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BioPharm International, November 2010 Issue (PDF)

Ensuring the integrity and quality of the biopharmaceutical supply chain has never been more difficult for life sciences companies or more scrutinized by regulators.  With globalization, the path that products take from source materials to consumers has grown increasingly complex. Many companies now must manage more relationships than ever with third parties, many of which operate in jurisdictions with weak regulatory systems. And as drug prices increase and supply chains become more complex, suppliers have more economic incentives and more opportunities to substitute ingredients or take shortcuts. 

Monitoring the supply chain is much more than simply testing the quality of raw materials or components. As all companies know, the US FDA expects sponsor companies to see that their contract manufacturing organizations (CMOs) are cGMP compliant, but with contract facilities often located far away and having complicated supply chains of their own, the challenge can be daunting. What is perhaps less well known is that you must also adequately monitor your third-party logistics and distribution providers. When those providers are the final link in the cold chain, effective monitoring is critically important. But because a typical cold chain involves moving a product through multiple storage locations and forms of transportation, which often include multiple flights, ground vehicles, and staging areas, monitoring these suppliers presents its own unique challenges.  

Meanwhile, regulators in many parts of the world, recognizing the increased vulnerabilities in the medical product supply chain, have stepped up their joint efforts at prevention and enforcement through increased information sharing, the leveraging of third-party auditors, and harmonization as referenced in the International Conference on Harmonization guideline Q10 

. In the US, the FDA has recently embarked on an ambitious supply chain safety initiative that includes, among other things, developing good importer practices, launching a Secure Supply Chain pilot program, setting out a foundation for a track-and-trace system, dedicating a larger portion of its resources to international inspections (opening offices in China, India, Latin America, and Europe), increasing domestic inspections, and increasing enforcement. Faced with this new emphasis on regulation and enforcement of supply chain security, biopharmaceutical companies would be well advised to re-examine their current practices in managing supplier quality.   

THE CONSEQUENCES OF POOR SUPPLIER QUALITY MANAGEMENT

Organizations that lack the resources, requisite capabilities, or determination to rigorously manage supplier quality may increasingly find themselves subject to the pitfalls of lax oversight. At the extreme, we have recently seen CMOs that have "show" factories where the product is purportedly manufactured when in fact it is being supplied by a "shadow" factory without the knowledge of the sponsor company. The sponsor's inadequate audit of the show factory finds nothing wrong, and the supplier continues to supply product from another location. Somewhat less blatantly, a supplier may move material from its initial place of production to a non-GMP compliant factory to save money, without notifying the sponsor company.   

Suppliers also may change their own suppliers of raw materials used to manufacture drug substance or other critical materials. For example, a supplier of vials might alter its manufacturing process, which in turn could lead to problems of glass breakage and contamination. If the sponsor has no mechanism in place for monitoring such changes, quality problems are difficult to anticipate and pinpoint.   

Lax oversight also may result in a decline in compliance over time. Because the supplier's quality performance has not been adequately or consistently tracked and measured, the sponsor company fails to notice when the supplier's compliance begins to trend downward. Quality problems that could have been prevented are instead allowed to fester until they eventually affect the quality of the sponsor's product. Sound quality supplier management also can save suppliers from themselves, helping them avoid regulatory actions—and helping the sponsor ensure an unhindered supply of needed materials or services.    

The consequences of poor supplier quality management can be profound. In addition to exposing companies to enormous financial liability and reputational damage, defective products can cause unquantifiable human tragedy in harmed patients. Valuable product also can be wasted or lost, resulting in unreliable supply to market, lost revenue, regulatory action, and the high costs of finding and fixing problems.    

Despite the potentially catastrophic consequences of poor supplier quality management, many organizations neglect it. In some cases, they may lack adequate human and technical resources to adequately monitor their suppliers. In other cases, they may monitor suppliers but neglect to follow up on concerns. Or, despite having an entire department dedicated to supplier quality, as in some large companies, a particular site still may not have received adequate attention. In other cases, the company may not have established clear and standard operating procedures (SOPs) and organizational mechanisms for supplier quality monitoring. 

According to the FDA's July 2009 report to Congress, "The cornerstone of a prevention-focused approach in the sourcing of ingredients or components and the manufacturing and distribution of medical products is the implementation of a quality management system (QMS)." As with many FDA requirements, the agency does not explicitly codify how to institute such a system, but provides only general guidance about what is expected, namely, that the QMS addresses "safety, quality, and security responsibilities of all persons who manufacture products, including starting materials . . ."  In our experience, a robust supplier quality management system (SQMS) should include, at a minimum, some key elements.   

 This is the foundation of an SQMS. It should define cGMP requirements and SOPs for suppliers of all kinds: CMOs, third-party logistics providers, and component and raw materials suppliers, and should take into account through due diligence and quality agreements these suppliers' suppliers. The supplier agreement should spell out quality standards and expectations, the communication of out-of-specification results, and a schedule of supplier audits. The agreement also should include a requirement that suppliers report to the company any changes in the way they do things, including their manufacturing methods and their sources of supply and transportation. Suppliers also should be required to report to the company any FDA observations that could affect the company's product.

It's not necessary to treat all suppliers equally. Given the sheer number of suppliers that many companies employ, enforcing the same audit schedule for all is extremely difficult. Label suppliers, for example, would not need to be audited as frequently as the suppliers of more critical elements like raw materials or equipment.  This approach not only makes the task more manageable but also allows the company to concentrate resources and effort in areas where the risk and the return are greatest. 

  One should also note that when a supplier is qualified, it is qualified for a particular material or service. Just because a supplier meets the requirements to provide one particular product, does not necessarily mean that it  meets the requirements to supply another.  

  Metrics should be focused solely on the quality and compliance performance of suppliers and considered separately from financial measures. These metrics might include the number of rejected items, temperature excursions, right-the-first-time rate, as well as observations from audits.  Particular metrics will of course depend on the nature of the goods or services being supplied. 

 Most companies do some level of monitoring, but sometimes fail to follow up on poor performance trends or observations made during audits. But if something is worth writing down, it is worth following up.  Follow-up audits should be conducted to determine if deficiencies have been corrected. 

  Some companies often execute an internal supplier qualification program. These programs allow companies to formally qualify, rank, and monitor suppliers and their products or services. Sponsor companies can have a high level of trust in suppliers who meet and maintain the rigorous requirements of these certification programs. Conversely, these programs should contain a mechanism for de-certification of a supplier if compliance problems arise.

For large companies, with dedicated supplier quality departments, establishing a more effective SQMS may entail only formalizing and enforcing existing policies and procedures. For other companies, it may be a matter of expanding oversight to take into account suppliers' suppliers or to include logistics providers in the program. Companies that lack resources may want to add them or look for ways to use their existing resources more efficiently.  Many companies, of all sizes and in all circumstances, will draw on external consultants to do continuous monitoring and conduct audits. The advantage that these third-parties offer is they have nothing else on their plates; they are specialists, they are objective, and they may be less expensive than internal personnel. In any case, they should be as carefully qualified as any other supplier.  

