics’. This means special handling instructions to analyze the samples within the demonstrated stability window, or the use of additives in the collection tube to stabilize study samples.   

 Are there any ‘best practices’ procedures or steps you can recommend for beginning a bioanalytical study program for a new therapeutic?

 There are a few best practices to consider, including identifying an appropriate blank matrix pool, testing disease-state selectivity as early as possible, and having a good supply of excellent critical reagents identified and appropriately characterized. Addressing these considerations will go a long way to avoiding future analytical headaches. Understanding the mechanism of action of your drug and how it relates to the sample is a critical assessment. For instance, if the drug target is a soluble cytokine that is abundant in serum and plasma, don’t be surprised if your selectivity experiment fails. More importantly in that case, what matrix pool are you using for your standard curve? Is the pool stripped of the cytokine, or did you use a surrogate matrix that doesn’t contain the interfering molecule?  

 FDA’s 2018 guidance on bioanalytical method validation (2) is a great place to start. Additionally, while still in draft form, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M10 

 guidance (3) has been a decade-long collaborative initiative of analytical sciences and regulatory agencies around the globe; it is among the best resource currently available to plan for, and design bioanalytical studies.

 Why is it important, or even necessary, for some biopharma companies to partner with an outsourcing partner for the purpose of bioanalytic studies?

 Method development in bioanalytical studies is a black box to many, requiring intense training and somewhat intuitive understanding of how analytes behave in diverse biological matrices. It is important to note that, unlike standard analytical studies, bioanalyses involve highly complex and largely undefined biological matrices, with likely millions of compounds that can interfere with specific and accurate concentration evaluation. 

	The required knowledge and expertise to successfully navigate bioanalytical studies may not be acquired quickly in-house, particularly when the regulatory landscape around bioanalyses changes regularly. Additionally, most bioanalytical studies are moving towards mass spectrometry (MS)-based analyses, which involve instrumentation that is too expensive to acquire for many companies and require specialized training for use and data analyses. 

	Furthermore, most bioanalytical studies involve conducting the work under good laboratory practice (GLP), thus, the analytical laboratory must adhere to 21 

 58. This requires companies having a quality system in place, regular audit of the facility, maintaining documentation, training records, instrument qualification, and so on, all of which often becomes too cumbersome for many biopharma companies with limited operational budget. It is thus much simpler to partner with a CRO with expertise in bioanalyses, which already have an established quality system and the required experience in interaction and data presentation to FDA and other regulatory bodies.  

 Partnering with contract laboratories can be extremely beneficial, and there are a number of reasons for doing so. The capacity in your own lab may have been exceeded and the need to outsource work to a partner lab would be necessary. Your own lab may be lacking in certain analytical equipment or experience, and a contract lab would be able to provide that service and expertise. CROs are particularly useful to smaller biotechs as the CROs can provide valuable consulting services and an expanded scope of service offerings such that they can be a ‘one-stop-shop’ for all of your bioanalytical needs. CROs have a very deep understanding of bioanalysis based on the number and diversity of assays they have developed. This is reflected in their scientific expertise as well as their understanding of global regulatory practices, since they are more frequently audited by multiple regulatory agencies; these factors end up benefiting all of their clients.

 (Government Printing Office, Washington, DC). 
	2. FDA, 

Bioanalytical Method Validation Guidance for Industry


	Vol. 44, No. 6
	June 2020
	Pages: 54–58

	When referring to this article, please cite it as F. Mirasol, “The Importance of Partnering for Bioanalytical Studies,” 

Bioanalytical studies are a significant part of biologic drug development that may require partnering with bioanalysis experts.

Partnering for Bioanalytical Studies

Bioanalytical studies are an important aspect of biologic drug development that may necessitate partnering with bioanalysis experts.

The Importance of Partnering for Bioanalytical Studies

Though cell therapy is a recent innovation, with the first therapies approved by FDA in 2017, the use of human cells has been a standard of care for decades in hematology and oncology. A cell therapy can be derived from a variety of sources, including hematopoietic, skeletal muscle, neural, and mesenchymal stem cells (i.e., adult stem cells that differentiate into structures such as connective tissues, blood, lymphatics, bone, and cartilage). Given the urgency of ensuring good patient outcomes, it is essential to have a strategy in place for preventing contamination of source cells, as well as the resulting cell therapies.

