Exam 2

isoelectric focusing 1st dimension

separation by charge

at the isoelectric point…

protein has no net charge so it no longer moves

protein is positively charge at pH…

below isoelectric point

isoelectric focusing 2nd dimension

separation by size

pros of isoelectric focusing

* can separate closely related proteins
* isoforms
* PTM

cons of isoelectric focusing

* not good for hydrophobic proteins
* limited by pH range
* not good for non-abuntant proteins
* analysis and quantification difficult

immunoassay

* biochemical assay that measures the presence of specific proteins in complex solutions like serum and urine
* involves the use of:
* antibody
* antigen

epitope

specific area of antigen where antibody binds

paratope

parts of antibody that binds to epitope

direct immunoassay

* uses only 1 antibody
* enzyme linked to antibody acts on chromogenic or chemiluminescent substrate
* fluorescent dye (fluorescein isothiocyanate (FITC)) linked to antibody to do immunofluorescence

indirect immunoassay

primary antibody binds to antigen and secondary antibody with enzyme binds to primary antibody

protein detection that issues immunoassays include:

* western blot
* ELISA
* FACS

FACS

* for extracellular membrane proteins or secreted proteins
* can use multiple probes
* can also be used to quantify how many cells in each group

flow cytometry

output of FACS

LC-MS

* liquid chromatography-mass spectrometry
* follows IEF for characterization
* confirm molecular weight and aa sequence
* identify PTM

LC-MS process

* digest with trypsin
* peptides bind by charge in 1st column
* elute using increasing salt = separate based on charge
* peptides separated by hydrophobicity in 2nd column

trypsin

cleaves c-terminal of R and K

MS stages

* introduce sample
* generate ions in gas phase
* separate ions based on difference in m/z with mass analyzer
* detect ions
* match masses of peptide fragments to known and predicted protein sequences

polyvalent

interact with different parts of antigen

antibody diversity arise due to:

* chromosomal rearragements
* alternative splicing

somatic hypermutation

* occurs in activated B-cells
* mutations in variable regions of antibody

polyclonal antibodies

* isolated from serum of immunized animal
* diphtheria and botulism antitoxin
* from multiple activated B-cells
* recognizes different epitopes (polyvalent)

monoclonal antibodies

* from hybridomas
* recognize single eiptope but has high affinity

SP 2/0

mouse myeloma cell line that is deficient in selectable enzyme marker hypoxanthine phosphoriribosyl transferase (HGPRT-)

FL-653

mouse myeloma cell deficient in adenosine phosphoribosyl transferase (APRT-)

hybridoma production process

* immunize mice
* prepare myeloma cells for fusion
* fuse myeloma and spleen cells with PEG
* myeloma cells with mutation die
* spleen cells without mutation are safe
* clone screening and picking
* functional characterization
* scale up and ween off selection agent

orthoclone

* immunosuppressant
* 1st protein from hydridoma for human application
* mouse mAb directed ahainst T-lymphocytes to prevent host vs graft rxn in kidney transplant

anti-mouse antibody response (HAMA)

antibodies from mouse hybridoma causes this, which leads to destruction of circulating antibody, making treatment ineffective the 2nd round

solution to HAMA

* change constant regions to human
* knock in human antibody genes in mice

HEK293

* human embryonic kidney
* aggregate prone due to adherent tendencies
* immortalized by adenovirus vector
* widely used for transient and stable
* highest PEI-mediated transfection
* cell division time approximately 30 hrs

COS

simian fibroblasts

CHO

* can grow in suspension
* few human viruses can infect
* can grow serum-free and chemically defined media
* PTM, like glycosylation
* several gene amplification systems

PER.C6

* human embryonic retina cells
* grows serum free
* productive as CHO
* human glycosylation
* Lonza?

NSO

* mouse myeloma cells
* only in stable
* more difficult to transfect
* low Mab yields

BHK

* baby hamster kidney
* no immunogenic glycans

integrating plasmid

* integrate into genome
* random or targeted integration
* linearized before transfection

episomal plasmid

* maintained as plasmid
* high copy number
* own ori

lentiviral vectors

integrate at specific site in chromosome

popular mammalian cell promoters

* CMV early (cytomegalovirus)
* tet (tetracycline)

tet-inducible promoters

* no doxycycline = repress
* doxycycline = activate

transfection methods

* transduction (viruses)
* electroporation
* Ca-phosphate co-precipitation
* polyethylenimine (PEI)
* lipofectamine

types of viral vectors

* retrovirus
* vaccinia
* adenovirus
* BacMam

retrovirus

* stable cell lines
* do not kill host but may produce progeny virons
* broad host range

vaccinia

* related to small pox?
* high lvls of recombinant protein expression since theres 5000 virus particles per cell

adenovirus

* transient expression
* random integration

BacMam

* baculovirus that has mammalian expression cassettes
* can also work in insects

how to make stable-sorted monoclonal antibodies

* put expression vector into host
* select with single cell cloning
* evaluate clones and scale up good ones
* freeze in liquid nitrogen (-170C)

how to make stable transfection pool

* put expression vector into host
* no need for single cell cloning
* grow in presence of selection then freeze some of it

how to do transient transfection

* put expression vector into host
* grow in presence of selection
* no need to bank, just grow and isolate protein of interest

platforms for transient or stable transfection pool

* shake flask
* WAVE bioreactor
* shake very slowly (25rpm)

transient expression is useful for:

* generating reagents
* functional studies
* testing expression of different protein variants
* testing expression of different constructs
* testing in different cell lines
* preferred for cell surface or secreted proteins

pros of transient transfection over stable

* shorter time period to harvest
* genetic stability due to shorter growth period
* \`DNA manipulation is simpler

cons of transient transfection over stable

* low yield
* inconsistent
* transfection chemicals expensive

stable transfection pool is useful for:

