The biopharmaceutical industry is growing exponentially, driven by an ever-increasing demand for monoclonal antibodies (mAbs) and related products that are capable of treating complex, life-threatening diseases such as cancer, as well as other infectious and autoimmune diseases. With more than 200 recombinant proteins on the market today, the biopharmaceutical industry is expected to reach approximately $180 billion by 2016 (1). Therapeutic mAbs and their derivatives-such as antibody fragments (Fab), single-chain Fv (scFv), and diabodies-represent the fastest-growing class of approved therapeutic proteins because of their high specificity, increased serum half-life, and low toxicity (2). These products have been used in biosensors, protein purification, and bioimaging (3).

	Since the advent of an era of antibody-based therapeutics, the major focus of the industry has been on the generation of antibodies with glycosylation patterns similar to the naturally occurring immunoglobulin Gs (IgGs). Mammalian cells have been primarily applied as the hosts of choice for mAb expression. However, clinical success of mAbs has tremendously increased their demand, and this in turn has fueled interest in other cost-effective alternative production systems such as microbial hosts. Further, mAb production in microbial hosts such as bacterial cells completely abrogates the nuisance of glycosylation control and speeds up production timelines, further simplifying bioprocessing. The groundbreaking discovery of hybridoma cell generation technology leading to mAb production is one of the key developments in the past decade (4). Generation of antibodies in microbial hosts was initially met with skepticism due to potential immunogenicity issues. Recent developments in aglycosylated antibodies possessing similar antigen-binding affinity and engineered-specific FcγR binding has led to an enormous interest in these products. In this article, the authors review the status of expression of antibodies in microbial hosts and present the recent advances in the production of aglycosylated antibodies in bacteria. 

Structure of a monoclonal antibody and its derivatives


	Antibodies are glycoproteins consisting of two heavy chains and two light chains that are linked with disulfide bonds. This Y-shaped molecule possesses an antigen binding fragment (Fab) and a crystallizable fragment (Fc). Presently, different formats of antibody fragments, such as single-chain variable fragments (scFv), the fragment (Fab’)

, diabody fragments (dAb), and single-chain antibody fragments (scFab) have been generated, which possess similar antigen binding affinities, but different effector functions, due to the lack of an Fc region. These different antibody formats have been illustrated in 

Motivation for antibody production in microbial hosts


	Mammalian cells have been predominantly employed for the expression of antibodies and related products because of their ability to introduce post-translational modifications similar to that of human cells. However, use of mammalian expression system also has few disadvantages, which has propelled manufacturers to search for alternative host systems. One of the most important challenges is predicting the manufacturing behavior of cell lines at early stages and selection of clones with appropriate growth characteristics. Process improvements have generally been expensive and time consuming. Medium complexity, serum requirements, and shear sensitivity are some of the other drawbacks that characterize mammalian systems (5). Cell-culture process parameters affecting the glycosylation level of mAbs also need to be controlled to ensure consistent product quality (6). Selection of a stable antibody-producing clone is also a tedious and cumbersome exercise. In addition, viral contamination of a therapeutic protein preparation can be altogether avoided with cultivation in microbial system. While mammalian systems have been predominantly used for the expression of therapeutic proteins, advances in cellular engineering of microbial hosts have resulted in an increasing acceptance in the use of these hosts as an alternative for the production of mAb therapeutics.

Challenges associated with the production of antibodies in microbial hosts


	Microbial cells such as yeast and bacteria possess many advantages, such as fast growth, well-known genetics, and low cultivation costs. Glycoengineering platforms, display technologies and library creation, and robust manufacturing process development support the use of microbial cells as suitable candidates for pharmaceutical development. Although bacterial cells do not have the glycosylation machinery necessary for mAb production, recent successes in the expression of antibody-related products demonstrate the application of a bacterial host as an alternative choice. A number of bacteria, such as 

 have been used for the production of recombinant proteins (7). Of these, 

is the most popular host and two of the commercially available antibody-fragment products-Lucentis (ranibizumab injection) and Cimzia (cerolizumab pegol)-are expressed in 

	Antibody molecules contain disulfide bonds in their structure, and proper formation of these bonds is highly crucial for folding, solubility, and epitope binding. Protein folding and disulfide bond formation require an oxidizing environment, which is provided by the periplasm, the main compartment for the production of functional therapeutic proteins. Co-expression of foldases and molecular chaperones (Skp, FkpA, DsbA, and DsbC) helps in disulfide bond formation, prevention of misfolding, and aggregation of heterologous proteins (9). 

 has several cytoplasmic endogenous proteases that can cause proteolytic degradation; this represents a critical disadvantage of 

as a production vector. However, this problem has been successfully obviated by either use of protease-deficient strains or secretion of protein into periplasm, where there are fewer proteases. In 

, the Sec-dependent pathway has been used for protein secretion because of its abundance, although accumulation of protein aggregates may occur due to the generation of premature proteins before secretion. The formation of the inclusion bodies can be avoided by co-expression of cytoplasmic chaperones and utilization of a signal recognition particle (SRP)-dependent pathway. In addition, periplasmic inclusion bodies may also form due to incorrect folding of translocated proteins in the periplasm. Based on the twin-arginine translocation system (TAT), a new transport system enabling translocation of fully folded proteins in bacterial cells has been developed (10). Many advances such as translation region initiation (TIR) optimization, engineering of secretion pathways, and co-expression of molecular chaperones have been made for enhancing antibody productivity in bacteria and these have been reviewed elsewhere (7, 9, 11). Major focus in antibody production in 

 has been on the generation of scFv and Fab fragments. Full-length antibody (IgG) production continues to be a challenge due to low productivity, and various strategies have been employed to counteract this issue.

