UNIT OPERATION / PRODUCTION PROCESS


Cell/Tissue Therapy Processing

Advanced cellular therapy products represent a new paradigm of bio-regenerative medicine using MSCs as one of the cell populations to meet with unmet medical need. The application of MSCs for cellbased therapies is in cognitive phase of development and their ability of multipotency, immunomodulatory, proliferation capacity and genomic stability are critical for achieving therapeutic success. On the other hand, global stem cell market expectations are raised constantly and providing more conducive environment for future growth. The effectiveness of MSC therapy is based on identification of CD markers and the relationship of these markers to the functional status of the cells as well as the culture conditions used to isolate or scale-up of MSCs product. Furthermore, due to extensive expansion potential and their broad, mesodermal differentiation as well as their immunomodulatory capabilities and secretion of numerous paracrine tropic factors which modulate inflammation, remodelling and regeneration of damaged tissue.

Cell therapy bio-processing involves the usage of numerous in-put materials to produce a complex and viable stem cell product. Therefore, developing a unique bio-processing model with novel production process is challenging for the nascent cell therapy manufacturing industries and requires utilizing bioprocessing experiences and solutions for the progressing towards GMP compliance large-scale MSC production. To date, large-scale production is typically achieved using two dimensional (2D) tissue culture vessels-an expensive, open and time-consuming process. Traditional bioreactors may be the right solution for closed production process of MSC but scale-up protocols still require optimization, and bioreactors cannot be used for adherent stem cells unless microcarriers become a viable technology. The researchers are evaluating the utility of a stirred-tank bioreactor in combination with microcarriers for Mesenchymal stem-cell expansion and comparing the characteristics of the manufactured cells with those grown in standard 2D cultures.

Production Process

The recent success in the use of MSC therapy has increased the demands to produce several hundred to thousand billions of MSCs per batch to satisfy the clinical trial requirements via large-scale expansion, while adhering to GMP compliances. These GMP practices ensure that the product is safe, consistent, effective and of good quality. Although substantial progress has been made in the production process for MSCs but still there is great need to identify and fill the gaps to meet successful GMP compliances.

In terms of GMP requirements, these processes are not fully closed and require a class A cabinet for each manipulation step. Therefore, the development and use of closed automated devices are an important step for facilitating MSC expansion under GMP compliance. For simpler and safer processes, a fully closed and automated bioreactor must be used. The main criteria for growing MSCs in bioreactors are a large ratio of surface area to volume, a closed system, automated inoculation and harvesting, and automated control of culture parameters.

Seed Preparation for Clinical-scale Production Batch

When it comes to large scale production for cell therapy, one of the most important challenges is how to scale-up its manufacturing process in a cost effective manner. For a pre-defined batch size during production, a constant number of culture vessels with good growth surface area single are seeded by using QC released WCB vials for clinical scale production. A methodical screening (microscopic observation) at different interval of culture age is also necessary to monitor the cell confluency as one of the process variables. Along with screening feeding schedule needs to be standardized base on the process. Over confluent cultures usually tend to undergo replicative senescence and preferable choice of confluency within 80-90% may minimize the cellular senescence and cell cycle growth arrest.

Clinical-scale Production

For clinical or commercial purposes, it is necessary to produce a very large number of cells per production run. Scale up can be achieved by increasing the batch size or number of layers per vessel to a manageable size for manufacturing. In order to scale up the production, a tide motion bioreactor in a cell therapy aseptic isolator can be used throughout the whole process. It will ensure the quality of cells produced that will be used in the clinical trial.

The seed harvest should be immediately followed by expansion of designated set of lot size in multiple layers of culture vessels of 10 or 40 units as clinical scale batch. Once the cultures reach required confluency cells are detached through washing the culture vessels with DPBS and followed by incubation with 0.2% of trypsin EDTA at 370 C. The detached cells are neutralized through trypsin neutralization solutions (TNS) which neutralize proteases or trypsin. Trypsinized cells are recovered by centrifugation and re-suspended in complete medium at a concentration of 1-3 million per mL. Seeding in all units of production vessels should be completed within 10-20 minutes after process initiation to prevent cell attachment to the walls of the vessels.