	When referring to this article, please cite it as F. Mirasol, “Detecting Contamination in Cell Therapies," 

Pharmaceutical Technology Biologics and Sterile Drug Manufacturing

A variety of assays should be used to detect bacterial, fungal, and viral contaminants in the human source cells used for cell therapies.

Detecting Contamination in Cell Therapies

Cleaning validation for biologics, particularly those that are manufactured in commercial stainless-steel equipment, has always been tricky. One worrisome issue has been the potential loss of product as the result of the cleaning process. Cleaning processes can degrade protein molecules, and biologic products-such as therapeutic antibodies-have been known to degrade and denature under extreme conditions, such as high heat and high or low pH. This can often result in loss of pharmacological activity. Yet, the cleaning of biomanfacturing equipment often requires that equipment surfaces be exposed to extreme pH and temperatures to ensure sterilization.

	FDA expects manufacturers to have written procedures on how their cleaning processes will be validated (1), and expects the validation procedure to specify the personnel responsible for performing and approving the validation study. Companies must also indicate staff members who will be responsible for establishing the acceptance criteria for the validation and the timing for when revalidation will be required. 

	The agency further expects companies to prepare specific, written cleaning validation protocols before carrying out studies that they expect to perform on each piece of manufacturing equipment. The written protocols should address important issues, such as the company’s sampling procedures, the analytical methods that will be used, and the sensitivity of those methods.

	Fortunately, advances in analytical technology have made it possible to detect even very low levels of residue left behind from manufacturing and cleaning processes, according to FDA. However, when residue or contamination is not detected, that may be due to a limitation in the sensitivity of the analytical method used. 

	Absence of detectable levels of residue or contamination is not itself a guarantee that a piece of equipment or the entire manufacturing system is clean. Because of this, the agency recommends that companies challenge the analytical method used for detection by combining it with sampling methods. This allows manufacturers to show that contaminants can still be recovered from equipment surfaces after cleaning. After all, a negative test (i.e., where no contaminants are detected) could be due to poor sampling technique.

	Sampling techniques are important for determining contamination levels, and FDA accepts two general types: direct surface sampling and rinse solutions (1). Direct surface sampling allows accessible but hard-to-clean areas to be evaluated, so that data can be used to establish an acceptable residue or contamination level per given surface area. 

	Dried residue, or residue that is insoluble, can easily be sampled by physically removing it. However, FDA has cautioned manufacturers that, in order to do direct sampling, they must first determine early on in their cleaning validation program what type of sampling material they will use and how the material will affect test data because there is potential for the sampling material to interfere with testing. 

	For the rinse sample method, advantages include use of a larger surface area for sampling and ability to test inaccessible systems (e.g., ones that cannot be routinely disassembled). However, the residue or contaminant may not be soluble, or may be obstructed by the physical structure of the equipment. As a result, evaluation of any system’s cleanliness should not rely solely on evaluation of the rinse solution, but rather evaluation of the equipment itself. The rinse solution should not simply be tested for water quality, but rather for the presence of specific contaminants. 

	Data must be recorded and documented during the sample testing procedures. Testing uncleaned equipment will establish what an unacceptable result would look like when indirect testing methods are used. 

	Alternative cleaning validation methods involve using a gauge other than permitted daily exposure (PDE) or acceptable daily exposure (ADE), or changing the limits set by regulators for these standards. It has been argued that protein molecules are degraded by the cleaning processes used to meet PDE or ADE limits (2), which has led to a search for alternative cleaning validation methods. 

	In the European Medicines Agency’s (EMA’s) guideline on setting health-based exposure limits (3), the agency states that it would be acceptable to use approaches other than PDE/ADE limits to determine health-based exposure limits, provided that the alternative approaches are “adequately and scientifically” justified. EMA also understands that, because the cleaning methods for “therapeutic macromolecules and peptides” can result in the degradation or denaturation of those molecules due to their exposure to extreme heat and/or pH, “the determination of health based exposure limits using PDE limits of the active and intact product may not be required” (3).

