Process Steps from Cell-Line Preparation to Manufacture of Personalized Medicines

Compliance with Good Manufacturing Practice (“GMP”) is mandatory for all medicinal products that have been granted a marketing authorization. Likewise, the manufacture of investigational medicinal products must be in accordance with GMP. Advanced therapy medicinal products that are administered to patients under Article 3(7) of Directive 2001/83/EC1 (so called “hospital exemption”) must be manufactured under equivalent quality standards to the manufacturing of advanced therapy medicinal products with a marketing authorization.¹

As outlined by the Alliance for Regenerative Medicine global sector report;²

• There are an estimated 300 million patients suffering from rare diseases worldwide.
• There are currently 647 ongoing clinical trials utilizing regenerative medicines.
• There are more than 400 companies worldwide active in developing regenerative medicines and advanced therapies for rare diseases.
• Companies developing regenerative medicine for rare diseases raised more than $6Billion in total financings globally in 2019.

Value Propagation - Drug Development Space

Alongside validation, the concept of applicability domain (AD) is probably one of the most important aspects which determines the quality as well as the reliability of the established quantitative structure– activity relationship (QSAR) models. A variety of approaches for AD estimation have been devised which can be applied to particular types of QSAR models and their practical utilization is extensively elaborated in the literature; Figure 1 and 2 depicts the value propagation.

Starting Cell to Produce and Populate Cell Line for Master and Working Cell Banks

Cell–based advanced therapies starting material for Advanced Therapy Medicinal Products (ATMPs) span the donor selection and screening, procurement, processing, immunological matching and clinical transplantation seen in cells and tissues transplantation and the level of manufacturing seen in the pharmaceutical and biotechnological industries. These therapies vary from minimally manipulated autologous products through to potentially largescale allogeneic products derived from pluripotent stem cells of considerable complexity. They are rightly subject to stringent regulatory control.

Depending on their classification, they may be under one or more of the blood, tissue, and cells or medicines pieces of legislation and their approved guidance documents. However, there are a number of open issues beyond mandatory requirements. The extent of donor screening for infectious agents and genetic abnormalities, the nature and extent of informed consent and the duty of care to the donor should findings arise which are of relevance to their health, family or public health. There are also risks associated with the manufacturing process itself, the characterization of the cellular product and its behavior in the recipient post administration. The endogenous risks associated with cellular therapies, particularly with respect to donor selection, consenting and testing, and to make recommendations how these can be optimized in order to support the development of cellular therapies and maximizing donor and patient safety. The possibility of infection from administered cell therapies remains one of the greatest risks to potential recipients. The majority of infectious agents will have a cytopathic effect on the cell line and be detected by mandatory product (Quality Control) testing so that their existence will be recognized and the cells discarded before use. There are some potential, and possibly some as yet unknown pathogens, which may be able to incorporate themselves into cells and establish persistent yet non-evident infection. These infective agents may originate from the donor cells themselves, contamination at the time of harvesting, or during the propagation process.

There are two options envisaged/employed in obtaining starting cells as an autologous starting cell line to process and produce the medicinal products;

1. The use of patient own cells as starting material, Figure 3.
2. The use of compatible (other than the patient) cells as a starting material, Figure 4.

What is Transfection and How to Transfect Cells

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It includes the introduction of DNA, RNA, or proteins into eukaryotic cells and is used in research to study and modulate gene expression.

Transfection techniques serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development. Transfection assays enable the advancement of cellular research to enhance drug discovery strategies. Strategies such as viral transfection or viral transduction, utilize, for example, lentiviral (see Table 1) particles to insert foreign material into eukaryotic cells.

Types of Transfection

There are a wide range of transfection methods being utilized including physical, chemical and biological techniques. These techniques generally involve the use of transient or stable transfection methods to incorporate nucleic acids into cells.

