By David Loong
The first article in this series gave a broad overview of what to look out for when sourcing or manufacturing your experimental medicine for first-in human (FIH), or Phase I trials. It highlighted important considerations regarding Good Manufacturing Practices (GMP), and quality. In this article, we examine specific recommendations for the most common therapeutic modalities: small molecules, antibodies or biosimilars, and cell and gene therapy products.
Manufacturing novel small molecules
Small molecules are manufactured by organic synthesis using chemical reagents. The science and engineering of manufacturing chemical APIs is now well established since the first pharmaceutical factories were set up in the 1850s. A key requirement is for all suppliers of raw materials to provide a Certificate of Analysis and statement indicating their suitability for pharmaceutical use.
Characterization and analytical methods for novel small molecules
Identifying your active ingredient is straightforward using techniques such as nuclear magnetic resonance (NMR), infrared spectroscopy, mass spectrometry, or X-ray diffraction. You can generate gold-standard reference material to use in routine high performance liquid chromatography (HPLC) analysis to establish identity, purity and potency. HPLC is a robust technique widely used in pharmaceutical manufacturing.
Recommended strategy for small molecules
As a lean start-up or research group, it makes sense to outsource the process development, manufacturing, and analytical development of your small molecule to an established contract development and manufacturing organization (CDMO) with a good track record of chemical process development and GMP manufacturing. There are many such CDMOs around the world, especially in India and China, including WuXi AppTec, AMRI Global, and Jubilant. Formulation development, dissolution testing, and final dosage form manufacturing can also be outsourced to specialist service providers such as Syngene, GVKBio, and WuXi AppTec.
Monoclonal antibodies (mAbs) and biosimilars
Therapeutic mAbs are secreted by living cells, usually stably transfected and monoclonal CHO cells in suspension. Unlike small molecules, the active ingredient will be a range of variant molecules and the undesired variants should be identified as early as possible. Upstream process optimization should result in a process biased to minimize the production of undesired variants, while downstream purification optimization should remove any remaining product-derived impurities. Most therapeutic mAbs are formulated for intravenous or subcutaneous administration so a suitable buffer for storage, freezing, and administration will need to be identified during process development.
Characterization and analytical methods for mAbs
A mAb active ingredient will be a mixture of isoforms, glycoforms and minor sequence variants. Initial characterization of the active ingredient by enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE will need to be complimented with a battery of other orthogonal physicochemical techniques such as capillary electrophoresis and mass spectrometry to make gold-standard reference material. This material can then be used to develop robust HPLC analytical methods for routine use.
Recommended strategy for monoclonal antibodies and biosimilars
For small start-ups or research groups, it makes sense to outsource the cell-line, analytical and bioprocess development, as well as GMP manufacturing to a CDMO as they will have “platform processes” that streamline all stages of process development. This is a quick way to get a workable process, albeit one that is perhaps not completely optimized. Ongoing process optimization can occur in parallel with the FIH trial, resources permitting. Globally, there are many CDMOs with established track records for mAb process development and manufacturing, including Lonza and Thermofisher/Patheon. Analytical development and characterization can also be outsourced to specialist contract research organisations (CROs) such as BiopharmaSpec, which can provide analytical data compliant to Good Laboratory Practices (GLP) to compliment any non-GLP analysis conducted at a research institute’s facilities.
It is worth noting that biosimilar mAbs now only need to be proven “interchangeable” with the originator compound, so a single clinical trial switching study to examine interchangeability in patients, along with thorough physicochemical characterization is all that is generally needed for market approval.
Cell and gene therapies (CGTs)
CGTs encompass both living cells and gene delivery systems, such as ex vivo genetically modified cells, viral or non-viral vectors, and other complex formulations containing nucleic acids. There are a wide range of sub-modalities because of the range of therapeutic cell types and vectors for delivering genetic material. The most common ones will be highlighted in this article.
Starting material for cell therapies may be from cell and tissue banks. In such cases, the donors used must be genetically screened prior to cell or tissue harvest. After characterization by sequencing or other cytometric analysis, the material may be expanded in vitro, genetically modified or transformed in some other way, then reformulated prior to administration. Autologous therapies refer to products derived from the patient’s own, potentially highly variable, cells, necessitating different manufacturing strategies depending on the cell profile. Allogeneic therapies are “off-the-shelf” cell therapies that can be administered to anyone. The cells used to manufacture such “universal” cell products need to be carefully screened for immunogenicity.
Cell-based products harvested from humans, e.g. peripheral blood mononucleocytes (PBMCs), pose an additional challenge due to the need for patient confidentiality and laws regarding organ trafficking. Customs clearance and transportation of human cells, genetically modified or otherwise, will be complex. Thus, CGT manufacturers must ensure that they have a reliable cold-chain logistics provider to maintain the quality of their products in the event of any logistical delays.
Viral vectors are commonly used to deliver genetic material into human cells for gene therapy. Adeno-associated viruses (AAVs) make up an overwhelming majority of such products and are preferred because of their superior safety profile as compared to other viruses, according to a statement by former FDA Commissioner Scott Gottlieb. A significant minority of gene delivery systems consist of lentiviruses (LV) as these can carry more genetic information and integrate into the host genome. A key requirement for manufacturing viral vectors is the use of separate facilities or isolated workflows to prevent contamination.