	Loss of biological product during cleaning processes can be costly, and especially challenging when a company is testing its cleaning validation method on a potentially new biologic product. To help mitigate expensive losses, companies may look for alternatives to their molecule that they can use as a “worst-case product” scenario for their cleaning validation methods.

	In one study, a worst-case product scenario was tested for the cleaning validation of brolucizumab (4), Novartis’ Beovu, prior to its approval by FDA in October 2019 for treating wet age-related macular degeneration (5). In the study, five molecule candidates for worst-case product were subjected to cleanability and solubility tests, with the caveat that a worst-case product must be more difficult to clean than the actual biologic product. This would ensure that acceptable cleaning settings would be established for the bioreactor when manufacturing the actual biologic product (4). 

	Going through this exercise helped the researcher demonstrate the multidisciplinary nature of cleaning validation, which is one aspect of good manufacturing practices (GMP) regulations that is still not well understood or gets little attention (4). FDA’s requirements largely focus on the need to record and document all steps used in cleaning processes. To that end, a company must be meticulous in its cleaning validation documents, including defining the equipment that is cleaned as well as the equipment used in cleaning processes; demonstrating understanding of the given drug’s properties; and describing analytical methods used to determine the level of cleanliness (or presence of contaminants). Sample residue collection from surfaces must also be recorded. The overall approach to cleaning validation, therefore, requires expertise in various disciplines and cooperation among those disciplines.  

	Air-liquid residues can be detrimental for biologics manufacturing and can come from a variety of sources, including hydrocarbons, polymers, mineral silicates, lubricants, and siloxanes (found in valves, gaskets, and tubing) (6). Hydrocarbons such as steramide, erucamide, and oleamide, are mold-release agents used to prevent caking of powders. They are often used in manufacturing the bags (e.g., those used for storage and transport) and biologics equipment. 

	Polymers such as nylon, polytetrafluoroethylene, and silicone are necessary components of equipment used in raw material manufacturing and are also used in bags or containers meant for raw material storage. These substances can be a source of raw material impurities. Likewise, mineral silicates, lubricants, and siloxanes can also be sources of raw material impurities, all of which can appear in bioprocess preparation tanks (6). 

	Common cleaning approaches that pharmaceutical manufacturers use to combat these residues include increasing spray (e.g., employing a rotating spray device), raising cleaning temperature to around 75 °C–85 °C, and using a formulated cleaning agent. In addition, the industry may increase the concentration of the cleaning agent and/or use an oxidizing cleaning agent or a detergent additive combined with an alkaline cleaning solution (so long as temperature is kept between 50 °C and 65 °C) (6). 

Validation of Cleaning Processes (7/93), Guide to Inspections Validation of Cleaning Processes

, accessed March 16, 2020.
	2. A. Walsh, 

Guideline on Setting Health Based Exposure Limits for Use in Risk Identification in the Manufacture of Different Medicinal Products in Shared Facilities

(CHMP, CVMP, November 2014). 
	4. D. Steyaert, “Cleaning Validation of Biologicals: Determination of a Worst-Case Product for RTH 258,” Master’s Thesis Paper (2016–2017).
	5. Novartis, “Novartis Receives FDA Approval for Beovu, Offering Wet AMD Patients Vision Gains and Greater Fluid Reductions vs Aflibercept,” Press Release, Oct. 8, 2019.
	6. B. Kroeger, “Current Trends in Cleaning Validation,” presentation for Parenteral Drug Association, 


	Vol. 44, No. 4
	April 2020
	Pages: 50–51

	When referring to this article, please cite it as F. Mirasol, “Alternative Cleaning Validation Methods for Biologics,” 

Conventional cleaning methods can risk product loss of biologics, prompting biopharmaceutical manufacturers to seek alternative criteria to validate cleaning processes.