With the number of suppliers companies use, the increasing complexity of the supply chain, and increasing regulatory scrutiny, biopharmaceutical companies can no longer afford anything less than world-class supplier quality management.

 is a managing consultant at  Tunnell Consulting, Inc., King of Prussia, PA, 610.337.0820, 

Regulatory Beat

Best practices to strengthen supplier quality management.

Time to Revisit Supplier Quality Management

On March 21, 2010, the US House of Representatives passed the landmark Patient Protection and Affordable Care Act, which includes the Biologics Price Competition and Innovation Act. Finally, a path has been set for the approval of biosimilars through the US FDA by an abbreviated biologics license application that, in theory, should broaden patient access to these new drugs. As stated by Jim Greenwood, Biotechnology Industry Organization (BIO) president and CEO, "...patients living with debilitating diseases will have expanded access to safe and effective cutting-edge medical therapies at lower costs."   

But how true will Greenwood's statement ring in the years to come? 

Some might say it has taken too long and will probably take even longer for US patients to benefit. With recent announcements from across the pond such as the approval of Nivestim (filgrastim) in Europe, a biosimilar to Amgen's very successful Neupogen, US patients may question the delay. Nivestim is the fifth European approval of a biosimilar of Neupogen. The EMA is a step ahead, leading the way forward with a regulatory framework for the emerging biosimilars market that already is being put into practice. The results can clearly be seen with the approval of five biosimilars to Neupogen alone. Is the new act welcome? Of course, but it has fallen short of providing a streamlined approval pathway that broadens access to cutting-edge medical therapies.  

Countless issues have been raised with the dawn of biosimilars, such as patient safety, comparable efficacy, and the substitution of originator biological products, to name a few. Patient safety should be paramount and should remain so. It is well known that variations in the quality and efficacy of biologics can be seen at different production facilities despite every effort being made in process transfer. With this in mind, biosimilar production is made even more complex when processes are not readily disclosed for originator biological products. Therefore, safety and efficacy testing become paramount through the costly and time-consuming route of clinical trials.  

But where does this leave the US patient? In Europe, patients already can take advantage of the supply of biosimilars, as can patients in many developing countries around the world. In the near term, it would appear that US patients may have to access more advanced affordable therapies overseas, which could put US patients at risk.  

The goal of the act must be to provide the impetus to make a competitive market, which encourages more affordable medicines in a dynamic, innovative, and fast growing sector of the healthcare industry. However, the competition will be muted because only a few companies will have the financial pockets deep enough to fund the regulatory requirements to deliver a biosimilar to market. In addition, any targeted originator product market will need to be of a size capable of providing acceptable returns on the time and investment. Consequently, smaller product markets may well not see accessibility increase. Of course, companies are already looking at solutions to the significant investment required. Joint ventures, partnerships, and alliances will no doubt proliferate as shown by the likes of Teva and Lonza, which have an alliance to exploit their relevant expertise to develop biosimilars.  

Ultimately, regulators must remember that the aim is not only to deliver safe and efficacious medicines but also to provide a framework for affordable and equitable healthcare. However, what the biosimilars market has put into perspective is the decreasing return on R&D investment to provide new and innovative drugs. That is still the biggest challenge that we all face in healthcare from the innovator to the patient, and regulators have a huge and positive role to play in this. 

 is the business development and marketing manager at RecipharmCobra Biologics, Keele, UK, +44 (0)208 246 5895, 

Final Word

The pathway for biosimilar approval in the US has been set. But are US patients too far behind Europe?

Biosimilars Regulation in the US: The Challenges

Enhanced process and product understanding are the basic tenets of Quality by Design (QbD). Although significant advances have been made in this respect, appropriate characterization and management of raw materials remains a concern for the regulatory authorities. In view of the large number of raw materials that typically are used in biotech processes, a QbD-based approach for raw material management must be based on scientific knowledge and risk analysis. This will ensure that adequate characterization is performed on those raw materials that are expected to have the most effect on process consistency and product quality. Part 1 of this article will present an approach for raw material management following the QbD principles. Part 2 will discuss how to conduct risk assessments, and will give an example of the application of risk assessment tools for raw materials in biotech processes.

The contribution of raw materials to product quality, safety, and process performance is considerable. It is therefore important in designing processes and selecting raw materials that thought is given to understanding their purpose and suitability for intended use. Raw materials also are a major source of variability and steps must be taken to minimize any negative impact arising from materials and their sources. There are essentially two parts to this; prevention, through assessment, inspection, and control of incoming materials; and intervention, in which processing conditions are modified to account for the variability, sometimes through continued monitoring of materials and their impact on process performance. 

An initial assessment of materials must extend beyond the material specification and its direct effect on the process to include a host of additional issues such as the supplier's manufacturing processes, quality systems, and sourcing strategy. This is particularly true of biopharmaceuticals, where processes are highly complex and a wide range of different materials are used which can affect quality and process performance in both subtle and catastrophic ways, as with the recent experiences of heparin and melamine contamination. It is necessary to characterize and understand how these interactions can occur, and if they affect the process or product negatively, but it is unrealistic and impractical to explore every possible permutation of material variables and process parameters. Modern design approaches recommend taking a science- and risk-based approach to development

  and the following sections will focus on how risk management practices can be applied to prioritize the required studies. 

 series presents an approach for raw material management for biotech therapeutic products following the Quality by Design (QbD) principles. Part 2 will discuss how to conduct risk assessments, handle risk communication to stakeholders, and also will give an example of the application of risk assessment tools to raw materials for biotech processes.  

Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients (APIs)

 discusses the various sources of raw materials and makes recommendations on their handling.

  It defines an API starting material as "a raw material, intermediate, or an API that is used in the production of an API and that is incorporated as a significant structural fragment into the structure of the API". This guideline provides recommendations on a variety of topics, such as quality management, personnel, buildings and facilities, process equipment, documentation and records, materials management, production and in-process controls, packaging and identification labeling, storage and distribution, laboratory controls, validation, change control, rejection and reuse of materials, complaints and recalls, and contract manufacturers. Furthermore, the guideline provides specific guidance for APIs manufactured by cell culture or fermentation and also on APIs used in clinical trials. The guidance presented in this document can serve as the foundation of the raw material management program for a company.  

 introduces QbD as "a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management."

