BBAT Exam 2

Throughput

the amount of material or items passing through a system or process.

Comirnaty Class

mRNA vaccine encapsulated in LNP

Comirnaty Treats

prevention of COVID-19/SARS-COV-2 infection

LNP Ionizable lipids

pH dependent, acidic environment = positive charge —> attract negative mRNA for LNP encapsulation

LNP Cholesterol

fills gap between lipids, increases membrane stability

LNP Helper Lipid

Phospholipid native to membrane, increases membrane stability

Polyethene glycol (PEG)-lipid

reduces immune responce, prevents LNP aggregation

Zeta potential

measure of charge of particles surface when suspended in fluid

Ultrafiltration

high pressure membrane filtration to concentrate product

Diafiltration

pressure driven membrane process to exchange buffer

Recombinant Protein

engineered proteins produced by inserting a specific gene, via plasmid, into a host organism

E.coli pros

fast growing, inexpensive, high expression

E.coli cons

no glycosylation, protein formed via inclusion bodies, no secretion

Glycosylation

post-translational modification where carbohydrates (glycans) are covalently attached to proteins or lipids

Inclusion bodies

dense aggregates of misfolded proteins

CHO pros

correct folding, Glycosylation, secretion

CHO cons

grow slowly, expensive, lower expression, viral safety concerns

Yeast pros

moderate cost and growth, some glycosylation

Yeast cons

Glycosylation different than mammalian cells

Humulin class

recombinant therapeutic protein/hormone replacement

Humilin Treats

type 1 and type 2 diabetes

cell breakthrough

high speed or insufficient density, particles fail to sediment, breaking through the separating medium or failing to be retained in the pellet

Equivalent Settling Area

area required to achieve the same solid separation as a centrifugation

Homogenization

cells pass through a narrow valve at higher pressure

Bradford Assay

measures protein concentration based on shift of colors

Chaotropic Agents

chemicals that disrupts protein structure via denaturation

Enzymatic Conversion - Trysin

cleaves Arg and Lys residues

Enzymatic Conversion - carboxypeptide B

removes basic AAs, Arg and Lys, from C-terminus

Capacity Factor

how strong you species binds

Lypholization

low-temperature dehydration process that freezes a product and lowers pressure to remove water via sublimation

Triple point

temperature and pressure where water exists in all three states

Circular Dichroism (CD)

measures difference in left handed and right handed protein side using circularly polarized light

Polarised Light

all light directed in one direction

CD - LCP

counterclockwise

CD - RCP

clockwise

Chiral

molecules that are non-superimposable on their mirror images

Mean Residue Ellipticity

measure used to compare protein secondary structure, independent of molecule length or concentration

CD Limitations

measures average structure so unable to find location

Mass Spectrometry

measures molecular mass of molecule

Electrospray MS

converts liquid into ions and measured based on various ionization

Matrix Associated Laser

protein mixed with UV abs matrix that ionises and vaporises by being pulses with a laser

RP-HPLC

chromatography separated based on hydrophobicity

RP-HPLC Tests?

Potency

Mass Spec Tests?

Identification

LAL Assay Tests?

quantify endotoxin bacteria

LAL Assay

gene therapy

single dosage to deliver corrected DNA into nucleus to be translated

Gene Therapy Requires

correct DNA sequences, Delivery Vehicle, way to express gene (promoter)

Viral Vector

genetically modified virus

Non-viral vectors

synthetic vector

HEK293 3 required plasmis

gene of interest, AAV replication, helper genes

SF9+ Baculovirus

infect SF9 with BEV carrying AAV gene

Benzonase

digests DNA and cuts into small fragments

MWCO

measures pore size for ultrafiltration membrane

Affinity Chromatography

uses resin with ligand specific to targeted product

cryoprotectant

sugar added to prevent freeze damage

Polysorbate

surfactant prevent adsorption to vial walls and inhibits aggregates

AAV structure

capsid outer protein shell, inside single stranded DNA

AAV pros

no integration into genome, long term expression

AAV cons

small packaging size, one time dosage, high yield loss

qPCR

laboratory technique used to amplify and simultaneously quantify targeted DNA molecules

ELISA

sensitive, plate-based laboratory technique used to detect and quantify substances like proteins, antibodies, hormones, and antigens