	Eukaryotic cells such as yeasts offer additional advantages of post-translational modifications-such as disulfide bond formation, which facilitates proper folding. While 

is the major strain used for expression of recombinant antibodies, other strains such as 

 have also been used to a lesser extent. Yeast cells tend to hyperglycosylate the recombinant proteins (even at non-native positions), and this may result in altered pharmacokinetics, activation of complement system, and generation of anti-glycan antibodies. 

 cells-cells that have been glycoengineered and express antibodies with superior volumetric productivity-have been developed to mitigate this problem (12). Yields of up to 1.4 g/L of humanized IgG have been reported with glycoengineered 

 (13), which is far superior to mammalian systems. Still, the use of this strain in antibody bioprocessing suffers from disadvantages, such as the occasional addition of heterogeneous O-linked glycans and slight differences in the N-glycan structure. A few examples of antibody and antibody derivative expression in microbial hosts that appear in current literature are presented in 

Aglycosylated antibodies as alternate therapeutic immunoglobulins

Antibodies constitute an important class of serum glycoproteins, and the IgG isotype is the most common isotype used for pharmaceutical applications. Natural IgG antibodies are glycosylated at the Asn297 amino acid position of the Fc fragment. The complex biantennary glycan structure depends on multiple glycosyltransferases and glycosidases located in the endoplasmic reticulum and Golgi bodies. These glycan molecules impart effector functions-such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)-to the antibody molecules. Absence of post-translational modifications in bacterial host cells leads to a complete abolition of glycosylation of antibodies, resulting in structural changes such as open conformation, enhanced flexibility, and complete loss of effector functions. In early experimental studies on X-ray structures of wild-type glycosylated IgG molecules and truncated Fc domains, glycan molecules were considered to provide the necessary flexibility to the C

2 domain of an IgG molecule, and removal of a glycan moiety was thought to be associated with the “closed IgG” conformation (40). 

However, recent small-angle X-ray-scattering experimental observations by researchers have suggested that aglycosylated Fc has a more flexible C

3 interface than previously imagined. This is due to their larger radius of gyration than their glycosylated counterparts, and the crystal packing in aglycosylated structures may result in a “closed upper C

2 region” in their X-ray crystal structures (41). 

 illustrates the conformational difference between glycosylated IgG and bacterially expressed aglycosylated IgG. The wild-type aglycosylated IgGs show almost no affinity towards FcγRs and don’t display any effector functions, but their engineering have enabled them to selectively bind to receptors. The need of aglycosylated antibodies with engineered Fc regions is highly crucial for anti-tumor therapies, where tumor cells exhibit many features similar to normal cells and selective engagement of FcγR receptors is essential to prevent deleterious effects (such as multi-organ failure or septicemia) resulting from nonspecific activation of dendritic cells. This effect has been predominantly observed in the case of the aglycosylated form of trastuzumab (Herceptin), which exhibited a 160-fold superior binding affinity to FcγRIIa than clinical grade drug and displayed enhanced antibody-dependent cellular phagocytoxic activity (42). This work deserves a special mention as FcγRIIa and FcγRIIb have high sequence identities (96%) but completely opposite roles. Aglycosylated antibody fragments showing selective binding to FcγRIIa were isolated by yeast surface display and flow-cytometry screening. In another study, researchers employed a knob-into-hole technique to assemble 

-derived aglycosylated half IgG and CHO-cell derived glycosylated half IgG to generate hemi-glycosylated IgG. It was observed that hemi-glycosylated IgG with a defucosylated glycan was able to display ADCC activity that was two-fold more potent than hemi-glycosylated IgG with a fucosylated glycan (43). Advances in engineering of effector functions in aglycosylated antibodies are summarized in 

, and these efforts have paved the way for possible emergence of aglycosylated antibodies as next-generation therapeutics with high selectivity and novel effector functions.