  The QbD approach involves identifying product attributes that are of significant importance to the product's safety and efficacy, design of the process to deliver these attributes, a robust control strategy to ensure consistent process performance, validation and filing of the process demonstrating the effectiveness of the control strategy, and finally ongoing monitoring to ensure robust process performance over the lifecycle of the product. Although the Q8 guidance is intended for drug product, some of the concepts are applicable for raw materials as well and should be taken into account when creating a raw material management program.  

guideline provides principles and examples of tools for quality risk management that can be applied to different aspects of pharmaceutical quality.

  These aspects include development, manufacturing, distribution, inspection, and submission or review processes throughout the lifecycle of drug substances, drug (medicinal) products, and biological and biotechnological products. As will become evident from this article, risk analysis and management pay a critical role in management of raw materials and as such, the ICH Q9 guideline serves as a useful reference when creating a raw material management program. 

Besides the ICH guidelines mentioned above, specific guidance also has been provided by the US Food and Drug Administration,

  and the Japanese Ministry of Health, Labor, and Welfare (MHLW).

  Although they provide regulatory expectations from the different jurisdictions, they all share the foundation laid by the ICH guidelines.  

CHALLENGES ASSOCIATED WITH RAW MATERIALS USED IN BIOTECH PROCESSES

Certain raw materials used in biotechnology processes are complex in nature as they often are not well-defined and known to exhibit lot-to-lot variability with respect to their impact on the process (for example, soy hydrolysate). There are a wide range of raw materials and components which have the potential to affect product quality or process performance. It includes process materials, process aids, materials contacting process fluids, excipients, devices, and primary and secondary packaging. A list of material categories is given in Table 1. 

Table 1. Material categories used in bioprocessing. The list is not meant to be exhaustive but rather used for illustration. 

Some of the several key challenges that the biotech industry faces with respect to raw material management are:

1. With respect to complexity of raw materials, a particularly challenging problem is that of the various components that are mixed to form the media for microbial fermentation or mammalian cell culture steps. A large number of such components are used, often a mix of chemically defined raw materials and complex raw materials such as hydrolysates.

  The performance of the process step is known to be sensitive to small changes in some of these components and even to changes in the procedure used to produce the media from them.  

2. A typical biopharmaceutical manufacturer may have anywhere from 20–50 vendors that are sourcing raw materials for a given process. Because the manufacturer is legally responsible for the quality of the raw materials that it procures and uses, raw material management is a complex task. 

3. Biotechnology processes are known to use a large number of different raw materials (typically 50–100). It is not feasible to examine the effect of each raw material experimentally.  

Variability in product quality caused by variability in the quality of raw materials has been highlighted as a concern by the regulatory authorities. It is widely accepted now that a robust raw material management system must be in place to facilitate implementation of QbD. This is greatest towards the end of the process, where the effect on the drug product quality is significant. 

ADVANCES IN RAW MATERIAL CHARACTERIZATION

With the large number of raw materials that are used in typical biotech processes, characterization of raw materials requires use of advanced analytical tools in combination with chemometrics. In this section we review some of the approaches that have been proposed in the literature: 

1. Proteomic analysis has been proposed as a tool for assessing complex raw materials such as the fetal bovine serum.

  Proteomic techniques were used to understand the lot-to-lot variability with respect to impact on growth properties of the cell culture process. A time course study was performed to monitor specific changes in the fermentation medium.  

2. An approach for routine testing of packed raw materials used in pharmaceutical processes has been recently proposed.

  The chemometrics-based approach consists of three steps. First, the initial calibration objects are divided into two classes using a global principal components analysis (PCA) model. Next, two separate PCA models are constructed. Finally, soft independent modeling by class analogy (SIMCA) is applied for calculating acceptance area. The approach was successfully used to analyze fourier transform-near infrared (FT-NIR) spectra of taurine samples.  

3. In a recent publication, a combined approach of near-infrared (NIR) spectroscopy and chemometrics for screening of lots of basal medium powders based on their impact on process performance and product attributes has been proposed.

  A combined NIR and chemometrics approach was able to finger print the raw materials to distinguish between good and poor performing media lots. 

4. A recent book chapter described a number of upcoming analytical techniques, including high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), inductively coupled plasma- mass spectrometry (ICP-MS), and liquid chromatography–mass spectrometry (LC-MS) and described experience managing these methods and results as part of a retrospective investigation.

  A case study also was presented using NMR as an analytical tool and PCA for analyzing data for characterizing raw materials for a cell culture step.  

With the above mentioned advances in characterization of raw materials, it is possible to fingerprint critical raw materials and ensure that only those lots that have acceptable quality are used.  

QBD-BASED APPROACH FOR RAW MATERIAL MANAGEMENT

Figure 1 presents an overview of one approach that could be implemented for managing raw materials in the QbD paradigm.

  As illustrated in the figure, raw materials are initially selected during early-stage or platform development, before detailed characterization during late-stage development, they are then subject to continual evaluation throughout the product lifecycle. The initial risk assessment is performed early in the development process to prioritize areas of concern and focus further research, and is repeated as greater knowledge is obtained. Typically, knowledge is limited in the early stages to general information about the material and its performance in similar processes. The process typically is not fully characterized until process characterization is initiated (typically after end-of-Phase 2 clinical studies are successful). Stage-appropriate risk assessment practices are followed at each point, with the level of detail increasing as more knowledge becomes available. Based on platform knowledge and product-specific knowledge, the raw materials can be categorized into three categories: (1) critical raw materials, (2) key raw materials, and (3) non-key raw materials. A combination of mechanistic modeling and multivariate analysis can be used to analyze the effect of the various raw materials on the different process and product quality attributes. The approach could be used to identify raw materials that have the most potential to impact and hence need the most characterization.

Figure 1. Raw material management approach in the Quality by Design paradigm 

 are known to significantly affect product quality and these raw materials are therefore thoroughly characterized and their mechanisms of process interactions well understood. A relationship is developed among the relevant raw material attributes and the quality attributes that are being affected. For these raw materials, acceptance criteria must be developed within which their attributes can vary without leading to unacceptable product quality. Furthermore, analytical tests are developed to monitor raw material attributes to ensure that each raw material lot meets the respective acceptance criteria before its use in the process.  

 do not significantly affect product quality but do impact process consistency. These raw materials can be characterized as thoroughly as critical raw materials, but this is a risk-based decision and will depend on the platform and the product. They might or might not require routine monitoring based on the significance of their impact. If a new key raw material is added to the platform, it should undergo the thorough characterization of a critical raw material before being used in the platform.  

 include the remaining (and majority of) raw materials. These materials are handled through the internal quality system of the biopharmaceutical manufacturer as in traditional pharmaceutical manufacturing.  

For all raw materials, the biotech company should have an internal quality system that would ensure appropriate raw material management. Some key aspects of this system would be:

1. Review of the certificate of analysis from vendor and confirmation that the raw material lot meets the internal specifications. Materials specifications are set based on materials meeting the performance requirements of the process in question. In the vast majority of cases, material specifications are predetermined by the supplier. It is still up to the manufacturer to determine if they are adequate for their intended use, and in some cases it may become apparent over time that alternative or additional specifications are required. 