Transient transfection techniques involve the introduction of DNA into cells, but in this method, the DNA does not integrate with the cellular chromosomes. This technique facilitates high transfection efficiencies and the gene transcripts can be analyzed after a period of one to four days. For large-scale transient gene expression (TGE) in mammalian cell cultures, transfection vehicles such as polyethylenimine (PEI) and calcium phosphate (CaPi) can be used. Large-scale TGE methods have also been developed using Chinese Hamster Ovary (CHO) cells in the absence of serum.¹

Some of the commonly used transfection techniques include calcium phosphate precipitation, lipofection, electroporation, and viral delivery. These techniques involve the simultaneous delivery of two distinct nucleic acids into the same cell and are often used to achieve stable transfections. Transfections methods have evolved to include several new methods such as the biolistic delivery systems that use high velocity microparticles to deliver nucleic acids into cells, and in vivo transfection protocols that facilitate systemic delivery of siRNA molecules.

Viral Transfection

This method involves the use of viral vectors to deliver nucleic acids into cells. Viral delivery systems such as lentiviral, adenoviral and oncoretroviral vectors can be used for transferring nucleic acids, even in hard-to-transfect cells.

Although viral delivery methods are highly efficient, they can be quite laborious. Most viruses require containment and careful monitoring of biosafety levels. Before performing viral transfections, it is also important to consider several limiting factors such as the lytic nature of viral vectors, cell line packaging and host-cell specificity.

mRNA Transfection Process

The process of mRNA transfection is simpler: mRNA is directly delivered and expressed in the cytoplasm and thus does not require to cross the nuclear membrane. After delivery, it can immediately be translated into a protein in the cytoplasm.

Since this gene expression method does not require the mRNA molecule to enter the nucleus, this is an ideal solution for transfection of stem cells, neurons, lymphoma and many more hard-to-transfect cell types (Figure 5). The transient nature of mRNA transfection is desirable for a number of applications, including cellular reprogramming, genome editing (CRISPR/Cas9) and vaccines.

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Advantage of mRNA Transfection mRNA transfection has some advantages over DNA transfection, which are especially helpful when working with difficult cell types in gene editing research:

• mRNA tends to transfect more efficiently with higher efficiencies reached (>80%) for hard-to-transfect cell types.
• mRNA transfection results in more rapid protein expression, as it doesn’t need to be transcribed first.
• Protein expression is easily adjustable by changing mRNA concentrations or transfection repetition.
• mRNA transfection does not require nucleus entry and is therefore perfectly suited for non-dividing cells.
• No risk of insertional mutagenesis, hence no genome modification of the transfected cell.

Transduction Process

• Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector.¹ An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer.² Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is Dnase resistant (transformation is susceptible to Dnase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome (both bacterial and mammalian cells), see schematic depicted by Figure 6.
• Note: A deoxyribonuclease (Dnase, for short) is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Deoxyribonucleases are one type of nuclease, a generic term for enzymes capable of hydrolyzing phosphodiester bonds that link nucleotides.

Type of Transduction

Transduction has three forms:

1. Generalized transduction: It occurs in the lytic cycle of phage virus. DNA of phages virus enter into E.coli bacteria. This DNA replicates and develops many new DNA and capsids. The DNA of bacteria is broken. Some pieces of DNA also enter into the capsid of virus. Bacteria burst and release new phage viruses. Now this phage enters into recipient bacteria and transfer the DNA of donor bacteria into the DNA of recipient bacteria. Bacterial endonucleases enzymes destroy the phage virus. Now these bacteria incorporate genes of donor bacteria and replicates.

1. Specialized transduction: It occurs in the Lysogenic cycle of phage virus. In this cycle viral DNA incorporate into bacterial DNA as prophage. It remains peacefully there. But sometimes, it becomes lytic. It comes out of bacterial DNA. Some part of bacterial DNA remain attached to it. Viral DNA with a piece of bacterial DNA replicates and lops new capsids. Bacteria burst. Virus infects other bacteria and transfer the genes of the donor bacteria to recipient bacteria.
2. Restricted transduction: Certain phages carry out a more restricted kind of transduction. They carry only a specific section of bacterial genetic material. They transduce only a few genes. Retroviruses carry out specific or restricted transduction. These viruses can cause the formation of tumors (oncogenesis) in animals. It is now known that these viruses exchange a small portion of their genome for a mutant cellular gene that has a role in gene regulation or replication. These viruses carrying mutant genes infect cells. They transform these cells into tumor cells.