	Aglycosylated antibodies are similar to their wild-type glycosylated counterparts in terms of bioavailability, pharmacokinetics, and epitope binding. Several aglycosylated antibodies are under clinical trials and to date, no immunogenicity issues have been reported. Tolerx (MA, USA) has developed a series of humanized aglycosylated antibodies (TRX1, TRX4, TRX518) by knocking out asparagine at the 297th position. TRX1 is a monoclonal IgG1 anti-CD4 mAb (N297A mutation) that is being exploited for the suppression of autoantibodies in autoimmune disorders and neutralizing antibodies induced by enzyme replenishment therapy (44). Onartuzumab (MET-mAb, Genentech) is the first full-length monoclonal humanized and affinity-matured aglycosylated antibody expressed in 

for the treatment of lung cancer. It inhibits hepatocyte growth factor (HGF)-mediated activation and receptor signaling and possesses similar pharmacokinetic properties as that of its glycosylated counterparts. Similarly, yeast-expressed aglycosylated monoclonal antibody ALD518 (Alder Biopharmaceuticals) exhibited a half-life of 25 days (comparable to the wild-type glycosylated form) and significant improvements in patients with rheumatoid arthritis (45).  Recent success in aglycosylated IgG production and clinical efficacy data indicates that they may have even more applications in the future.


	The development of microbial expression systems that are capable of delivering projects that are not immunogenic and retain antigen specificity could pave the way for the cost-effective production of mAb products. Though substantial progress has been made in cellular engineering and in the secretion pathway of microbial cells, further progress in metabolomic and proteomic techniques is required to improve the understanding of microbial systems and the generation of host strains with enhanced potential.

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	ALL FIGURES ARE COURTESY OF THE AUTHORS.

Anurag S. Rathore* (pictured) is a professor, Department of Chemical Engineering, Indian Institute of Technology, New Delhi, India.
	Jyoti Batra is a post-doctoral fellow at the Indian Institute of Technology Delhi, New Delhi, India.

 When referring to this article, please cite it as A.S. Rathore and J. Batra, "Antibody Production in Microbial Hosts,"

Articles

Upstream Processing

The authors review the status of expression of antibodies in microbial hosts and present the recent advances in the production of aglycosylated antibodies in bacteria.

Antibody Production in Microbial Hosts

Extensive testing is required throughout the drug-development process and during manufacturing to ensure the safety and efficacy of marketed medicinal products. Numerous assays for the characterization of biopharmaceuticals and determination of any biologic contaminants have been developed and are highly effective for most biotherapeutics. For many cell-based therapies, such as chimeric antigen receptor (CAR) modified T-cells (also known as CAR-T), however, these conventional methods often take too long and require excessive sample quantities. Consequently, developers of these novel treatments have been faced with a number of challenges. The development of new rapid methods designed to provide comparable results while meeting the need for high-throughput performance show significant promise for addressing these issues.


	CAR T-cell therapies are produced by harvesting blood cells from a patient, selecting and growing the desired T-cell population, and then transducing them with a viral vector (typically lentivirus) carrying the CAR-T gene cassette. Transfecting cell lines with plasmids produces the viral vectors, and special care is taken to ensure that no replication-competent lentiviruses (RCLs) are generated. After CAR T-cell expansion, the cells are reintroduced into the patient.

	Microbial and viral testing is performed to determine whether any microorganisms (e.g., bacteria, viruses) are present during the pharmaceutical process, including in intermediates, active ingredients, the manufacturing environment, and formulated drug products. Test methods are employed for detection, screening, enumeration, and identification purposes. Examples include sterility testing for detection, screening for specified microorganisms, determination of the total aerobic microbial count for enumeration, and analysis to identify specific microbes. Other tests to determine the long-term stability (e.g., genomic and epigenetic stability, X-chromosome inactivation) and quality and function (e.g., potency, efficacy, lot-to-lot variability) are also required.

All raw materials must be sourced from approved suppliers and subjected to extensive testing to ensure there is no presence of microbial or viral contamination, as are master and working cell banks (MCBs and WCBs, respectively). Plasmids produced in bacteria are tested for sterility and endotoxin levels, and viral vectors are subjected to identity, purity, adventitious agent, and potency testing before they can be released for the transduction of cells. While no screening of patient blood cells for adventitious agents is required, the transduced cells are tested for sterility, mycoplasmas, RCLs, and endotoxins. The specific tests required for the control of critical raw materials and throughout the production process are determined by regulatory guidelines and are designed to ensure that cell therapy products are well characterized and free of microbial contamination. The required level of testing depends on the phase of the drug-development cycle and the step of the production process; for instance, cell banks and plasmids differ in their testing requirements. 

 presents a schematic of the manufacturing steps and associated testing regimes for a cell therapy production process.

	FDA, the European Medicines Agency (EMA), the US Pharmacopeial Convention (USP), and the European Pharmacopoeia (Ph. Eur.) have all published guidance materials related to the production and testing of cell-based therapies. Some examples include:

		EMA guidance on the development and manufacture of lentiviral vectors (1)

 use of retroviral gene therapy vectors (2, 3)

 (5) general guidance on the manufacture and quality control of therapy viral vectors

		FDA supplemental guidance on testing for replication-competent retroviruses in retroviral-vector-based gene therapy products (6)

		FDA guidance on potency tests for cellular- and gene-therapy products (7)

		FDA guidance on preclinical assessment of investigational cellular- and gene-therapy products (8).


	A broad range of standard, well-recognized methods that are accepted by regulatory agencies around the world are employed to characterize raw materials, biologic actives, and formulated drug products. For cell-based therapeutics, in addition to the analysis of typical raw materials such as media, assays must also be performed to characterize any cell lines and viral vectors used.