Biologics Risk Product Loss Under Conventional Cleaning Validation Methods

	Over the years, many advances have been made in bioprocessing as biomanufacturers strive to increase yield, improve product recovery, enhance product purity, and streamline manufacturing. Innovations in technology and equipment for both upstream and downstream processing have led to more integrated and efficient processes, but there still remain manufacturing challenges that drive the need for further innovation.

	Successful upstream bioprocessing innovations have focused on key areas, including:

		Increasing volumetric productivity through process intensification (high seed fed-batch, perfusion, and continuous culture technologies)

		Increasing cell-specific productivity and control of critical product quality attributes (CPQAs) via novel cell expression and inducer technologies with greater molecular and cellular biology understanding through systems biology

		Accelerating the overall process development timeline.

	“These improvements have resulted in the ability to quickly advance fromcell-line generation to clinical currentgood manufacturing practices (cGMP) manufacturing with bioprocesses that are more productive and reproducible,” observes Brian Follstad, director, upstream process development, Catalent Biologics.

	“There have certainly been a number of revolutionizing, innovative approaches in bioprocessing in different product areas,” says Vasily Medvedev, process development manager at Univercells. “If we consider monoclonal antibody (mAb) manufacture, one of the key bioprocessing advances seen over the years relates to the use of high cell density prefusion cell cultures. This technology reduces the size of the bioreactor needed, directly influencing the overall footprint of operations, capital expenditure (CAPEX), and, subsequently, cost of goods.”

	Furthermore, Medvedev says the use of design of experiment (DoE) techniques is a promising trend in the industry, when these principles are correctly applied to process development and scale-up studies. “In order to fully benefit from the use of DoE studies, representative scale-down models are paramount to ensure identification of high-performing small-scale process design with seamless transfer to commercial manufacturing scale, reducing development timelines (time to market), and costs.”

	Looking at the past five years in upstream bioprocessing, Thibaud Stoll, global head of operations, biologics, at Lonza Pharma & Biotech, notes three main innovations that have impacted bioprocessing operations:

		Continuous improvements in cell expression systems, leading to processes with increasing titers, reaching or even increasingly exceeding the 10 g/L-level

		Continuous development and improvement of disposable bioreactors and associated equipment

		Development of digital tools as part of the digital transformation of biomanufacturing.

	“These innovations have contributed to improving process robustness in general, lowering cost of goods and services and enhancing flexibility to respond to fast-evolving demand,” Stoll states.

	Other innovations, such as media development, cell line selection, improvements to host cell lines, optimized vectors, and the introduction of high-throughput screening technologies, have also revolutionized upstream bioprocessing, adds Atul Mohindra, senior director, research and development, Lonza Pharma & Biotech. Mohindra also counts improved processmodeling tools, which have enabled a better understanding of the cell culture process as well as the development of more advanced technical equipment (e.g., single-use technologies [SUT], inline testing technologies) as important upstream innovations. “This has enabled the industry to develop more complex molecules, to significantly shorten the time taken to manufacture a first-in-human batch as well as to reduce the costs of a development program,” Mohindra says.

	“Besides the latest generation of single-use stirred tank and rocking motion (RM) bioreactors, one of the most significant recent introductions has been alternating tangential flow (ATF) filtration,” remarks Gerben Zijlstra, global technology consultant, continuous and intensified biomanufacturing, Sartorius Stedim Biotech, who notes that ATF provides for much more efficient and cost-effective perfusion culture than previous methods. “It is also gentler on cells, resulting in higher cell viability and lower levels of impurities to process downstream,” Zijlstra adds.

	“This technology has enabled tremendous intensification of mammalian cell cultures by allowing 5–10-fold higher cell densities compared to traditional fed-batch processing,” Zijlstra explains. By applying different ATF filter pore sizes, continuous upstream manufacturing can be performed as either a dynamic or continuous perfusion, where the product passes the filter and is direct captured using continuous chromatography; or it can be performed as concentrated fed batch (CFB), where the product is retained in the bioreactor and is harvested batchwise, he says. “The productivity of these intensified cell culture processes greatly surpasses those of existing fed-batch platforms. For CFB, titers of around 30 g/L have been reported, while for dynamic perfusions, 60 g/L (equivalent) titers were reported,” Zijlstra says.