2. Analysis of raw materials as necessary. The testing plan should ensure that while critical quality aspects are examined, superfluous testing that is not providing useful information is not performed. 

3. Review of any changes to the vendor's manufacturing process with respect to change in raw material quality and its impact on process consistency and product quality. 

4. Vendor site audits at appropriate frequency to ensure vendor qualification as needed during product lifecycle. These may include technical audits by subject matter experts to assess supplier capabilities and competencies. 

5. Appropriate supply chain management system in place to avoid failure of incoming raw material lots in meeting specifications.  

6. Ensure traceability of raw materials.  

7. Raw materials of animal origin should be checked with respect to requirements with animal feed, animal age, manufacturing practices of the vendor, and other relevant aspects.  

8. Custom-synthesis based raw materials need to be thoroughly characterized to ensure the process that is used to make them is well controlled and the raw materials are comparable across lots and scales that were used over product lifecycle.  

9. A robust change control program should be in place to assess potential changes of the raw material to evaluate the potential to impact product quality.  

Raw material risk assessments are focused principally on the effect on the process in terms of quality and process performance, but additional risk factors should be considered in selecting and a material and its source, such as material risk (e.g., toxicity, immunogenicity, viral safety), and risks posed by the supplier (quality systems, business, and technical capabilities). This is presented in Table 2. Raw materials are required to meet industry standards where relevant, and should undergo testing to confirm compliance with user specifications. Materials should be fully traceable to the site of origin and handling procedures should be in place to prevent cross-contamination. Deliberate adulteration of raw materials for economic or other reasons is a particular concern. Its detection and prevention requires a combination of analytical controls and supplier relationship management. 

Process risks arise from the stage in the process where the material is used and the effect the material has on product quality or process performance at that point and at subsequent steps. The risk posed by the material, rather than by processing conditions, is assessed. As in all risk assessments, the evaluation should be performed by a multifunctional team consisting of the development team (experience of the specific molecule) together with subject matter experts with experience of similar molecules and unit operations, or packaging and delivery devices as appropriate. Process risks can affect product quality, and process performance (supply continuity, variability) and end-user satisfaction. 

Features

Adequate characterization of materials protects product quality.

Managing Raw Materials in the QbD Paradigm, Part 1: Understanding Risks

Developing chemically defined (CD) media suited for multiple Chinese hamster ovary (CHO) cell lines can be a challenging and extensive process because of the complexity of cellular metabolism and the associated phenotypic variations observed in cell lines, even when derived from the same parental cell line. In this article, we present certain systematic approaches to develop CD cell culture media. Two feed media development applications have been explored, each with distinct goals. First, a systematic approach was used to develop a platform CD feed medium, which can be used for many antibody producing CHO cell lines without any major cell line-specific optimization. Second, an iterative optimization approach was applied to develop an enriched feed media tailored to individual cell lines, to achieve higher titers.  

As cell culture technology has matured, platform processes have become the standard in the biotechnology industry, particularly in early-stage development, to enable the rapid production of toxicology and Phase 1 and 2 clinical material with acceptable product quality attributes. The cell culture platform often consists of a standard host cell, expression vector, transfection and selection methods, cell culture media and culture techniques with appropriate process control, and scale-up methodologies. An ideal cell culture platform medium not only enables rapid cell culture process development, but also could provide predictable process performance at different production scales and thereby aid in the development of downstream process platforms as well. 

The majority of the biopharmaceutical industry currently is using fed-batch cultures of animal cells as the platform technology to synthesize biopharmaceutical proteins because it offers the benefits of high yields and operational simplicity.

  A fed-batch process requires a basal medium to support cell growth and productivity and a feed medium to prevent nutrient depletion, extend culture longevity, and maintain high volumetric productivity. A typical basal medium contains 50–70 chemical components, including carbon sources, amino acids, vitamins, salts, and trace elements. Feed media usually contain fewer components than the basal media, but at higher concentrations.

  Cell culture media have evolved over the years from serum-containing to chemically defined (CD) formulations.

  Recent advances in cell culture media optimization have resulted in higher volumetric productivities in fed-batch processes. Cell culture media optimization is an integral part of mammalian cell culture process development and optimization, which support all activities from cell line development and clone selection to process optimization and scale-up. Cell culture process development starts with cell line generation and selection, followed by process and media optimization in scale-down systems, including 96-well plates, shaker flasks, and bench-scale bioreactors. After the process conditions are defined, the process often is transferred to the pilot scale to test scalability and produce material for preclinical toxicology studies. Subsequently, the process is further scaled-up to generate clinical material under current good manufacturing practices (cGMP) regulations (Figure 1). The ideal cell culture medium should support robust cell growth, productivity, and product titer and quality goals, both during development and process transfer to different scales and manufacturing sites. 

Figure 1. A platform cell culture medium, integrated from early cell line development activities to clinical production, streamlines process development to meet aggressive timeline 

Historically, bovine serum had been a popular supplement in cell culture media because it improves cell growth and productivity. Regulatory agencies have encouraged the industry to avoid animal-derived supplements to mitigate the risk of introducing adventitious agents such as viruses and prions.

 Protein hydrolysates, which can provide similar benefits as serum, can be animal-derived from bovine milk or tissues, or non-animal–derived from yeast, cotton seed, soy, wheat, or other plant sources. Some disadvantages of hydrolysates include lot-to-lot variability leading to process and product inconsistencies. The undefined chemical composition of hydrolysates can lead to challenges in process understanding, making further medium optimization difficult.

  Therefore, many biopharmaceutical companies are transitioning to CD media formulations and a few vendors now also offer CD basal and feed media.

 We observed that the current version of our peptone-containing media, when used with most cell lines, lead to robust cell culture performance at different scales and generated titers greater than 1 g/L for most of our in-house cell lines. After removing the peptone from our platform feed medium, a systematic approach was applied to develop a suitable CD platform feed medium. An iterative optimization approach was then applied to develop enhanced feeds tailored to individual cell lines to achieve higher titers.  

A SYSTEMATIC APPROACH TO MEDIUM DEVELOPMENT

Establishing Criteria for Platform Medium and Model Cell Lines

Several criteria need to be considered before developing a CD platform medium. Robustness of the medium must be demonstrated for cell line development applications, clone selection, seed and inoculum trains, and in production cultures using representative cell lines. These model cell lines should be identified to include diversity in properties such as host cell, expression plasmid, selection and amplification methods, growth, metabolism, and specific productivity (Qp). In addition, manufacturability and scalability criteria must be met, including consistent preparation, filtration methods, and stable shelf life. Moreover, titers should be comparable to that achieved in the peptone-containing platform medium, with acceptable product quality profiles (Figure 2). 