	Standard identity tests for cell lines include cytochrome oxidase or short tandem repeat (STR) profiling and DNA fingerprinting, while those for vectors include determination of genetic identity by sequencing of transgenes and restriction enzyme digestion.

	The absence of microbial contaminants in cell lines and vector products (bulk harvest [BH] purified) is confirmed through sterility and mycoplasma testing. Due to the large number of different viruses that can potentially contaminate biologic drugs, numerous assays, both 

, must be conducted on cell lines and vectors to demonstrate the absence of adventitious viral agents. These tests include those that detect ranges of viruses (broad specificity) and those that target specific viruses that have been known to be an issue (e.g., bovine, porcine). Virus-specific testing of cell lines is typically accomplished using polymerase chain reaction (PCR)-based methods and are often required if there is a known risk of contamination associated with the components used in a given process. Cell lines may also be subjected to transmission electron microscopy or product-enhanced reverse transcriptase assays.

	For CAR T-cell therapies produced using lentivirus as the viral vector, the absence of replication-competent vectors, particularly RCLs, must also be demonstrated. These assays are performed on vector products (BH, purified vector, and 

	Purified vectors are subject to further tests, including determination of the viral titer, residual bovine serum and plasmid, osmolality pH, endotoxin, host-cell DNA, and protein assays. The vector titer and endotoxin tests are also conducted for the 

Unique testing needs of CAR and other cell-based therapies


	Although cell-based therapies have been in development for more than two decades, they still face a number of challenges. Regulatory scrutiny is particularly high. Because these drug products contain live cells, terminal sterilization is not possible; therefore, demonstration of the absence of contaminants is essential. Early failures and questions about the safety of initial treatments have also led to intense interest from regulators.

	Testing can be difficult, however. Often, there are limited supplies of the key raw materials required for process, product, and test method development. In addition, drug-substance and drug-product lot sizes are often quite small; thus, sample volumes are typically small, leading to the need to use modified test protocols, which must be validated for the same specificity and sensitivity as the original test.

	Further complicating the issue is the limited shelf life of most cell-based therapies. In some cases, the transduced cells can be frozen, allowing for completion of testing prior to product release. It is not possible, however, to freeze most CAR T-cell therapies. The difficulty lies in the lengthy nature of most conventional sterility assays and tests for determination of the absence of bacteria, adventitious viruses, and RCL.

	Most assays and tests are cell-culture-based methods with extended incubation times to allow turbidity formation in liquid culture and colony formation on solid media, and require up to two to four weeks to complete. In addition, they involve many manual procedures (e.g., sampling, dilution, dispending, incubation, reading, recording, subculture, and microorganism identification), which all take time. Overall, these tests can take as many as 40 days from start to finish, assuming up to one week for sample delivery to the test lab, two to four weeks for testing, and up to an additional week for delivery of the final report.

	Rapid tests (PCR- and rapid-cell-growth-based microbiological and RCL assays) must be performed on cell-based therapies that cannot be frozen. Generally, conventional assays must also be run for confirmation of the results of the rapid tests, even though the results won’t be received until well after the treatment has been administered.


	The potential benefits of effective rapid-test methods have led to interest in their implementation for many more therapies than those that are just cell-based therapies that cannot be frozen. Not only do these assays enable reduced product-release cycle times, they generally require small sample volumes and provide higher-quality results. In addition, most can be automated and combined with electronic data capture, reducing opportunities for human error and the introduction of contaminants, and further decreasing the overall test time to approximately nine days (one day for sample delivery, seven days for testing, and one day for delivery of the final report).

	New microbiological testing methods achieve the same results as corresponding classical methods, but within a shorter time period, for example, less than three days for sterility testing, and 24 hours or less for microbial counts and ID tests. The ultimate rapid tests are completed in real time (e.g., one to three hours). The ultimate goal is to develop methods than can be completed in hours rather than days.

	Faster access to test results can also improve the manufacturing process, because potential problems can be investigated/addressed much sooner than is possible when conventional methods are employed. Very rapid methods may also enable in-process and raw material testing.

	There are, however, several key requirements that must be met by any rapid-test method that is intended to replace an existing compendial method. Most importantly, a rapid method must meet or exceed the performance of the existing assay in terms of both specificity and sensitivity. Extensive validation is required by regulatory agencies to support the use of a rapid method by demonstrating comparability to the standard method. Parameters that are considered part of such an evaluation can include accuracy, precision, linearity, specificity, the detection limits, operational range/sample volume, robustness, repeatability, and intermediate precision.

	Particular laws, regulations and guidance regarding rapid testing for biopharmaceutical manufacturing include:

		EDQM’s “EP 2.6.1, Sterility”, which discusses approaches to sterility testing (9)

		EDQM’s “EP 2.6.7, Mycoplasma” includes information on nucleic acid detection for mycoplasma in Europe (10)

		The Parental Drug Association (PDA) published 

PDA Technical Report Number 33 (TR33): Evaluation, Validation and Implementation of New Microbiological Testing Methods

’s “EP 5.1.6 Alternate methods for control of microbiological quality” (13).