Pre-harvest Preparation

A day prior to harvest, cryo-labels and cryobags need to be air evacuated and labeled appropriately and tube sealers needs to be charged properly. If the product presentation is in vials then ensure various vial container and appropriate stoppers and seals, hand sealer and sterilization of seals and printed labels are prepared a day before harvest. The filling machine need to be check and qualified at least a day before using. Another important step for clinical scale harvesting is the preparation and filtration of TNS and freezing media. All these activities need to be performed a day before or few hours before the harvest of the IMP in GMP production suites. A pre-harvest process and material check list verification will synchronize the system and process during large-scale harvesting

Harvest Process

Ideally production batch should have single harvest of pooled cells of all the culture vessels that goes in all the cryobags or cryovials of single dose. This ensures the product uniformity and consistency in all the cryobag or cryovials pertaining to that batch. Clinical-scale harvesting of 10 or 40 layers cultures vessels starts with DPBS washing after decanting or aspirating the spend media from vessels. A 5-10 mL of spent media needs to be collected from each of the vessel and analyzed for the spent media characteristics. Warmed (37°C) 0.25% trypsin EDTA should be added to each of the production vessels and incubated for 3 to 5 minutes. The culture vessels are tapped gently to dislodge most of the cells to form uniform cell suspension immediately neutralized by adding half of the intended volume of TNS and the cells are collected in collection container. The production vessel is then rinsed with another half volume of TNS for efficient recovery of cells. The detached single cell suspension of MSCs collected in centrifuge tubes and pellet is recovered through centrifugation. It is important to note that harvesting process should be completed within 2 h otherwise the final product will have impact on viability and differentiation.

Post-trypsinization

Process Large scale cultured and expanded cells are harvested and washed with DPBS and centrifuged to reduce the BSA (Bovine Serum Albumin) levels in the IMP (Integral Membrane Protein). The optimization of post culture processing step need strategic volume reduction approaches to retain the viability and to minimize the BSA level in final product. It is essential to prepare a cell based product with high purity levels by reducing the BSA content to about <50 ng/mL. Residual FBS components in terms of BSA needs to be removed through one or two DPBS washing. Several technologies are available for downstream processing steps of cell therapy products and most of them are still being evaluated for commercialization purpose. In fact, automated washing machine (cytomate) has shown 95% post washing viability of cells, but low cell recovery of 85.3%. Tangential flow filtration (TFF) and sequential differential centrifugation technologies are other options for cell harvesting however these systems require extensive optimization and validation for processing large lot sizes to ensure that all microcarriers or particulates are removed from final product. In contrast, manual operation results in inconsistency, manual error and excessive shear stress due to centrifugation, contamination risk and are time consuming process. Future optimization studies needs sterile closed volume reduction post-harvest process either by low shear tangential flow or by continuous centrifugation or integration of both systems for efficient cell washing.

Fill and Finish

Commercial success of cell therapy requires a consistency in cell number, viability and efficacy of the product. The current transition from open system to closed automated end to end fill and finish needs to be performed in a replicable, controllable way. Conventional open process are still in use, because of usage of cryobag for cryopreservation of mesenchymal stem cell but the use of cryo-bag increase additional preparative steps and the manual filling activities are usually performed by more than one trained personnel in multiple biosafety cabinets within the GMP production facility. The most common manual errors during fill and finish are; filling inappropriate cell doses in cryobags because of volumetric error, and excess time taken to complete filling process (more than 30 minutes), delay or lack of coordination in placing cryobags for controlled rate freezer (CRF), selection of inappropriate CRF program for run. Recently closed semi-automated or full automation filling instruments are available for freezing cell therapy product and require a class 100 clean rooms. Integration of automation for fill and finish step in cell therapy products can be easily upgraded to vials, as many conventional pharma and bio-pharma products in vials has already proven safe and are in regular usage for clinical and as commercial application. The screw cap based cryovial of polypropylene container has challenges of container-closure integrity, extractable and scalability. The shift to pharmaceutival vial and automated compatible fillin systems would enable filling several hundred doses of cells per production batch.