* generating reagents
* testing expression of diff protein variants
* testing expression of different constructs
* testing in different cell lines
* can be sorted later to identify high producing single clones

pros of stable cell line

* high yields
* stable

cons of stable cell line

* gene to protein can take 6-12 months
* complex process

methotrexate (MTX) amplification tech

* MTX inhibits dihydroxyfolate reductase (DHFR)
* grow cells in hypoxanthine and thymidine free media (HT)
* only way to live is if cell has extra DHFR genes

glutamine synthase (GS) tech

* from Lonza
* methionine sulphoximine (MSX) inhibits GS
* grow in glutamine free media
* only cells with extra copies of GS can grow in presence of MSX

clonal selection

* after transfection, select in presence of drug (MTX or MSX)
* screen cells for high producer then do single cell cloning

how to isolate single clones

* serial limiting dilution
* FACS
* ClonePix2

serial limiting dilution

literally keep diluting cells in wells until there is only 1 cell

FACS

* can result in lower cell viability cuz it’s stressful
* selection is based on proteins on cell membrane
* high throughput and shorter time

ClonePix2

* use automated clone picker
* cluster of cells in each well
* label with fluorescent probes and then you can see which well is brighter and pick your clones from there
* higher throughput that serial limiting dilution, but lower than FACS

batch system

* closed system with limited nutrients
* inoculum added when cells in exponential growth phase
* final protein harvested as culture enters death phase
* good for transient expression
* high cell densities difficult to reach

fed-batch system

grown in batch, then nutrients are fed incrementally near end of exponential phase

pros of fed batch over batch system

* higher cell density
* minimize substrate inhibition
* less catabolite repression
* extended production time

continuous culture

* subtrate and other nutrients added to system at fixed, continuous rate
* products removed at continuous rate while cells and media are recirculated
* maintain culture in exponential phase

continuous culture advantages

* highest volumetric productivity
* savings in time, labor, and energy
* uniform product quality
* better automation, monitoring, and process control
* reduced manual labor and human error

continuous culture disadvantages

* cell adhesion to reactor walls and sensors
* difficult to maintain nonoseptic conditions
* mutation and instability
* large volume of media = expensive
* complex process validation

culture parameters that can be optimized:

* media
* aeration
* temp
* pH
* induction strategy
* protease inhibitors
* selection agent

you can stabilize proteins in solution by:

* buffered from pH extremes
* add protease inhibitors
* low temp
* antimicrobials

product recovery

* centrifugation
* filtration
* precipitation

how to precipitate proteins:

add salts ((NH4)2SO4), alcohols, acids

size exclusion chromatography

* use gel beads with pores of different sizes
* larger proteins move down faster

ion exchange chromatography

* proteins with opposite charge of resin will stay in tube
* elute proteins by changing charges with salts

affinity chromatography

proteins bind specifically and reversibly to ligands

hydrophobic interaction chromatography

sort proteins on basis of their repulsion of water

2-step protein purification of Mabs

* affinity chromatography w/ protein A
* anion exchange chromatography

virus removal

* __**H**__eat
* __**U**__V
* __**N**__anofiltration
* __**S**__olvent/detergent
* low pH during or after protein A chromatography

after viral removal…

concentrate product with diafiltration

tangential flow filtration

* ultrafiltration and diafiltration
* used to concentrate protein
* diafiltration removes cell contents, salts, buffers, and additives and replaces it with cleaner, suitable buffer that will stabilize proteins

concerns of biologics

* aggregation
* oxidation
* deamination

protein stabilizers for biologics

* sugars like sucrose and trehalose serve as cryoprotectant and lyoprotectant
* amino acids: histidine, arginine, glycine

surfactants for biologics

polysorbate 80 (tween 80): anti-foam, protect from freeze thaw and agitation-induced aggregation

antioxidant for biologics

methionine thiosulfate

preservatives for biologics

* benzyl alcohol
* m-cresol
* phenol
* too much preservative can cause protein aggregation

fill and finish

divides bulk formulation into single dosage forms

lyophilization

* freeze protein
* vacuum makes water evaporate faster
* maintains protein structure and can be stored at room temp for long time
* followed by stopper, capping, sealing

bioanalytical tests for lot release

* identity
* native size
* denatured size
* charge heterogeneity
* glycosylation
* potency

quantity of recombinant protein produced by process depends on:

* viable cell density
* specific protein productivity of the cells

how to increase viable cell density

* inhibit apoptosis
* delay cell-cycle progression
* telomerase overexpression
* metabolic engineering
* reduce toxic metabolite accumulation

how to increase protein productivity

* improve protein folding
* decrease degradation
* enhance protein secretion

caspase-3

protease that initiates apoptosis

MoA of anti-apoptotic proteins

* interfere with activator
* interfere with effector
* prevent effector from going where it needs to go

Bcl-2

* human proto-oncogene overexpressed in certain B-cell lymphomas
* membrane protein located in ER, nuclear envelope, and mitochondria
* high lvls protect B-cells from early death

Bax and Bak

form pores in mitochondrial outer membrane which releases cytochrome c which activates caspases and therefore apoptosis

overexpressing Bcl-2 would…

inhibit Bax and Bak which prevents apoptosis

p21 and p27

blocks phosphorylation of cell division kinases which arrests cell cycle at G1

p53

activates p21

non-genetic approaches to delaying apoptosis or cell-cycle progression

* biphasic growth
* use galactose instead of glucose
* grow under glutamine limitation
* addition of rapamycin
* caspase inhibitor