	Another recent innovation is the introduction of several online process analytical technology (PAT) tools, such as cell density monitors that use capacitance sensors, adds Thomas Erdenberger, also a global technology consultant, continuous and intensified biomanufacturing, at Sartorius Stedim Biotech. “These sensors allow real-time cell density monitoring of viable cells without having to measure density using traditional methods by taking a sample, risking contaminating the culture. This new method allows the automation of bioreactor feeding as well as cell bleeding using the sensor coupled with a supervisory control system. At Sartorius, we can place these monitors in any of our single-use bioreactors and RM bioreactors to fully automate the entire seed train and main bioreactor,” Erdenberger says.

	Another PAT tool gaining traction is Raman spectroscopy. Previously, the key difficulty of using this technology was the need to “train” the mathematical models, the correlation of individual metabolites with the complex Raman spectra. “To solve this challenge, Sartorius recently launched a scalable Raman probe interface so these models are already preset in the 15-mL ambr [Sartorius] high-throughput mini bioreactors. This allows deep process insight and improved process understanding from early development, onwards,” states Erdenberger.

	Meanwhile, the latest generation ofdepth filters has also been a powerful innovation for upstream bioprocessing, emphasizes Peter Levison, executive director of business development at Pall Biotech. “As bioprocessing trends have continued to evolve, SUT have offered an alternative solution to drug manufacturers to accommodate shifting drug profiles. Yet, this presented a new challenge when looking at the clarification step,” according to Levison.

	In traditional stainless-steel facilities, centrifugation has been a widely adopted solution for clarification, but when working in smaller facilities that deliver higher cell densities, such as the newer SUT installations, centrifugation is no longer as feasible, Levison explains. “Not only is it costly to implement, requiring large capital and process investments, it also has a larger footprint and does not scale down so easily. So, while centrifugation is well suited for 10,000-L stainless steel bioreactors and other large facilities, it is not an ideal solution for facilities based around 2000-L single-use bioreactors that many manufacturers use today,” Levison asserts.

	Initially, depth filters offered an alternative to centrifugation with someperformance limitations-traditionally handling cell densities of up to around 20 million cells/mL. “With advances in cell culture and titer increases, we are routinely pushing cell densities up towards the 30 million cells/mL mark, and this is where advanced depth filters deliver the next generation of clarification. The high-performance platform is flexible to support semi- to fully continuous bioprocessing, allowing users to process more product per unit of bioreactor volume. To achieve this performance improvement, two dual-layered depth filtration stages are combined into one clarification step with a flexible chassis to accommodate the capsule configuration needed to deliver consistent filtrate quality in a significantly decreased footprint,” Levison states.

	For gene therapy products, the traditional technologies for cell culture and virus production are not suited for commercial-scale manufacture, notes Tania Pereira Chilima, deputy technology officer at Univercells Technologies. “This is not only due to capacity constraints, as these flasks are only compatible with a scale-out approach, increasing costs, and CAPEX, but also due to the laborious nature of operations associated with these technologies and the lack of control over critical process parameters (e.g., pH, dissolved oxygen). This poses some regulatory concerns related to process reliability and reproducibility. Moreover, there is an industry-wide shortage of skilled labor, which means that labor-intensive processes are not as feasible due to resource constraints,” Chilima states.

	The use of bioreactors for viral vector manufacture poses several benefits, making cell culture and virus production possible in a highly controlled microenvironment. Moreover, these systems are highly scalable and can benefit from the incorporation of PAT to increase the level of process controlwhile simplifying operations. The most advanced bioreactors for cell culture and virus production incorporate principles of process intensification to enable high-titer and low-footprint virus production, Chilima says. 