Figure 2. Considerations for developing a robust platform chemically defined medium 

Because of the complexity of cell culture media formulations, it was not practical to use Design of Experiment (DOE) approaches to simultaneously optimize all compositions. Instead, we applied a step-by-step optimization strategy by starting with media screening, followed by subgroup titration, single and multiple component titration, and finally conducting confirmation studies (Figure 3). 

Figure 3. A systematic medium development strategy 

 An in-house chemically-defined feed (CDF), previously used in combination with peptone, was chosen as the starting point after wide-ranging screening experiments that compared this with other in-house and commercially available feed media. This medium had a simple proprietary formulation, which made optimization less complex. The overall strategy involved enriching this feed with essential nutrients previously shown to improve performance for many cell lines. The same CD basal medium, which was chosen after extensive small-scale screening studies in batch mode, was used for all experiments conducted for feed optimization.  

 To quickly optimize the current CDF, we used an optimization strategy that involved enriching the feed medium with subgroups of media components. Approximately 30 media components were divided into three different subgroups, namely amino acids, vitamins, and an "others" sub-group that included nucleosides, fatty acids, amongst other components. Instead of working with individual components, these subgroups were titrated into the CDF in shaker flasks to reduce experimental complexity. Traditionally, components have been individually titrated at various concentrations and optimized. Our subgroups strategy was less labor intensive, demonstrated the importance of the vitamin subgroup concentration, and helped identify the optimal vitamin subgroup concentration. The newly adjusted concentration of vitamins resulted in improved cell growth and extended viability, even for a cell line that exhibited poor growth and high lactate synthesis (Figure 4). 

Figure 4. For two different model cell lines tested in shake flasks, vitamin subgroup titration studies demonstrated similar growth compared to the peptone-containing process 

Identification and optimization of key components:

 To complement shaker flask studies, a modified version of the CDF was evaluated in 2-L scale-down bioreactors using a cell line that exhibited robust growth and productivity. Although shaker flasks or high-throughput systems are more powerful for screening a larger number of medium components or cell lines, small-scale bioreactors with pH and dissolved oxygen control capabilities are more representative of conditions encountered in large-scale processes used in clinical or commercial manufacturing. The bioreactor process using the vitamin-enriched feed medium was conducted for 14 days, and supernatant samples were collected daily to quantify specific components in the spent medium and to determine the specific amino acid consumption and production rates. Based on specific amino acid consumption rates, key amino acid concentrations were adjusted in the feed medium. Supplementing CDF medium with vitamins and amino acids resulted in higher cell density, specific productivity, and overall final titers. Key amino acids asparagine, tryptophan, and valine were not depleted during production.  

Figure 5. For 10 different model cell lines tested in 2-L bioreactors, the average titer in the chemically defined (CD) medium was 30% higher than the peptone-containing process  

 Multiple cell lines previously used in early-stage and late-stage projects were tested in 2-L scale-down bioreactors for confirmation that the newly developed CDF was optimal for a variety of cell lines. Compared to the peptone containing medium, the CD medium resulted in an average titer improvement of 30%. Among the 10 cell lines tested, only three had lower titers in CD media (Figure 5). It was worth pointing out that the cell lines shown in Figure 5 also were preselected in a peptone-containing medium. To implement the new CD medium in a manufacturing facility, there are many process scalability items to be tested, such as large-scale medium preparation and cell culture process performance. An example of such a scale-up run using a late-stage cell line at a 400-L scale is shown in Figure 6. In this case, titer, cell growth, and metabolism profiles were comparable between the 2-L and 400-L scales. 

Figure 6. Scale-up of the chemically defined medium from 2 to 400 L (data shown for 2 Ã 2 L and 1 Ã 400 L runs) 

: Changing medium formulations may introduce challenges in comparability of product quality. It is necessary to ensure that the transition from peptone-containing to CD media did not cause major product quality changes, especially for the molecules already in the clinical development stage. Our results show that the product quality differences were acceptable in terms of protein aggregation level, glycosylation pattern, and charge variants (Table 1). Furthermore, we have demonstrated that the CD medium gave consistent product quality profiles at different scales throughout cell line development, process development, and manufacturing stages. 

Table 1. For five different model cell lines tested for product quality, aggregate levels, glycosylation patterns, and charge variants have minor and acceptable differences 

Enhanced Feed Medium and Process Development

Although high titers are typically not required for early-stage development because of relatively low material requirements in early clinical stages, they often are desirable for some late-stage processes because of market demand or manufacturing capacity constraints. There are two reasons to develop enriched media. First, to gain a better understanding of the determinants for high volumetric productivity in cell culture fed-batch processes. Second, to develop a general cell-line–specific medium optimization methodology to achieve higher titers.  

Although a platform process emphasizes simplicity of feeding strategy and ease of implementation during manufacturing, it might not lead to the maximum possible volumetric productivity that might be achieved with any specific cell line. The aim of developing the enhanced process was to achieve higher titers with a specific cell line through feed media optimization and the development of feeding strategies. 

 Enhanced feed medium optimization was achieved through an iterative process to customize the formulation for specific cell lines. In this approach, metabolism and other key performance indicators were monitored to determine levels of lactate, ammonia, and osmolality. Nutrient and metabolite consumption rates from fed-batch bioreactor experiments were analyzed and used to adjust the feed formulation and feeding strategy in accordance with the nutrient requirements for a specific cell line. To achieve this, daily supernatant spent medium samples were collected and used to obtain amino acid and vitamin consumption profiles. With accumulating components reduced or omitted, more essential components could be added. The feed medium formulation was adjusted when components were either increased or reduced. In this case, amino acids and vitamins were further enriched. Because of variability associated with cell growth processes and the measurement of nutrient consumption rates, this had to be conducted iteratively to adequately optimize the culture medium. Thus, the feed medium was reformulated for each subsequent study, with the goal of maintaining a high specific productivity by supplementing key nutrients at desired levels. In our study this was achieved by 2–3 rounds of optimization studies (Figure 7).  

Figure 7.  Development of enhanced feed medium and feeding strategy in an iterative optimization process 

The enhanced fed-batch process was operationally more complicated than the platform fed-batch process. In both cases, however, the timing of specific volume additions needed to be optimized for each cell line, but for the enhanced process, the feed volume was calculated daily based on a combination of viable cell counts, cell size, and viability. This iterative process for reformulating feed and feeding strategy was tested on two model cell lines with different growth and productivity characteristics. MAb A exhibited high growth but average specific productivity, whereas MAb B exhibited low growth but high specific productivity. The customized enhanced feed resulted in improved cell growth and significant titer increase. Titers as high as 9 g/L (MAb A) and 6.7 g/L (MAb B) were achieved compared in the platform medium to 4.2 and 4.6 respectively (Figure 8). Our results indicate that compared with the platform feeding strategy, the enhanced feeding strategy prevented excessive osmolality increases, nutrient depletion, and enhanced volumetric productivity. 

