Upstream Process Technology I

• Upstream processes I is about Understanding Cell Biology and its implications in cell line development for manufacturing of protein biologics
• The learning outcomes include reviewing gene expression (transcription and translation), understanding gene organization and structure, and gaining an overview of tools and techniques for amplifying and cloning genes.

Recombinant Protein Synthesis: A 6-Step Process

• Gene cloning is the first four steps in this process and are called upstream processing:
• Isolation of gene of interest
• Introduction of gene to expression vector
• Transformation into host cells
• Selection of the required sequence and propagation of cells
• Isolation and purification of protein and formulation of protein product are called downstream processing

Revision of Key Terms

• Chromosome: A molecular "package" for carrying DNA in cells
• Locus: The region on a chromosome where a gene is found
• Gene: A locatable region of genomic sequence, corresponding to a unit of inheritance
• Allele: One of multiple forms of a single gene

Gene Structure in Eukaryotes

• Important regulatory sequences, enhancers, silencers, are associated with the sequence of DNA that is responsible for specifying the codons in a protein-coding gene
• Transcription start site includes the Promoter: which has a sequence of 7 bases (TATAAAA), also called the TATA box. It is bound by a large complex of some 50 different proteins
• Specific start point (Tc)
• Stop site (tc)
• Exons can be found on the mRNA, they are sequences that encode the polypeptide.
• Introns are sections that must be removed from the primary RNA transcript before mRNA can be translated
• Enhancers and silencers are associated with the sequence of DNA

Gene Expression (Central Dogma)

• In the nucleus, DNA goes to mRNA
• In the cytoplasm, mRNA will become Protein

Translation Details

• Translation, the peptide is assembed due to the information provided by mRNA
• mRNA, ribosomes, tRNA, amino acids, aminoacyl synthases and translation factors, work together in a coordinated manner for protein synthesis
• mRNA serves as a template for translation into a polypeptide by ribosomes
• Triplets of the genetic code encode amino acids
• AUG is the start codon for translation in mRNA
• UAA, UGA, and UAG are stop codons

Isolation of the Gene of Interest (GOI)

• Messenger RNA (mRNA) is isolated from cells, the cloned from mRNA using reverse transcriptase RTase (DNA polymerase that transcribes single strand RNA into single strand DNA)
• It's not possible to clone mRNA directly. It must be converted into cDNA (complementary DNA) before being inserted into a suitable vector
• RTase synthesizes a copy of mRNA which makes a cDNA-mRNA hybrid
• mRNA breakdown with alkali or RNaseH

In Vitro Amplification of the GOI

• The polymerase chain reaction or PCR copies gene of interest
• Appropriate primers for PCR, designated restriction sites are introduced to the 5' and 3' flanking ends of the GOI

Restriction Enzymes Overview

• Restriction enzymes, act as molecular scissors that cut DNA at specific sites
• Bacterial enzymes that recognize specific 4- to 8-bp sequences, called restriction sites, and then cleave both DNA strands at this site
• Results in blunt or protruding ends, called sticky ends.
• Sticky ends, also known as cohesive ends, are complementary and base pair with other fragments generated with the same restriction enzyme
• DNA ligase covalently ligate fragments with the building of phosphodiester bonds between nucleotides therefore working opposite to restriction enzyme cut sites

Ligation Enzymes Overview

• Joining of DNA ends catalyzed by enzyme DNA ligase acts as molecular glue
• Ligation is ATP-consuming, leads to formation of covalent phosphodiester bond
• Ligation reactions are less efficient in joining blunt-ended DNA fragments together which means a higher DNA conentration is required

Overview of Introduction of DNA into Production Host

• Transformation is the process to introduce vectors (containing GOI) into microbial cells.
• Transformation efficiency is determined by the number of colony forming units or cfu produced through the transformation of 1 μg of plasmid into a given volume of competent cells
• Method 1: Heat shock -Competent cells in solution of calcium chloride incubated at 0°C
• Competent cells need to be coincubated with plasmids briefly for 1-2 minutes
• Cell exposure to heat shock move cells from 0°C to 42°C to create porous membranes
• Cell return to cold temperatures to close pores and trap up DNA
• Method 2: Electroporation
• Cells coincubated with plasmids are subject to current which forces pores to open in cell walls

Transfection Overview

• Transfection is the introduction of vectors into animal eukaryotic cells.
• Methods
• Lipofection: A cationic lipid forms liposomes-DNA complex which enters cells by endocytosis, releasing DNA into the cytoplasm to travel to the nucleus
• Polyfection: Cationic polymers or dendrimers complex with DNA and works similarly to lipofection.
• Electroporation: An electric pulse is applied to create pores in the plasma membrane for DNA to enter the cell. Has a high efficiency for CHO cells but a lower viability.

Plasmid Replication Strategies Overview

• Non-Integrative Plasmid: Remains seperate from the cell genome
• Integrative plasmid is integrated into cell

Transient vs Stable Transfection

• Transient: DNA taken up by host cell is maintained/replicated as extrachromosomal units and is useful for rapid but small production
• DNA is gradually lost and cell proliferation decreases which decreases product yield and quality
• Stable: DNA integrated into the cells genome, uses a selection step with markers to select cells containing vector DNA from the pool
• Host cells retain expression longer although the genome's position for the DNA integration can have unexpected changed

Gene Cloning Summary

• Gene cloning allows segments of DNA from diverse sources to be reassembled into a single construct
• Basic outline: Foreign DNA, Recombinant DNA, DNA insertion, Bacterial chromosome, Bacteria cell

Plasmid Vector: Overview

• Small circular DNA that is in bacterial cells
• Allows for the transfer of genetic material between cells a process called conjugation
• Advantages
• Replicates independence from it's host genome
• Exists in high copy numbers in bacterial cells
• They can be inserted directly into the host genome

Plasmid Overview

• Promoter: located 5' upstream of gene of interest
• ORI (Origin of Replication): Plasmids can replicate independently from the host cell's chromosomes
• MSC or Multiple Cloning Site: Contains multiple restriction sites, allows for easier insertion of DNA
• Antibiotic resistance genes: Allows for selection of recombinant clones

Colonies Overview

• Cell colonies each formed from a single cell, on the correct media those with the target transform can be selected for
• Each transformed colony in theory should be different although there may be similarities

Cell Lines

Cell bank creation in Upstream Processing Overview

• Biopharmaceutical production uses characterized recombinant clones for stable cell lines that can grow and produce high yields

Dihydrofolate Reductase (DHFR) selection system

• A study on the creation of selection marker systems used for selection of cell lines.
• Delivery of gene of interest: is carried in a vector into host cell's nucleus for chromosomal integration, the integration site is random
• Selection: Cells stably expressing co-transfected DHFR enzyme are selected
• Recovery and expansion: Surviving cells that express the prortein of interset are recovered, single cloning or limiting dilution is technically essential
• Amplification: After surviving cells are exposed to amplification via concentrations of MTX that increase, the CHO cells selection pressure they undergo genomic rearrangement and amplification
• Screening: Individual clones with the highest possible growth rate as well as best product quality of clones screened in limiting dilutions plates
• Expansion and Evaluation: selected chosen Clones expanded by subculturing are evaluated against a bioreator
• Banking of Cell: frozen vials banked as a master cell line