Cryopreservation

Cryopreservation media plays an important role to maintain stability, increase shelf life period without losing any functional characteristics. Many cryopreservation protocols uses 10% DMSO as cryoprotective agent for MSCs. The usage of 10% DMSO enhances post-thaw manipulation during administration of drug. In house cryopreservation formulation require USP grade or cGMP grade reagents to satisfy the safety requirements when used as therapeutic parental drug. Thus cryopreservation media used for cryopreservation of MSC requires GMP compliances, which can take care of all regulatory hurdles for cell therapy products. It can be possible by using the commercial available GMP compliance USP grade; serum free, protein free media that contain low percentage of DMSO for therapeutic applications. Several serum-free cryopreservation media are available in the market as GMP-compliant products. 

Quality Control
  1. Raw Material Testing

The quality control of cell therapy production system should ensure the suitability of raw materials for specific activities in a production setting. In most cases, raw materials testing of components is not possible, as there is no definite pharmacopeia method to test the identity of materials which are used in cell therapy manufacturing. In most of the processes raw material release criteria rely on Certificate of Analysis (COA) verification for consumables. However, appropriate quality testing and release criteria are needed for cell culture reagents and consumable as safety and purity of the cell based product would ultimately be dependent on the consumables and reagents used. There is a need to develop robust testing methodologies specifically to qualify and release the critical raw materials like cell stacks, FBS, starting cell banks apart from doing general microbiological testing. The raw material like bovine serum, trypsin can be tested for their identity, purity and safety with relevant specification.

  1. Intermediate Product Testing

The Quality control (QC) of cell therapy production system is increasingly gaining importance, as the product needs to meet safety and efficiency in preclinical and clinical trials. Apart from the standard characterization of MSCs as plastic adherent cells, specific immune phenotypic marker expression and multi-lineage differentiation capacity, it is necessary to check immunomodulatory function, cytokine profile, stability and molecular signature through microRNA expression analysis of these cells. The testing and determining specification for starting material, intermediate products and final product should be analysed during the production process.

  1. Criteria for batch qualification

The basic characteristics to qualify MSCs for clinical trials includes identity, purity, potency, tumourigenicity and genomic stability of these cells. Apart from extensive safety evaluations like sterility, endotoxin, residual FBS related component like BSA removal at the end of the production batch, evaluation of cryopreserved final product for potency assay, in vitro-toxicity, immune-suppressive and immunogenicity analysis are very important.

Quality Assurance

                The need of continuous quality assessment program for cell therapy manufacturing to maintain overall standards in assuring patient safety and drug and system efficacy through internal audits, validations, due diligence, strategic assessment, accreditations, certifications, clinical approvals etc. QA ensures the demonstration of safety standards through implementation of safe controllable, consistent GMP amenable manufacturing and drug delivery procedures. Maintaining standards for process assurance through implementing Standard Operating Procedures (SOP), Process Procedures (PP), Standard Testing Procedures (STP), Out of Specifications (OOS), Corrective and Preventive Actions (CAPA). QA also need to conduct Management Review Meetings (MRM), vendor auditing and developing a system and procedures for process deviation management, risk management (failure mode effect analysis), archival systems, batch records, calibration records, manpower training and assessment, equipment and facility qualifications and logs etc. One of the major requirement in cGMP that will contribute to the quality of the cell production process is the use of cell therapy aseptic isolator during the cell production. This isolator will make sure that sterility is maintained on and during the cell cycle production. The main responsibility of QA is enforcing the cGMP standards, release of batches, and monitoring the process for maintaining the cGMP compliances. Some of the errors or deviations in cell therapy manufacturing can be controlled through regular training, evaluations, and corrective actions to ensure the quality performances. Analyzing the notable events of deviations, manual errors, batch failures through developing a framework of developing online database and quarterly review will ensure deviation trends for process improvements, identifying recurring events to minimize the potential risk and ensures the successful function of quality assurances.