Due to the relatively recent emergence of CGTs, no one platform has had time to become the industry standard for viral vector production, unlike suspension CHO culture for mAbs. Proprietary transfection systems, bioreactors and affinity columns for downstream purification are still being developed by companies such as GE, Thermofisher, Sartorius, Pall and Merck Bioreliance. This, ensuring that the manufacturing platform you adopt for making FIH material can be readily scaled-up for your pivotal clinical trials avoids complications in the future.
In contrast to mAb production, making virus material for FIH trials requires the use of transient transfection because developing a stable virus packaging cell line is a lot more time consuming and challenging compared to that for mAbs. Transient transfection requires GMP-grade plasmid DNA which can become a bottleneck in terms of supply and cost. Another difference is that adherent cell culture processes are favoured over suspension culture due to their superior transfection efficiencies which generally translates to higher virus titer and productivity. However, there is limited global expertise in process development and scale-up for adherent cell culture systems compared to suspension ones.
Aseptic processing is required from the beginning to the end of a CGT manufacturing process
Since CGT products cannot be terminally sterilized by filtration, microbial contamination is controlled by the quarantine and sterility testing of cells and other raw materials, and by conducting the entire manufacturing process using aseptic processing techniques. Manipulations should be carried out using closed loop devices with single-use cartridges and sterile connectors as much as possible because if your process requires the opening of T-flasks or centrifuge caps, these activities need to be performed in a Grade A environment.
Challenges of characterising CGTs
Robust, non-destructive and label-free analytical methods for characterizing CGTs are still in development and are especially necessary for in-process control. Current draft guidances for CGT manufacturing recommend a range of orthogonal techniques to establish identity, potency, safety. For FIH material, titer and virus copy number can be measured by PCR as a surrogate for assessing potency. The digital drop technique allows measurement at the single cell level and is now the industry standard for manufacturing quality control using PCR. Cytometry techniques based on fluorescent labels also require a GMP-source for the antibody-based detection reagents, which can be challenging to secure. Image cytometry is a microscopy technique that offers similar capability to flow cytometry. Images or videos are processed with machine learning algorithms for cell counting, characterization, and morphology analysis. An example is digital holographic microscopy, which can be used to characterize live adherent cells on a surface. Cell electrical impedance spectroscopy is also an emerging non-destructive technique.
Recommended strategy for cell and gene therapies
There is currently a global shortage of CDMO capacity for making CGTs. Although scale-out manufacturing strategies using segregated incubators and biological safety cabinets in clean rooms should be adequate for making the small amounts of material required for FIH trials, and already exist in many hospitals to process hematopoietic stem cells from bone marrow for transplant, the USFDA recommends planning for commercial manufacturing and comparability studies during Phase I/II trials. Companies like GE, Thermofisher, Pall, Sartorius, Lonza and Merck Bioreliance are all vying for their proprietary systems to become the industry standard for CGT manufacturing, so there may be opportunities to establish mutually beneficial collaborations even at an early stage to manufacture your CGT product.
Since manufacturing platforms for CGTs are still in development, establishing a potency assay as early as possible will allow the impact of process or platform changes on the material to be assessed rapidly. Without a robust potency assay, any changes in manufacturing platforms will require a new clinical trial. In the case of Zolgensma, a gene therapy product that treats spinal muscular atrophy, a murine potency assay was used to establish process equivalence and gain regulatory approval when changing from using T-flasks to a packed bed system between Phase II and Phase III trials.
Ensuring product safety, identity and process scalability are universally important for product manufacturing at all stages of development. Identifying important impurities and setting limits whilst minimizing all other impurities ensures safety. The product must also be translationally equivalent to material used in animal studies, so its identity must be determined as early as possible. Lastly, the process must be scalable to make the amount of material needed upon launch, with only “insignificant changes” anticipated. Otherwise, a plan will be required to demonstrate product equivalence between new processes or process changes, for example analytical or functional characterization of the product.
 The opinions in this article are meant for general information and should not be taken as professional advice.
 GLP refers to a set of analytical test protocols and guidelines agreed upon by OECD countries and affiliates. Standardized practices, protocols and audits of GLP certified labs by the OECD enable mutual acceptance of laboratory test data from laboratories in different countries.
 A change in the manufacturing process of CGTs may result in a different product. Regulatory authorities will request a demonstration of product equivalence before and after such process changes.
 There are specialist CDMOs, such as Cevec, that have service platforms using their proprietary cell lines.
 You can avoid the high cost of renting a Grade A cleanroom complex by using a closed-system isolator glovebox to house equipment such as an incubator, centrifuge and microscope. If the isolator-glovebox internal environment is qualified at Grade A, draft guidance for CGT manufacturing suggests that locating the closed-system device or isolator-glovebox in a Grade D room will meet regulatory requirements.
About the Author
David has a PhD in Synthetic Organic Chemistry from the Australian National University. He has over ten years of experience in the pharmaceutical industry including positions at GlaxoSmithKline Manufacturing (Singapore), AMRI Global Discovery Services (Chemistry), Hummingbird Bioscience (preclinical development) and Esco Aster (bioprocessing services). He can be contacted at firstname.lastname@example.org.