Figure 8. Feed optimization by the iterative process (enhanced feed) resulted in significant titer increases for two model cell lines compared to the platform feed 

Using a CD medium in cell development workflows could potentially lead to more comparable results between cell line development and early-stage process development by minimizing changes or eliminating cell adaptation when using a different production medium. Some cell lines also can adapt slowly to CD media during the later stages of development for inoculum expansion and protein production.

  To transition from early-stage to late-stage processes, cell-line–specific process optimization is more straightforward in CD media compared to media containing undefined raw materials, as exact concentrations of all components are known. 

It was essential to balance solubility and osmolality of the platform basal medium for ease of use, manufacturability, and long-term storage. The enriched feed medium, on the other hand, was formulated to minimize the dilution of culture volume during feeding without resulting in an excessive increase in osmolality. Excessive concentration of feed media should be avoided because this could lead to the precipitation of specific media components if their concentration exceeds their solubility.  

Developing a CD feed platform and enhanced feed formulations for fed-batch processes resulted in overall benefits with more favorable metabolic profiles and culture growth. Interestingly, tested cell lines that were lactogenic in the peptone-containing medium exhibited a shift from lactate production to consumption during the later stage of cultures in the CD medium. At the end of these processes, most cell lines had almost undetectable lactate concentrations that resulted in higher peak cell densities and extended viability during production. The importance of the newly adjusted amino acid and vitamin concentrations were clearly identified to improve lactate metabolism, enhance growth, extend viability, and increase volumetric production rates.

Systematic feed medium development approaches were successfully used to enable the transition from a peptone-containing process to a process using chemically-defined basal and feed media. An iterative process was used to increase titers based on the metabolic needs of specific cell lines. In addition, we have successfully conducted filterability, medium preparation, and scale-up studies. These CD formulations have the potential to increase process robustness, generate higher titer processes with acceptable product quality, and be used for further medium optimization efforts. 

The authors would like to acknowledge the contributions of Gayle Derfus, Roberto Flores, Jennifer Autsen, Joana Caria, Dacia Brooks, Masaru Shiratori, and Robert Shawley for their guidance, ideas, and support.  

 is a senior engineer, all at Oceanside Pharma Technical Development,  Genentech, Inc. Oceanside, CA, 760.231.2127, 

 is a senior scientist, both at late stage cell culture, US Biologics Pharma Technical Development, Genentech, Inc., South San Francisco, CA. 

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2.  Fike R. Nutrient supplementation strategies for biopharmaceutical production, Part 2 Feeding for optimal recombinant protein production. BioProcess Int. 2009;7(11):46–52.  

3.  Vijayasankaran N Li J, Shawley R, Chen A, Shiratori M, Gawlitzek M, Li F, Kiss R, Amanullah A. Animal cell culture media. In: Flickinger MC, ed. Encyclopedia of Industrial Biotechnology, Bioprocess, Bioseparation, and Cell Technology. New York: John Wiley & Sons, Inc. 2010; p. 261–75. 

4.  Heidemann R, Zhang C, Qi H. Larrick Rule J, Rozales C, Park S, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D. The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnol. 2000;32(2):157–67. 

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7.  Hodge G. Media development for cell Culture. Biopharm Int. 2005;18(5):54–57 . 

Systematic Approaches to Develop Chemically Defined Cell Culture Feed Media

Monoclonal antibodies represent a significant portion of sales in the biopharmaceuticals market. Ever-growing biotechnology pipelines have greatly increased demand for higher throughput, multi-product protein characterization methods. By taking a holistic approach to improving the analytical process, one can identify strategies to increase throughput and reduce development times and the costs of analytical methods. This article discusses recent advances in high-throughput and multi-product liquid chromatography and electrophoretic methods for protein characterization. In addition to advances in analytical methodology, technological advancements in sample preparation throughput and data handling automation add value and time savings to the overall characterization process, which will enable increased productivity and faster product development.

Monoclonal antibodies (MAbs) are an important class of therapeutic proteins in biotechnology and have been developed to treat a variety of indications to fill significant unmet medical needs.

  MAbs generally are target-specific and well tolerated with a relatively long half-life, contributing to their success in drug development. Of the classes of immunoglobulins, IgG1 is the most commonly used for pharmaceutical and biomedical purposes.

In recent years, increased understanding of disease and advances in drug discovery have resulted in continuously growing pipelines for potential therapeutic antibody products. To keep up with the increasing demand for protein characterization, high-throughput and multi-product characterization methods are being developed. If used correctly, these methods can reduce analysis and development time and thus accelerate the progress of therapeutic proteins toward the clinic.  

REGULATORY EXPECTATIONS FOR THE ANALYSIS OF MABS

Several guidance documents have been issued by regulatory agencies recommending approaches for protein characterization.

  Although guidance documents are valuable to the industry, other publications have emerged that more clearly identify methods that are useful for characterizing MAbs.

  These orthogonal assays include potency, identity, and impurity assays that evaluate critical quality attributes (CQAs) such as size and charge heterogeneity.

  These CQAs are part of the overall target product profile, which is based on the desired clinical performance. Appropriate control of CQAs is a common review concern for both investigational new drug (IND) and license applications.

  Product quality characteristics encompass a wide variety of product variants that include product-related species and product-related impurities.

  For example, aggregation is a carefully monitored product variant from the earliest stages of clinical development because of the possibility of eliciting an immunogenic response in the patient.  

Many of the recommended protein characterization assays are based on liquid chromatography methods such as ion exchange chromatography (IEC) for charge heterogeneity analysis, size exclusion chromatography (SEC) for size heterogeneity, and reverse-phase high performance liquid chromatography (RP-HPLC) for peptide mapping.

  This article primarily will focus on multi-product and high-throughput charge-based methods for analyzing charge heterogeneity and size exclusion methods for quantifying size heterogeneity, in particular aggregates or high molecular weight species (HMWS). These methods are among the most frequently used tests for lot release of drug substance and drug product,

  as well as during formulation and process development. Several multi-product analytical methods for MAb size and charge assessment are shown in Table 1. 

Table 1. Multi-product analytical methods for monoclonal antibody size and charge assessment 

Before an analytical method can be incorporated into a characterization platform or a quality control system, it must first demonstrate that the method is suitable for its intended purpose. Guidelines for validating analytical methods have been published in the 

  by the US Food and Drug Administration,

  The guidelines published by the International Conference on Harmonization (ICH) have established a uniform understanding of the performance characteristics that are evaluated in the course of validation.

