cGMP Compliance: What Does It Mean for Your Cell Lines?

As the promise for cell-based therapy grows, the interest in making clinically relevant cell lines has skyrocketed for industrial and academic researchers alike. For translation into human therapies, cell-based products must be made following current Good Manufacturing Practice (cGMP). Many groups have already claimed to generate cell lines that are “cGMP-compliant,” “cGMP-ready,” or “certifiable under cGMP.” But what does it take to be truly cGMP-compliant, and what practices can you introduce in your lab to comply with cGMP standards?

A common misconception in the United States is that a facility is granted a ‘cGMP license’ from the government to manufacture cGMP-grade products. Rather, the Food and Drug Administration (FDA) evaluates the manufacturing process for each product to determine if it is compliant with cGMP standards. The primary concern when it comes to deriving cell-based products for therapies is making sure that the product is derived in a safe and reproducible manner. To ensure maximum quality assurance, researchers should

• choose reliable, xenogeneic-free raw materials,
• establish and monitor a clean environment,
• qualify all equipment and software,
• remove variation in laboratory procedures by creating detailed Standard Operating Procedures (SOPs) and by providing rigid process validation at each step.

Nevertheless, even establishing robust quality assurance does not imply that the process is scalable for commercial production. In the world of biologics, “the product is the process.” A requisite step to ensure a smooth transition to cGMP practice is to ensure that the process of manufacturing is not altered due to changes in production scale. For example, depending on the therapy, millions or billions of cells may be required for a single patient. Therefore, it is in the best interest of the researchers to develop a scalable method at the beginning to avoid revamping the entire process (e.g., changing from adherent cells to suspension). Along these lines, the quality control (QC) requirements of cell-based products should be carefully considered and not have to include difficult-to-assay tests. For example, some cell lines have been qualified as cGMP-compliant upon conversion from research-grade conditions to cGMP quality standards. Rigorous tests were performed on the converted lines to ensure that the cells were free of contamination. Even though strict measures were carried out to ensure cGMP compliancy, deriving cell lines in this manner makes scalability and reproducibility a challenge. Ideally, the entire process of deriving cell products for clinical use should be performed under cGMP conditions: from the acquisition of human tissue to the manufacturing, testing, and storage of derivative cell products.

Another important consideration when instituting cGMP-compliance is documentation. Each process must be described with rigorous SOPs, the training of individual manufacturing operators must be well-documented, and the entire established process must be validated and well noted. Failure to document—in the eyes of the FDA—is often equated with failure to perform the underlying activity. It is equally important to remain ‘current.’ The FDA expects manufacturing processes to stay up-to-date with current regulations, even as policies change.

For an academic lab, closely aligning with cGMP standards can ensure that the resulting cell lines are comparable to other truly cGMP-produced products used during clinical trials. It is in the best interest of academic researchers to establish rigorous SOPs and use qualified reagents and equipment, even if it is not possible to carry out all steps in a certified cleanroom. Whenever possible, it is advisable to acquire truly cGMP cell lines from appropriate sources for preclinical projects; if prohibited by costs or other reasons, it is recommended to use a protocol that is as close to cGMP as possible.

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Researchers use GFP nano antibody to study organ growth

Single-domain nano antibodies have a broad range of applications in biochemistry due to their small size, high affinity, and high specificity. Now, a team of researchers from the University of Basel and the University of Zurich has demonstrated that nano antibodies can be used for research in complex living organisms such as Drosophila, uncovering another new and exciting application for nano antibodies.

The team used nano antibodies to develop an assay for studying morphogens, molecules that regulate the pattern of tissue growth and the positions of various cell types within tissue. Morphogens form long-range concentration gradients from a localized source, ultimately determining the fate and arrangement of cells that respond to that gradient. Drosophila is a classic model system for understanding how morphogens regulate organ development. One morphogen called Dpp controls uniform proliferation and growth of the wing imaginal disc. Yet because Dpp is an extracellular, diffusible protein, it is difficult to immobilize in situ. Therefore, despite over 20 years of studying the role of Dpp as a morphogen, the lack of a dynamic system for controlling Dpp gradients has prevented researchers from understanding precisely how Dpp governs development of the wing disc.

By developing a novel synthetic system using nano antibodies, the researchers were able to modulate the concentration gradient of Dpp at the protein level. Their system—coined “morphotrap”—uses a membrane-bound GFP nano antibody to “trap” GFP-tagged Dpp at different locations along the wing imaginal disc. By tethering Dpp in a controlled spatial manner, researchers were able to determine how Dpp gradients affect wing disc development. They discovered that the gradient of Dpp is required for the patterning of the wing disc but not for lateral growth, disproving one of the field’s popular theories that address the role of Dpp. In addition to resolving the controversy with respect to the role of Dpp as a morphogen, this study pioneers a new method for using nano antibodies in situ.