	“Technology improvements in process intensification and connection of unit operations have enabled manufacturing updates to run more efficiently and produce higher yields in less time and space, reducing the amount of capital investment,” concurs Darren Verlenden, head of bioprocessing, MilliporeSigma. “In upstream, we’ve seen that utilizing perfusion technology can increase cost efficiencies, decrease risk, and enhance manufacturing flexibility. Between 50– 60% of companies are already exploring or have implemented perfusion technologies for seed train or production bioreactor steps,” he explains.

	Innovations in technology and equipment have also benefitted downstream bioprocessing. Recent innovations, which include acoustophoresis (ultrasonics) cell separation and high-precision microfluidics for label-free cell selection, in-line cell washing, and rapid gene delivery, have resulted in significant productivity gains, states Jenna Balestrini, head of precision medicine and cell bioprocessing at Draper, a Cambridge, MA-based not-for-profit engineering firm.

	To accomplish cell separation on a clinical blood sample, for example, Draper developed a system that performs acoustophoresis in a high-performance microfluidic device compatible with a range of patient materials and input volumes. This system bypasses the need for centrifugation. “The module continuously and rapidly removes interfering cell contaminants without compromising cell health. With less handling than conventional approaches, acoustophoresis improves end-to-end yield of cells and accelerates delivery to downstream steps in the process,” according to Balestrini.

	In the gene therapy space, Draper has developed a microfluidic transduction module that can co-localize viral vector around cells, increasing viral-cell interaction while using about half the viral vector typically needed to achieve high transduction efficiency. This allows for more controlled viral gene delivery. The system can transduce at standard efficiency levels in 90 minutes using a wide range of vector sources, Balestrini says.

	And finally, to allow for a variety of payloads, such as ribonucleoprotein, mRNA, or DNA to be introduced into the cell without the need for viral vectors or even activation steps, Draper has engineered a practical continuous-flow electroporation module and in-line buffer exchanger that uses high-precision microfluidics to tightly control cells exposure to electrical signal, increase throughput, reduce manual-touch labor, and allow for in-line wash steps, Balestrini explains. 

	“Groundbreaking innovation is seen across different product classes in downstream processing,” adds Medvedev. “In mAb manufacture, the advent of continuous purification processes- namely, chromatography-has enabled significant improvements in process productivities, yields, and utilization of key materials (e.g., protein A resin).”

	Chilima further adds that in the gene therapy field, the use of alternative media (e.g., monolithic or membrane chromatography, as opposed to traditional bead-based separation) has proven to increase the dynamic binding capacity (DBC) and reduce processing times as this alternative media can be operated at higher flowrates. “Moreover, the use of membrane and monolithic chromatography systems with advanced separation modalities has enabled a consistent increase of resolution in the separation of empty and full capsids to be achieved with this type of media, which is critical in gene therapy manufacture,” Chilima says.

	The innovations in downstream processing have had similar impacts as in upstream processing innovations, namely the development of continuous manufacturing-in particular, continuous chromatography-that can increase facility throughput while reducing costs and the further development of disposable equipment and digital tools, Stoll says.

 modeling tools, which, when combined with high-throughput screening technologies, can increase our capabilities and reduce time,” says Mohindra. Further downstream processing innovations he identifies are the introduction of end-to-end, single-use solutions for clinical manufacturing (e.g., prepacked columns, single-use flow paths) and the development of new, high-binding capacity resins.

	Erdenberger further highlights the development of newer resins as a particularly beneficial innovation. “Firstly, for the capture step, improved (Protein A) resins with higher binding capacity and improved caustic compatibility have allowed for substantially improved downstream processing productivity, reduced cost of goods, and improved bioburden control,” Erdenberger observes. “Currently, even ‘closed, sterile’ chromatography seems to be within reach with gamma-irradiated pre-packed columns, enabling continuous, no/low bioburden chromatography operations. In parallel, continuous downstream technologies such as multi-column and simulated moving-bed chromatography have matured and are increasingly implemented to intensify downstream processing,” Erdenberger adds.

	For the polishing steps, substantial improvements in mixed mode resins, membrane adsorbers, and associated equipment are now, and increasingly, allowing for flow-through polishing as a highly efficient mode of operation, Erdenberger states. He further explains that, for the virus removal steps, methods that allow for continuous virus inactivation and virus filtration are being introduced and that continuous methodologies are even available for crossflow filtration.