	In 2011, FDA’s Center for Biologics Evaluation and Research (CBER) investigated matrix effects through the evaluation of three rapid microbial test systems: Milliflex Detection (Millipore), BacT/ALERT (bioMerieux), and BACTEC (BD) (14).

	It should be noted that when rapid test methods are approved, they are approved as part of the filing for a specific drug product. In addition to equivalent performance and significantly faster turnaround times, rapid methods should also be easy to use and be less costly than the corresponding standard methods. They must also be designed to address potential matrix effects.

	The technologies on which rapid test methods are based are divided into four categories for convenience:

		Cellular compound- or artifact-based technologies

	Examples of rapid microbiological assays and rapid methods for the detection of adventitious agents are presented as follows.


	A variety of technologies have been developed for rapid microbiological assays. Methods derived from blood-culture methods employed in clinical microbiology are attractive because they are based on techniques that have been approved by regulatory authorities, albeit for different applications. Examples include methods that rely on carbon dioxide sensors (pH-sensitive fluorescence and colorimetric response) and the use of a pressure-sensitive transducer to measure changes in headspace pressure.

	Many methods have also been developed that are based on technologies that have not previously been used in a clinical setting. Examples include determination of the electrical impedence of the media supporting growing microorganisms, solid-phase fluorescence laser scanning microscopy, flow cytometry of fluorescently labeled organisms, and ATP bioluminescence.

	ATP bioluminescence is of interest because the sample preparation is similar to that of compendial methods: It is compatible with a wide range of product types, and the results can be read and reported automatically using compliant software. Nonsterile product release is possible within 23 to 48 hours; however, this method does not provide enumeration of contamination levels. Automation of current compendial cell-culture methods is, however, making rapid microbial enumeration possible. Detection of positive cultures is faster with automated interpretation of culture results (via image processing), the general workload is reduced, and computerized data management provides documentation control. Microscopy, solid-phase fluorescence laser scanning, and ATP bioluminescence have also been applied for total aerobic microbial count enumeration.

	Nucleic-acid-based methods have also been developed for rapid sterility and mycoplasma testing. One concern with NATs is that nonviable DNA can provide false positive results. Careful design of test systems to ensure sterile environments for samples is crucial. Such methods can be run after immunomagnetic separation to achieve targeted separation using magnetic beads linked to antibodies or lectins that bind specific organisms. Real-time detection is possible with such systems given that within 20 minutes, 36 to 48 nucleic acid amplification cycles can be achieved for a typical bacterium using PCR.

	DNA contamination issues can be minimized through the use of NAT methods based on RNA. Several such methods have been developed for microbial identification. Genotypic identification of bacterial organisms can be completed in eight hours to three days depending on the specific technology. However, the equipment required for these tests is expensive, and specialized skills are necessary to perform them. Biochemical methods and methods involving gas chromatography analysis of the fatty-acid content in cell membranes have also been developed for rapid microbial identification.

Rapid methods for adventitious agent detection

	As mentioned previously, due to the large number of potential adventitious viral agents that are possible, many different assays are required to ensure detection of all likely viruses. In addition to being lengthy, cell-based methods may give false negatives, because in some cases, replicate viruses may not give any signs of cytopathic effects, or an infectious virus may not replicate in the cell lines chosen for the assay.

	PCR-based assays are much simpler and more rapid, with turnaround times of hours compared with weeks. Each PCR test, however, detects the DNA sequence from a specific virus. While accurate, the results include both viable and nonviable DNA, and are not specific to live virus particles. In addition, PCR alone is not applicable for the detection of multiple viruses in a single assay.

	Combining PCR with degenerate probes enables the detection of a broad range of viruses. Information on the virus family, subfamily, and genus is obtained using these methods. However, these degenerate/virus-family PCR assays are limited by the fact that contaminant DNA (nonviable) can influence the results.

	PCR is also being combined with mass spectrometry and microarrays for lot-release testing. New cell-based methods in development using engineered cells are designed to detect viral contaminants within 48 hours. Massive parallel sequencing, or deep sequencing, can be used to detect multiple DNA sequences from different viruses. Notably, multiplexing allows the rapid detection of virus families. These next-generation sequencing technologies help to minimize the risks associated with conventional and simple PCR-based methods for adventitious agent detection.

	Automation is also playing an important role in advancing rapid methods for the detection of adventitious viruses. For nucleic acid extraction, specially designed software can aid in primer/probe selection, while bioinformatics tools help facilitate multiple sequence alignments. Automated PCR assembly leads to improved design principles and chemistries, and real-time PCR amplification and analysis dramatically reduce assay times, as does the ability to immediately compare results to existing viral sequence records in databases. Furthermore, these databases are continually updated with information on new viruses soon after they are identified, enabling more comprehensive analyses.


	The development of CAR-T and other cell-based therapies has created opportunities for patients with diseases that previously had no treatment options. However, extensive safety testing of such drug products is necessary, as they are based on live cells that cannot undergo a final sterilization step.