Bacterial Transformation

What is Bacterial Transformation? Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. It was first reported in Streptococcus pneumoniae by Griffith in 1928.⁵ DNA as the transforming principle was demonstrated by Avery et al in 1944.⁶

The process of gene transfer by transformation does not require a living donor cell but only requires the presence of persistent DNA in the environment. The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Such bacteria are termed as competent cells.

The factors that regulate natural competence vary between various genera. Once the transforming factor (DNA) enters the cytoplasm, it may be degraded by nucleases if it is diff erent from the bacterial DNA. If the exogenous genetic material is similar to bacterial DNA, it may integrate into the chromosome. Sometimes the exogenous genetic material may co-exist as a plasmid with chromosomal DNA.

Reasons for Transformation

The phenomenon of natural transformation has enabled bacterial populations to overcome great fluctuations in population dynamics and overcome the challenge of maintaining the population numbers during harsh and extreme environmental changes. During such conditions some bacterial genera spontaneously release DNA from the cells into the environment free to be taken up by the competent cells. The competent cells also respond to the changes in the environment and control the level of gene acquisition through the natural transformation process.

Competence of Bacteria

Not all bacteria are capable of taking up exogenous DNA from their environment. The practical approach to acquire competent cells is to make the bacterial cells artificially competent using chemicals or electrical pulses.

• Chemical induction of competence involves the following steps:
  • chilling the cells in the presence of calcium phosphate to make them permeable
  • incubation with DNA
  • heat shock treatment at 42°C for 60-120 seconds that causes the DNA to enter the cells. To endure the heat shock treatment, it is important the cells used are in the log phase of growth
• Alternatively, the bacterial cells are made permeable by subjecting them to electrical pulses, a process known as electroporation.

What Are the Applications of Transformation?

The phenomenon of transformation has been widely used in molecular biology. As they are easily grown in large numbers, transformed bacteria may be used as host cells for the following:

• make multiple copies of the DNA
• in cloning procedures
• express large amounts of proteins and enzymes
• in the generation of cDNA libraries
• in DNA linkage studies

What is Required in a Typical Transformation Reaction?

• Competent cells
• Supercoiled plasmid DNA
• Transformation medium
• Selection marker (antibiotic and/or chromogenic substrate)

The transformation efficiency is defined as the number of transformants generated per μg of supercoiled plasmid DNA used in the transformation reaction.

References

1. EudraLex - Volume 4 - Good Manufacturing Practice (GMP) guidelines, Part IV – GMP requirements for Advanced Therapy Medicinal Products, https://ec.europa.eu/health/documents/eudralex/vol-4_en
2. The Alliance for Regenerative Medicine Global Sector Report, 2019
3. Kenneth Lundstrom, Ph.D., Gene Therapy Applications of Viral Vectors, Technology in Cancer Research & Treatment, ISSN 1533-0346, Volume 3, Number 5, October, 2004, Adenine Press. https://journals.sagepub.com/doi/pdf/10.1177/153303460400300508
4. Westburg, Efficient mRNA transfection for gene expression of difficult-to-transfect cells. https://www.westburg.eu/efficient-mrna-transfection-for-gene-expression-of-difficultto-transfect-cells#:
5. Griffith, Fred. (January 1928). “The Significance of Pneumococcal Types”. Journal of Hygiene. Cambridge University Press. 27 (2): 113–159. doi:10.1017/S0022172400031879. JSTOR 4626734. PMC 2167760. PMID 20474956.
6. Avery, Oswald T. (1944-02-01). “Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a Desoxyribonucleic Acid fraction isolated from Pneumococcus Type III”. Journal of Experimental Medicine. 79 (2): 137–158. doi:10.1084/jem.79.2.137. PMC 2135445. PMID 19871359. Retrieved 2008-09-29.