	“These technologies result in continuous or semi-continuous product streams to the next unit operation in a process and in much more efficient chromatography at reduced media cost. Processing is also more rapid. When connected with certain upstream operations such as perfusion, the downstream process can be directly connected for a continuous transition from upstream to downstream,” Erdenberger notes.

	These advancements in both upstream and downstream processing have had a mixed impact, but overall a beneficial one. While creating the requirement for more well-thought-out and nuanced processes, they have also allowed for closer integration between upstream and downstream. “Although these innovations have increased bioprocess complexity, they have contributed to substantially reduced overall costs, time, and risk in generating drug substance and product. In some bioprocesses, for example, initiating culture harvest while the production culture is still running over the course of a few days (or weeks in continuous) allows purification to begin earlier when compared to historical approaches. Furthermore, on-line analytics and product attribute control strategies permit the measurement and real-time adjustment of CPQAs during a batch, allowing for a more efficient method of reproducibly producing drug substance,” remarks Follstad.

	“Continuous improvement and interplay between bioreactor productivity and advancements in downstream unit operations are increasing efficiency and streamlining manufacturing,” adds Verlenden. “An initial response to upstream intensification includes single-pass tangential flow filtration for downstream debottlenecking. In new facilities, implementation of continuous capture and flow-through polishing can remove process constraints while enhancing facility fit by reducing buffer requirements by up to 47%,” Verlenden states.

	Continuous biomanufacturing will play an integral role in better integrating upstream and downstream operations over the next five years as manufacturers look to suppliers for integrated solutions because they are thinking holistically about their processes while visualizing future scale up, Verlenden explains. “Our customers expect that, in five years, 40–50% of their processes will incorporate continuous capture and flow through polishing technologies, though adoption of fully continuous processes from end-to-end is likely to be further out,” he estimates.

	Hearkening back to traditional batch processes, Levison notes that traditional processes are inherently more disconnected step by step. In the upstream, more effective clarification with advanced depth filtration offers a flexible solution. “Users can work towards a semi- or fully continuous processing approach with continuous streams of feedstock, which also impacts the ability to integrate downstream processes.

	With the ability to integrate the downstream, manufacturers can create more efficient processes, increasing product quality, saving time and money, and maximizing overall productivity and facility utilization,” Levison says.

	Another development of note on the cell-therapy biomanufacturing front, meanwhile, is the integration of a complicated multistep process into a closed, modular, automated benchtop system that enables effective, safe biomanufacturing that can be used in a hospital (point of care) or in a central manufacturing facility, says Balestrini. Current instrumentation and methods for manufacturing cellular therapies are expensive, time consuming to use, difficult to scale, and limited in their ability to effectively deliver genetic material, Balestrini notes. “A modular system is emerging as an industry gold standard,” she states. 

	Yet, despite these advances in bioprocessing, additional work is still required to ensure that upstream and downstream processing are smoothly integrated, interjects Medvedev. He points to the struggle that current single-use downstream processing technologies for antibody purification are coping with-namely the increasing titers achieved in upstream processing.In gene therapy, on the other hand, the high performance of membrane, beads, and monolithic chromatography systems has caused a mismatch between what upstream processing is able to deliver and what downstream operations are able to purify.

	“This is caused by the fact that current technologies for viral vector purification were adapted from the protein industry, which makes them extremely oversized for viral vector applications.The DBC achieved using the current resins available is high with respect to the product harvested in upstream processing. This may cause manufacturers to pull different batches together with intermediate freeze steps, which has disadvantages in terms of yield loss, cold storage space, batch-to-batch variability concerns, and overall processcomplexity,” Medvedev explains.


	Vol. 44, No. 5
	May 2020
	Pages: 16–21

	When referring to this article, please cite it as F. Mirasol, “Technology Advances Streamline Bioprocessing,” 

Bioprocessing advances improve product yield, cut costs, and streamline integration between upstream and downstream processes.