	Not only is comprehensive characterization of the cells necessary, rigorous testing to demonstrate the absence of any microbial or viral contamination is paramount. In addition, testing strategies must be designed to meet the unique requirements of each cell-based therapy. Advances in rapid testing methods for use throughout the entire manufacturing process for CAR-T therapies will not only provide even greater assurance of drug product safety, but also may facilitate the further development of novel, effective treatments for patients with unmet medical needs.

Guideline on Development and Manufacture of Lentiviral Vectors

Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy 

Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs) 

, USP General Chapter <1046>, “Cell and Gene Therapy Products” (US Pharmacopeial Convention, Rockville, MD, 2011).
	5. EDQM, 

, Gene Transfer Medicinal Products for Human Use 5.14 (EDQM, Strasbourg, France, 2010).
	6. FDA, 

Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors

Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products

Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products

, Sterility 2.6.1 (EDQM, Strasbourg, France, 20601, 04/2011) 10. EDQM, 

, Mycoplasma 2.6.7 (EDQM, Strasbourg, France, 20607, 01/2008), 11. 

 Title 21, Part 610.12 (Government Printing Office, Washington, DC), pp. 70-75.
	12. PDA, Technical Report No. 33, 

Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods

, General Text 5.1.6. (EDQM, Strasbourg, France, 2011).
	14. FDA, Center for Biologics Evaluation and Research, “Identifying Faster Sterility Tests for Biological Products: Regulatory Research Seeks to Reduce the Time Needed to Ensure the Safety of Critical Products,” (Rockville, MD, 2011), 

www.fda.gov/downloads/BiologicsBloodVaccines/ScienceResearch/UCM266975.pdf


	Alison Armstrong, PhD, is senior director, development services at BioReliance, UK.

 When referring to this article, please cite it as A. Armstrong, "Advances in Assay Technologies for CAR T-Cell Therapies," 

Rapid methods to test CAR-T therapies for potential contamination are on the horizon.

Advances in Assay Technologies for CAR T-Cell Therapies

	FDA set a 19-year record in 2015 in approving more new drugs and biologics, and agency officials expect this pace to continue. Manufacturers are testing a full pipeline of important, new therapies to treat both rare diseases and widespread serious conditions such as high cholesterol, diabetes, multiple myeloma, and a range of cancers. More biosimilars are poised to come to market in 2016 following FDA approval of the first such therapy in 2015. At the same time, though, the development of such innovative and targeted therapies heightens the importance of establishing production systems and processes capable of fast scale-up of high-quality complex products.

	The surge in new drug development will be encouraged by increased public support for biomedical research and innovation, led by President Obama’s campaign to cure cancer. Presidential hopefuls are championing medical cures, as seen in Hillary Clinton’s proposal to boost Alzheimer’s research. The 2016 budget bill approved by Congress at the end of 2015 increased funding significantly for the National Institutes of Health (NIH) and other government research programs, while providing a small, but encouraging increase in FDA’s appropriation.

	The spending measure directs NIH to allot portions of its $2 billion budget boost to Alzheimer’s disease research, precision medicine, the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) initiative, and development of antimicrobial medicines. The development of new antibiotics gained from several spending provisions: in addition to $100 million to NIH for that purpose, the Centers for Disease Control and Prevention (CDC) received $160 million to prevent and monitor superbug outbreaks, and the Biomedical Advanced Research and Development Authority (BARDA) received nearly $100 million to help test new therapies and diagnostics to protect against infectious disease threats.

	FDA also was instructed to improve its oversight of antibiotics as part of its $2.7 billion appropriation for 2016. Most of its $132 million budget increase goes to expanding food safety programs, but small amounts are allotted to ensuring the accuracy of genetic tests important to precision medicine, expanding foreign inspections of high-risk operations, and supporting orphan drug development. The 

 further instructs FDA to finalize guidance on biosimilar development and to do more to prevent drug shortages (1). And it puts a hold on FDA consideration of new therapies that use genome-editing tools capable of modifying the DNA of human embryos.

	Legislative action further made permanent the federal R&D tax credit, a long-sought change expected to boost private sector investment in biotech companies. Medical device makers gained a two-year delay in paying a new tax authorized by the Affordable Care Act (ACA), which manufacturers say will promote device innovation. And the legislators extended the rare pediatric disease priority review voucher program until September 2016 to keep it going until Congress can review and reauthorize it.
	
	


	These developments generate optimism that scientists finally will begin to transform the treatment of disease based on decades of important genomic research. FDA’s Center for Drug Evaluation and Research (CDER) approved 45 new molecular entities (NMEs) and biotech therapies in 2015, beating the near-record set in 2014. FDA’s Center for Biologics Evaluation and Research (CBER) also approved more than a dozen new biologics, vaccines, blood products, and diagnostics.
	
	In the process, FDA met nearly all the review timeframes and goals set by the Prescription Drug User Fee Act (PDUFA V), reported John Jenkins, director of CDER’s Office of New Drugs (OND), at the FDA/CMS Summit in December 2015. Jenkins noted that most NMEs were approved in one review cycle; more new drugs reached the market first in the United States; and CDER continues to receive hundreds of requests from manufacturers for breakthrough drug designations, a 

 that has led to the approval of more than 20 innovative products (2).