  Although high-throughput and multi-product methods can save time and add value to a business process, these methods also must be evaluated considering regulatory requirements and validation procedures. In other words, the validatability of these methods must be assessed before implementation. 

HOLISTIC VIEW OF SAMPLE ANALYSIS FOR PROTEIN CHARACTERIZATION

A generalized sample analysis workflow is shown in Figure 1. Typically, protein samples are submitted, prepared, and analyzed, and then results are reviewed and reported. When developing high throughput or multi-product analytical methods, it is important to keep a holistic viewpoint of the sample analysis workflow to optimize the total process. For example, if a new method is developed with reduced run time but with increased sample preparation steps, then the value of reduced run time could easily be negated.  

Figure 1. Schematic of a generalized sample analysis workflow 

When exploring high-throughput and multi-product methods, investigation into cost savings is aided by a holistic view of the organization. Methods that use the existing analytical equipment in an organization may be more advantageous to develop than methods that require extensive capital investment on new instruments and consumables. In addition to capital costs, analyst experience should be leveraged when implementing new methods. Many biotechnology and pharmaceutical companies are well equipped with many types of liquid chromatography instruments and analysts that are trained to use them. Thus, the development and implementation of high-throughput and multi-product liquid chromatography methods that leverage existing facilities may result in greater time and cost savings compared to novel methods that do not use existing resources. Over-engineering a solution should be avoided; an existing technology should not be overhauled when a minor update would suffice to meet business needs. 

MULTI-PRODUCT OR HIGH THROUGHPUT MAB CHARACTERIZATION METHODS

As mentioned previously, heterogeneity of MAbs is monitored as part of the ongoing control system that ensures product quality and manufacturing consistency throughout the commercial life of the product.

  Capillary electrophoretic separations, such as capillary electrophoresis with SDS (CE-SDS) and capillary iso-electric focusing (CIEF), have been shown to have significant potential for multi-product usage, negating the need for method development for each product.

  CE-SDS analysis can serve as a replacement for silver-stained SDS-PAGE to detect low-level impurities, MAb size variants, and to determine lot-to-lot consistency.

  The high separation power of CE-SDS has been demonstrated by showing baseline resolution between the heavy chain and nonglycosylated heavy chain species present at a low level (2%).

  Recently, quantitative CE-SDS was validated for MAb quality control and stability monitoring.

  CE-SDS also can be performed on microfluidic chip systems for MAb analysis.

  Microfluidic systems offer advantages compared to conventional CE systems, including increased speed of separation, reduced band broadening effects, and low sample consumption.

  Chip-based CE systems can be used for high-throughput analysis because several chips can be applied in parallel. 

CIEF is an orthogonal tool used for assessing the charge heterogeneity of MAbs. CIEF usually is performed on commercial capillary electrophoresis instruments, which have a 20–60 cm long capillary and on-column ultraviolet (UV) absorbance detector. Imaged CIEF, or iCIEF, which uses whole-column imaging detection through a charged coupled device (CCD) camera, recently has been commercialized by Convergent Biosciences.

  Although iCIEF is a relatively new technique, it has the potential to assess the overall charge distribution of a variety of MAbs. However, iCIEF methods require specific equipment, and fraction collection for further characterization is indirect. Samples for iCIEF also must undergo additional sample preparation steps, frequently including a buffer exchange before analysis.  

Figure 2. MAb samples analyzed by quaternary solvent IEC. Samples were digested with carboxypeptidase B before analysis. Flow rate was set to 0.5 mL/min. Sample loading was 20 Âµg. Column temperature was 40 Â°C. Solvent A: water. Solvent B: 0.5 M NaCl. Solvent C: 20 mM ACES, 20 mM MES, 20 mM Tris, 20 mM HEPES, pH 5.5. Solvent D: 20 mM ACES, 20 mM MES, 20 mM Tris, 20 mM HEPES, pH 8.5. 

Ion-exchange chromatography (IEC) is widely used for profiling the charge heterogeneity of proteins, yet despite good resolving power and robustness, ionic strength-based ion exchange separations are product-specific and time-consuming to develop. Some improvements have been made to decrease the development time of IEC, such as the development of a quaternary solvent system to simultaneously adjust the pH and salt concentration to reduce buffer preparations during development. In a quaternary solvent system, different combinations of the salt gradient and pH are achieved automatically by programming the HPLC pumps to deliver solvents from four different solvent channels. Figure 2 shows chromatograms obtained on a quaternary solvent IEC system for MAb charge heterogeneity analysis. Pumps A and B were used to adjust the salt gradient, and pumps C and D were used to maintain a target pH. By using the quaternary solvent system, method development time was greatly reduced, from a typical development time of weeks down to four days. Other advantages of developing a method using a quaternary solvent system includes ease of mobile phase composition control, which can greatly reduce the labor needed to make and change a variety of buffers, and the capability of automation for buffer screening during method development. Despite these advantages, the final developed IEC method is product-specific and carries the same disadvantages of conventional IEC, such as limitations in analyzing in-process samples caused by intolerance of the method to various sample matrices and low tolerance to buffer pH changes.

Figure 3. Elution profiles for five MAbs with different pI values using a single pH gradient ion exchange method. Column: ProPac WCX-10, 4 mm Ã 50 mm, 10 Âµm particle size. Column temperature: 25 Â°C. Flow rate: 1.0 mL/min. Sample load: 20 ug (20 ÂµL injection), with samples diluted to 1 mg/mL with mobile phase A. Gradient: 0â100% B in 16 min. Mobile phase: 9.6 mM Tris, 6.0 mM imidazole, 11.6 mM piperazine A) at pH 6.0, B) at pH 11.0. 

We have previously reported a novel pH-based separation of proteins by cation exchange chromatography (pH-IEC) that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH.

  Simple mixtures of defined buffer components were used to generate the pH gradients that separate closely related antibody species. This method separated MAb species with a wide range of isoelectric points through a complex retention mechanism, combining both ionic strength and pH. Unlike typical ionic strength elution methods, the pH gradient method was much more generic and could easily separate different components of a range of antibodies using a single method (Figure 3). The multi-product aspect of this method translates to less method development time for new IgG molecules. In addition, the ability of the method to assess charge-heterogeneity at a wide range of sample matrix salt concentrations and pH indicate the suitability of the method for use in evaluating in-process samples. Figure 4 shows chromatograms of samples collected from in-process pools and analyzed using the pH-IEC method. The data obtained from in-process pools using the pH-IEC method are reproducible at various salt concentrations, which is not the case for in-process pools analyzed by conventional IEC and icIEF. Table 2 compares the advantages and disadvantages of conventional IEC, iCIEF, and pH-IEC for charge heterogeneity analysis of MAbs. 

Figure 4. MAb samples from in-process anion exchange chromatography pools (Q-pool) analyzed by pH-IEC. Samples were diluted to ~1 mg mL-1, with no adjustment to pH. Protein A purified samples show equivalent profiles compared to samples analyzed directly from in-process pools. 