“Dpp spreading is required for medial but not for lateral wing disc growth.”
Harmansa S., Hamaratoglu F., Affolter M., Caussinus E.
Nature. 2015 Nov 19;527(7578):317-22. doi: 10.1038/nature15712. Epub 2015 Nov 9.

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Allele Biotechnology & Pharmaceuticals Forming Cell Banking Business for Personalized Medicine

Allele to Generate Human iPS Cells under Good Manufacturing Practice for Private Individuals for Potential Therapeutic Use and Future diagnostics; Cell Banking Scientific Advisory Board formed

October 06, 2015 11:25 AM Eastern Daylight Time
SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals, Inc. (“Allele”), a leader in the development of specialized cells for regenerative medicine and pharmaceutical drug discovery, today announced plans to form a commercial business for the banking of human induced pluripotent stem cells (iPSCs) by private individuals.

Allele is pleased to have Drs. Mahendra Rao and Joseph Paulauskis as the first members of its Scientific Advisory Board (SAB) for iPSC banking and cGMP production. Dr. Rao is a world-renowned scientist in the fields of stem cells and medicine, having served as the VP of Regenerative Medicine at Invitrogen, founding Director of the NIH Center for Regenerative Medicine, Chair of the Biological Response Modifiers Committee (BRMAC, now CTAGT) of the FDA, and he is currently Chief Strategy Officer at Q Therapeutics. Dr. Paulauskis is the Chief Operating Officer of Paradigm Dx as well as the Vice-President of Research and Biobanking for the International Genomics Consortium, and has previously held senior positions in pharmacogenomics at Pfizer. Allele plans to make additional appointments to the SAB as individuals with world-class experience and expertise are identified.

Human iPSCs are cells that can be grown to virtually infinite numbers and can become any cell in the human body, features which hold great promise for therapies that can alleviate or cure human disease. Allele’s business model recognizes that an individual can have their own cells ready for future therapeutic use via the generation and storage of iPSCs from that individual’s skin cells. While this is similar to what is done with newborn cord blood, iPSCs can be generated from humans of any age. The banking of human iPSCs for potential future therapeutic use is a relatively new industry with unprecedented potential, and Allele is benefitting from expert opinions internally and from current and future SAB members, ensuring a solid scientific and ethical foundation for this business.

In addition to serving its customers, Allele’s iPSC bank will be an unparalleled resource for biomedical research. Proper consent and privacy guardianship will allow thousands of iPSC lines with accompanying sequence database and health information to be made available from the bank to scientists and clinicians. Currently, iPSC banks are funded by government agencies at multi-million dollar costs per project; Allele’s model does not rely on tax dollars and provides potentially a larger bank of iPSCs of higher quality to aid research and treatment efforts. To this end, NIH Director Dr. Francis Collins recently announced the implementation of the Precision Medicine Initiative (PMI), the goal of which is for health care professionals to have the resources to take into account individual differences in genes, environments, and lifestyles that contribute to disease when providing treatments in the new era.

“We are happy to have the guidance from world leading experts in stem cells, biobanking, and cell therapy fields such as Drs. Rao and Paulauskis”, said Jiwu Wang Ph.D., President and CEO of Allele. “We believe that setting the bar high will be ultimately beneficial to future customers, fellow researchers, industry partners, and regulatory agencies alike. We are happy to see the recent release by the International Society for Stem Cell Research of a draft of ‘Guidelines for the Clinical Translation of Stem Cells’, whose principals we plan to follow closely. We also intend to obtain certification by the cord blood banking association AABB, if possible, and abide by other regulatory rules as they become public, such as the “Stem Cell Clinical Research Management (tentative)” by the Health Commission of China, if and when we move to operate under that jurisdiction”.

Towards the establishment of this business effort, Allele has recently purchased an 18,000 square-foot facility, located near its headquarters in San Diego, California. This new facility will be the center of cGMP-production of human iPSCs using Allele’s proprietary synthetic mRNA platform, a technology that generates cells with neither the random integration of foreign DNA nor the use of viruses or virus-based elements, drawbacks common to other technologies for making hiPSCs; thus, the “footprint-free” cells generated by Allele’s synthetic mRNA platform are optimally suited for therapeutic use, and Allele’s technology has been licensed for clinical trials by companies such as Ocata Therapeutics (formerly ACT). This effort received strong support from Yuan Capital and Yifang Ventures.

About Allele Biotechnology & Pharmaceuticals, Inc.

Allele Biotechnology and Pharmaceuticals, Inc. is a private, San Diego-based company that explores the mechanisms of biological processes to develop technologies and products for biomedical researchers. Allele utilizes proprietary non-integrating cellular reprogramming methods to generate human and non-human primate iPS cells, GMP-grade human iPS cells and their derivatives, and differentiated cell types. With additional expertise in genome modification and cell-based sensors/reporters, Allele provides advanced cell-based assays for drug discovery. Allele also has developed a wide variety of reagents including superior fluorescent proteins and camelid antibodies. The company has also been a leader in the RNAi field with its patents in Pol III promoter-driven siRNA, shRNA, and miRNA.