	Jenkins voiced concern, however, about the continued emergence of new therapies, noting the need for FDA to assist pharmaceutical manufacturers seeking to accelerate the production timeframes for more targeted and complex medical products. In developing and bringing to market important new therapies, Jenkins noted, it’s “often manufacturing and inspections that are the rate-limiting steps” for expedited approvals, not clinical development. CDER often faces difficulties in scheduling timely preapproval inspections within the six-month review timeframe for breakthrough drugs, he explained, and small biotech companies have run into difficulties with contract manufacturers that have quality and data problems uncovered during plant inspections.

	FDA seeks to avoid such issues by clarifying policies and offering new solutions to drug development and manufacturing challenges. A 

, for example, aims to clarify requirements for producing fixed-combination and co-packaged drugs and over-the-counter medicines (3). The agency also is supporting the development of innovative 

diagnostics and combination products through improved coordination of oversight for combination therapies. CDER further aims to encourage manufacturer adoption of cutting-edge pharmaceutical production technology by providing assistance in meeting regulatory requirements for innovative systems. A draft guidance document advises manufacturers on how to request meetings with experts on CDER’s Emerging Technology Team to discuss regulatory issues related to submitting chemical, manufacturing, and controls (CMC) data for new manufacturing systems involving both innovator and generic products. The aim is to avoid delays that could discourage the adoption of new production methods able to improve drug safety and quality (4).
	
	


	Further global harmonization of drug-development policies also is on the horizon following major changes at the International Council for Harmonisation (ICH). The organization now seeks more involvement of regulatory authorities and manufacturers in Asia, Africa, and the Americas in the ICH standards-setting process. FDA, the European Medicines Agency, and other established authorities, moreover, are collaborating more to avoid duplication in drug facility inspections and in the registration of innovative medicines for rare conditions and for children.
	
	FDA also is under pressure from Congress to expand its capacity for conducting foreign drug inspections. The House Energy & Commerce (E&C) Committee recently asked the Government Accountability Office (GAO) to examine the effectiveness of FDA foreign drug inspections and overseas offices in the face of ever-expanding imports of drugs and APIs. Both Republicans and Democrats 

 to update its 2010 report on FDA’s overseas regulatory capacity, with a focus on agency progress in implementing a risk-based inspection system and in staffing up its foreign offices (5). FDA’s ability to process drug applications efficiently and to ensure drug safety, efficacy, and quality will continue to draw scrutiny on Capitol Hill, along with drug pricing issues. A broad group of legislators has requested multiple documents from the agency on generic-drug approval times, action dates, user-fee payments, and other items to help assess market competition issues related to generic-drug price increases.

	Biosimilar development will remain in the spotlight, along with efforts to ensure the safety and quality of “complex” generic drugs that are not regulated as biologics, but are more diverse than conventional drugs. The legislators have 

 to ensure that FDA requires appropriate scientific analytic methodologies to fully identify the structure and properties of such products and to ensure their bioequivalence, safety, and efficacy (6).


	1. Division A-Agriculture, Rural Development, Food And Drug Administration, And Related Agencies Appropriations Act, 2016 Congressional Directives, 

http://docs.house.gov/meetings/RU/RU00/20151216/104298/HMTG-114-RU00-20151216-SD002.pdf

, accessed Jan. 5, 2016.
	2. J. Jenkins, MD, CDER New Drug Review: 2015 Update, FDA/CMS Summit, Dec. 14, 2015, 

http://www.fda.gov/downloads/AboutFDA/CentersOffices/Officeof MedicalProductsandTobacco/CDER/UCM477020.pdf

 Parts 300, 330, and 610, Fixed-Combination and Co-Packaged Drugs: Applications for Approval and Combinations of Active Ingredients Under Consideration for Inclusion in an Over-the-Counter Monograph, 

www.gpo.gov/fdsys/pkg/FR-2015-12-23/pdf/2015-32246.pdf

Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base Guidance for Industry, Draft Guidance

 (CDER, December 2015).
	5. US House of Representatives Energy & Commerce Committee, Bipartisan Committee Leaders Enlist Government Watchdog on FDA’s Foreign Inspection Program, Dec. 18, 2015, 

http://energycommerce.house.gov/press-release/bipartisan-committee-leaders-enlist-government-watchdog-fdas-foreign-inspection

, accessed Jan. 5, 2016.
	6. US House of Representatives Energy & Commerce Committee, Letter to GAO Requesting a Study on FDA’s Pathway to Review Generic Non Biologic Complex Drugs, Dec. 11, 2015, 

http://energycommerce.house.gov/letter/letter-gao-requesting-study-fda%E2%80%99s-pathway-review-generic-non-biologic-complex-drugs

:
	When referring to this article, please cite it as J. Wechsler, "Innovative Therapies Require  Modern Manufacturing Systems," 

Regulatory Beat

FDA and industry see progress and challenges in bringing cutting-edge medicines to patients.