Recent work demonstrates the excellent robustness of pH-gradient IEC.

  Robustness studies recently have been performed using two column manufacturers, three HPLC instruments, and two analysts over 13 days of analysis. The method performs very well despite changes to column temperature, protein load, and buffer pH. The data from these robustness experiments reflect the expected range for the system suitability results, which reflects the precision of the method that compares favorably to other charge heterogeneity methods (Table 3). The ability of the method to perform well using different column and instrument manufacturers poses a considerable business advantage compared to methods requiring specialized instrumentation and consumables. 

Table 2. Advantages and disadvantages of conventional ion exchange chromatography (IEC), imaged capillary isoelectric focusing (iCIEF), and pH-gradient ion exchange chromatography (pH-IEC). 

There are several ways to increase the throughput of an analytical method, including reducing method run time. We recently reported a practical SEC method for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually.

  Generally, size-exclusion separations occur in less than a single column volume. Thus, it is possible to minimize the lag time by injecting samples before the previous sample has eluted to increase the throughput, so that at any given instant approximately two samples are eluting through the column. By coordinating the injection and detection time windows, the samples can be kept discrete and significant throughput enhancements achieved, up to nearly two-fold, with a typical run reduced from 30 to 14 min. This approach can be used to increase the throughput for any size exclusion column. 

Table 3. Precision in terms of absolute standard deviation range (Â±3 SD) of main peak relative area for conventional ion exchange chromatography (IEC), imaged capillary isoelectric focusing (iCIEF), and pH-gradient ion exchange chromatography (pH-IEC) for three MAbs. 

Ultra-high pressure liquid chromatography (UHPLC) has recently been introduced as a liquid chromatography tool to improve separation resolution and reduce overall analysis time. UHPLC uses much higher pressures than conventional HPLC, and the required solvent usage and analyst time is has been reduced because of lessened run times. Until recently, UHPLC was limited to the application of small molecules, but new developments have yielded promising applications for large molecules such as antibodies. Figure 5 shows a chromatogram from size exclusion analysis using a Waters Acquity UHPLC BEH SEC column, in which the run time for analysis of a MAb was only 3 min, compared to typical SEC run times of 15 min or more.  

Figure 5. Size exclusion chromatography (SEC) analysis of a MAb using a Waters Acquity ultra high pressure liquid chromatography instrument. Waters UPLC BEH column dimensions were 4.6 Ã 150 mm, with 1.7 Âµm particles. The column was run at ambient temperature. The mobile phase consisted of 200 mM potassium phosphate, 250 mM potassium chloride, pH 6.2. Flow rate was set to 0.8 mL/min. Sample loading was 10 ug. Relative peak areas for high molecular weight species (HMWS), monomer, and low molecular weight species (LMWS) are tabulated. 

Two-dimensional separations systems involving SEC have been developed to analyze proteins. Recently, automated 2D Protein A–SEC methods for the purification and analysis of MAbs have been developed.

  In these methods, multiple protein separation steps were performed automatically. Samples containing antibodies in cell culture fluid were injected onto a Protein A column for antibody recovery, then the purified antibody sample was automatically injected onto the size exclusion column. These 2D methods assessed both titer and size heterogeneity of the MAbs, demonstrating a streamlined workflow, and can be used for a variety of applications, including molecule assessment and clone selection. 

AUTOMATED SAMPLE PREPARATION AND DATA HANDLING

In addition to high-throughput and multi-product analytical methods, the use of robotics for sample preparation automation may further reduce sample analysis time and cost. There are several companies that provide liquid handling automation instruments, including LEAP Technologies and TECAN. Figure 6 shows a LEAP Technologies CTC PAL liquid handling system, which is intended for HPLC and GC usage and capable of fraction collection and injection. The CTC PAL instrument is capable of on-the-fly sample preparation, such as protein dilution and digestion. On-the-fly sample preparations are viable if the sample preparation takes less time than the analytical method. For sample preparations that take longer than the analytical run time, batch sample preparation can be performed using robotic liquid handling systems such as the TECAN Freedom EVO, which can handle multi-well plates for increased sample throughput. Robotic liquid samplers can increase reproducibility, efficiency, and safety compared to manual handling of samples. 

Figure 6. LEAP Technologies CTC PAL sample preparation robotics system. The system interfaced with a Dionex U3000 HPLC, with both CTC PAL system and HPLC controlled by Dionex Chromeleon chromatography management software. MAb samples were digested and diluted by the CTC PAL and injected directly onto the HPLC for IEC analysis. 

The final steps to most characterization workflows include data analysis and report generation. Several software packages are available that are designed to reduce the time necessary to complete post-data acquisition tasks. For liquid chromatography applications, commercially available chromatography data software, such as Dionex's Chromeleon chromatography management software and Waters Corporation's Empower chromatography data software, include features such as automated peak integration and one-click report generation. The Chromeleon software also has an extension pack that includes validation report templates and sequences, enabling increased automation of method validation. More companies are implementing electronic laboratory notebooks, which have advantages over traditional laboratory notebooks such as ease of data sharing and collaboration, streamlined review and witnessing processes, standardized documentation, and long-term data preservation. 

MAbs are under development for a wide range of indications, and ever-growing biotechnology pipelines have greatly increased demand for high throughput, multi-product protein characterization methods. By taking a holistic approach to improving the protein characterization process, one can identify strategies to increase value and reduce the development time of an analytical method. Implementing high-throughput and multi-product liquid chromatography methods that use available knowledge and resources typical in a biopharmaceutical facility can address the increasing demand for protein characterization studies, and these methods can be readily validated following regulatory guidelines. Further technological advancements in sample preparation throughput and data handling automation adds value to the overall characterization process, which will enable faster product development and shorter timelines to clinic. 

We would like to acknowledge the many people involved in our work, including but not limited to Ed Bouvier at Waters Corporation, Mark van Gils and Guillaume Tremintin at Dionex Corporation, and Peter Smith at LEAP Technologies, for technical input and assistance. 

 is a senior scientist and associate director, all at Genentech, Inc., Protein Analytical Chemistry, South San Francisco, CA, 650.225.6378, 

These new analytical methods can reduce analysis and product development time.

BioPharm International-11-01-2010

BioPharm International, November 2010 Issue (PDF)

Time to Revisit Supplier Quality Management

Biosimilars Regulation in the US: The Challenges

Managing Raw Materials in the QbD Paradigm, Part 1: Understanding Risks

Systematic Approaches to Develop Chemically Defined Cell Culture Feed Media

High-Throughput Multi-Product Liquid Chromatography for Characterization of Monoclonal Antibodies

Biotech Manufacturers Anticipate CER Challenges

A Dizzying Course