Allele Biotechnology & Pharmaceuticals, Inc.
Matthew A. Singer, Ph. D.
Director of Business Development and Strategic Alliances
+1 858-587-6645, ext. 1

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A breakdown of your burning nAb questions

Allele Biotechnology just released its latest batch of nAbs (nano antibodies), the first wave on a long list of new antibodies to come! You might have a few questions about how these “antibodies of the future”, as we call them, can help your research:  What can I use them for?  How much should I use?  And how do they work compared to a traditional antibody? 
To answer these questions, we need to first discuss some antibody basics.  Conventional antibodies (your typical mouse or rabbit derived antibody) have a “Y” shape and tightly bind targeted antigens as a result of two factors.  The first is affinity between each monomer Fab fragment and the antigen.  The second is the fact that traditional antibodies are di-valent, i.e. they have two identical binding sites for each antigen, which is known as avidity. 
When developing a nano-antibody, we screen and select our clones to have extremely high affinity as a monomer.  This is because nAbs are mono-valent VHH fragments. The intrinsic high affinity VHHs possess for their antigens can make up for the lack of multivalency (avidity).  As a result, nAb binding is often superior to conventional antibody binding, which leads to superior performance in a variety of biological assays (immunoprecipitation, immune-staining, FACS staining, immunofluorescent imaging, etc.). 
Each nAb is roughly one tenth (1/10) the size of a traditional antibody.  The small size and stable conformation of nano-antibodies enable pinpointed localization of target antigens and allow access to antigen and cellular regions generally restrictive to larger antibodies. As a result of this smaller size, when measured by weight 1mg of a nAb is equivalent to 5 – 10mg of a traditional antibody (the lower end takes di-valency into account).  When substituting a nAb for a traditional antibody you can use as little as one tenth (1/10) the amount by weight. 
There are a couple of different ways to use nAbs.  The first is immobilizing the nano-antibody on a resin (i.e. magnetic-agarose resin) for immunoprecipitation.  The nano-antibody will not be released from the resin upon elution so you will not have contaminating bands.  The second method is direct labeling with a fluorescent dye or hapten.  nAb’s are compatible with standard NHS-ester amine chemistry binding.  This enables single or multiple fluorophore labeling per antibody.  Moving forward, additional platforms will be released that allow for a more flexible and adaptable labeling system, allowing you to harness nAbs for any biological assay you can imagine.  Have some suggestions? Don’t hesitate to let us know by emailing at Or call 858-587-6645 and ask for a nAb expert.

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Press Release: Allele Biotech Takes Major Step into Nano Antibody Leadership Position

SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals Inc., a San Diego based life sciences company with a focus on novel technology development, releases the first group of a brand new class of antibodies against crucial biological targets to the research market. This week, Allele launches nano-antibodies isolated from llamas against human bFGF, P16, VEGF, and TNFa, which are all important targets in the field of cancer biology.

Nano-Antibodies (also known as nAb™, Nanobodies®, Single Domain Antibodies, Camelid Antibodies and VHH antibodies) represent the future in antibody technology of Allele’s interest. “Camelid antibodies have been an area of intense research activities at Allele because they have desirable features that no other antibody has. These tiny antibodies outperform conventional antibodies in many ways and thrive in extreme conditions, eventually they will occupy a significant portion of the antibody reagent market,” said Dr. Jiwu Wang, CEO and founder of Allele Biotechnology. This first wave of novel reagents has been meticulously tested for immunohistochemistry (IHC) in human cancer tissues; some of these antibodies also performed well in cross-species reactivity in mouse and rat while others are highly suitable for advanced applications such as flow cytometry and antigen immunoprecipitation.

This is the first release in a long-term effort to generate and commercialize hundreds of nano-antibody derived capture tools. “Our nano-antibody project is based on years of internal technology development partially funded by the National Institute of Drug Abuse of the NIH,” according to Allele’s Marketing Director, Abbas Hussain. “The nAb product line will shortly encompass a wide range of high value targets that are applicable to both basic and clinically relevant research. It will also feature cutting edge conjugation technologies that enable fluorescent imaging and electron microscopy techniques being developed at Allele.”

Since the ability to generate monoclonal antibodies was discovered in 1975, antibodies have been used in virtually every branch of biomedical research and development. In the past decade there has been a shift toward harnessing antibody technology for therapy, as illustrated by large number of antibody-based drugs on the market today. Allele’s nAb development has been one of the targets of investment from Yifang Ventures and Yuan Capital.

“Nanobodies® is a trademark of Ablynx; nAb as nano antibodies is under copyright of Allele Biotechnology, all rights reserved.”

Allele Biotechnology & Pharmaceuticals Inc.
Abbas Hussain, 858-587-6645
Director of Sales & Marketing
6404 Nancy Ridge Dr.
San Diego, CA 92121

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