Potency Assay

                The potency is the ability of the product to affect a given therapeutic result. This is an extremely important characteristic of the cellular product, which should be taken into consideration during the product development phase. It is considered an essential aspect of the quality-control system for a CT drug substance and drug product. It is performed to assure identity, purity, potency and stability of products used during all phases of clinical study as well as for commercial products. Hence, potency is defined as “the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to affect a given result”.

Reference: Kolkundkar U, Gottipamula S, Majumdar AS (2014) Cell Therapy Manufacturing and Quality Control: Current Process and Regulatory Challenges. J Stem Cell Res Ther 4: 230. doi:10.4172/2157-7633.1000230

 

Gene Therapy Processing
Two Types of Gene Therapy
  1. Germ line gene therapy - where germ cells (sperm or egg) are modified by the introduction of functional genes, which are integrated into their genome. Therefore changes due to therapy would be heritable and would be passed on to later generation. Theoretically, this approach should be highly effective in counteracting genetic disease and hereditary disorders. But at present many jurisdictions, a variety of technical difficulties and ethical reasons make it unlikely that germ line therapy would be tried in human beings in near future.
  2. Somatic gene therapy - where therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only and will not be inherited by the patients’ offspring or any later generation.
Gene Delivery

In most gene therapy studies, a normal gene is inserted into the genome to replace an abnormal, disease causing gene. Of all challenges, the one that is most difficult is the problem of gene delivery i.e. how to get the new or replacement gene into the patient’s target cells. So a carrier molecule called vector must be used for the above purpose. The ideal gene delivery vector should be very specific, capable of efficiently delivering one or more genes of the size needed for clinical application, unrecognized by the immune system and be purified in large quantities at high concentration. Once the vector is inserted into the patient, it should not induce an allergic reaction or inflammation. It should be safe not only for the patient but also for the environment. Finally a vector should be able to express the gene for as long as is required, generally the life of the patient.

Two techniques have been used to deliver vectors i.e. ex-vivo and in-vivo. The former is the commonest method, which uses extracted cells from the patient. First, the normal genes are cloned into the vector. Next, the cells with defective genes are removed from the patient and are mixed with genetically engineered vector. Finally the transfected cells are re-infused in the patient to produce protein needed to fight the disease. On the contrary, the latter technique does not use cells from the patient’s body.

Some of the vectors that can be used in gene therapy are:

  1. Viral Vectors

One of the most promising vectors currently being used is harmless viruses. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the viral genome and replace them with working human gene. This altered virus can then be used to smuggle genes into cells with great efficiency. Some of the viruses insert their genes into the host genome, but do not actually enter the cell. Others penetrate the cell membrane disguised as protein molecule and enter the cell. Once the transplanted gene is ‘switched on’ in the right location within the cell of an infected person, it can then issue instructions necessary for the cell to make the protein, that was previously missed or altered.

  1. Non-viral Vectors

Simplest method of non-viral transfection is direct DNA injection. Clinical trials to inject naked DNA plasmids have been performed successfully. There have been trials with naked PCR products, which have had greater success. Research efforts have yielded several non-viral methods gene transfer such as electroporation (creation of electric field induced pores in plasma membrane), sonoporation (ultrasonic frequencies to disrupt cell membrane), magnetofection (use of magnetic particle complexed with DNA), gene guns (shoots DNA coated gold particles into cells by using high pressure) and receptor mediated gene transfer are being explored. Each method has its own advantages and disadvantages.

Among the several non-viral approaches, receptor mediated gene transfer currently holds the most promise. This application involves the use of DNA conjugated with specific proteins (viral structural protein), or with liposome, or both. Under experimental ex-vivo conditions, liposomes containing DNA have been shown to undergo cellular uptake through endocytosis, with subsequent transient exogenous gene expression. Nevertheless, the application of this approach will likely be limited until methods for the stable integration of the endocytosed DNA are devised and improvement in target ability, transfection efficiency and DNA carrying capacity are developed.

 

Reference

Sanjukta, Misra, Journal of the Associations of Physician in India, Human Gener Therapy: A Brief Overview of the Genetic Revolution, 2013