Innovative Therapies Require  Modern Manufacturing Systems

	Bio/pharmaceutical companies, and the companies that serve them, tend to think they are immune from broader macroeconomic and political developments. As populations age, emerging middle classes expand, and scientific knowledge progresses, research on new drugs and demand for new therapies seem to follow an inexorably upward trend.

	However, the global financial crisis of 2008 demonstrated that the industry is not isolated from macro events. Early-stage companies, which drive the early development engine, had great difficulty raising money as the availability of venture capital declined and the window for initial public offerings (IPOs) closed altogether. It took nearly five years for drug-development funding to become readily available again, and for the pharmaceutical services industry to once again thrive.

	It’s worth reminding industry participants of the vulnerability of the bio/pharmaceutical industry to macro events because memories tend to be short, and the global environment is becoming uncertain again. The coincidence of two events, in particular, threaten the global economic equilibrium: the collapse of commodity prices and the resumption of interest rate hikes by the central bank of the United States, the Federal Reserve. 

	Most people are keenly aware of the steep decline in oil prices, but prices have also collapsed for a broad range of commodities including industrial metals such as copper and agricultural products such as soybeans. The falling prices are a result mainly of increased supply (thanks to fracking for oil and robust harvests) coming at a time when demand, especially from China, has slowed considerably. In affected economies, government tax and royalty revenues have decreased sharply, as have employment, foreign currency reserves, and investment.

	The decision by the Federal Reserve to begin raising interest rates is exacerbating the problem for emerging markets. Many businesses in those countries had borrowed US dollars heavily to fund investment because the Federal Reserve had kept interest rates so low. Those companies, and some governments as well, are now facing a financial crisis brought on by rising interest rates, declining revenues, and rising repayment costs caused by the depreciation of their local currencies against the US dollar.
	
	


	So what do these macroeconomic developments have to do with the prospects of bio/pharma companies, CROs, and CDMOs? There are at least three negative implications of the deteriorating global financial outlook for the bio/pharma services industry.
	
	Bio/pharma industry growth has been hurt by the problems in emerging markets. Expansion in those countries, with their rapidly growing middle classes, was a major element of the post-patent cliff strategy of many global bio/pharma companies. Now, declining government revenues, skyrocketing debt service, declining foreign currency reserves, and depreciating currencies will severely limit the ability of those countries to import and distribute all but the lowest-cost generics.

	CROs and CDMOs have not been major participants in the emerging market expansion plans of their global bio/pharma clients, so their exposure to these developments will be limited. They could see lower product volumes, however, as emerging market countries limit imports and encourage more local production.

	A bigger concern is what happens to investor confidence. The banking and investment communities will take a big hit as the financial condition of companies and countries around the world deteriorates. Further, there is increased uncertainty surrounding even the strongest economies, as evidenced by the turmoil in world stock markets at the beginning of 2016. While few financial observers expect a crisis as severe as the 2008 financial meltdown, investor nervousness could reduce willingness to invest, negatively impacting company valuations, and the ability of companies to float IPOs and otherwise raise capital.

	That could be a big problem for the CRO and CDMO industries. Freely flowing investment capital for early-stage companies has been a huge driver of demand for CRO and CDMO services in the past three years. As seen in 2007–2008, just the fear that finding new capital could be difficult can make early-stage companies slow their spending (e.g., by reducing the number of candidates in active development).  
	US-based CROs and CDMOs have an additional concern as the US dollar has appreciated considerably against most currencies. The euro has lost 17% of its value relative to the US dollar in the past year and that can considerably alter the comparative advantage of CDMOs in the US and Europe. It means that a €1 million contract with a European CDMO, which would have cost a US client $1.3 million
	in 2014, will now cost that US client just $1.1 million. On the other hand, a European client considering a $1 million manufacturing or development contract with a US-based CDMO will be facing a €909,000 expense today versus €750,000 just a year ago. Moreover, the situation could get worse for US-based CDMOs in the next year or two as the euro is expected to depreciate to parity (€1= $1). US-based CDMOs that have benefitted from a weak US dollar for nearly 10 years are now facing strong foreign exchange headwinds.

	The point here is not to wallow in doom and gloom about the macro environment. CRO and CDMO executives should not take for granted the robust market conditions they have enjoyed during the past three years. They need to be fully cognizant of the macro environment in which the industry operates and the risks it presents. As learned in the 2008–2012 period, the bio/pharma industry is not immune from global economic and political developments.

:
	When referring to this article, please cite it as J. Miller, "Macro Matters," 

Perspectives on Outsourcing

Heightened global uncertainty could slow bio/pharma development activity.

BioPharm International-02-01-2016

Antibody Production in Microbial Hosts

Advances in Assay Technologies for CAR T-Cell Therapies

Innovative Therapies Require Modern Manufacturing Systems

Macro Matters

Fine Tuning the Focus on Biopharma Analytical Studies

Biopharma Innovation Born Again?

Failure Mode Effects Analysis for Filter Integrity Testing

The Emerging View of Endotoxin as an IIRMI

Use of an E. coli pgi Knockout Strain as a Plasmid Producer

Complementary Techniques for the Detection and Elucidation of Protein Aggregation

BioPharm International, February 2016 Issue (PDF)