Strategies to Obtain High-Quality Recombinant Proteins

- Protocols and Case Studies Contents

Recombinant Protein Expression Overview

Key Influencing Factors for Protein Expression

FAQs & Case Studies 1

Recombinant Protein Expression Overview Applications of Proteins

Protein Therapeutic Structural Cell Enzymes Therapeutics Studies Culture

Immunogen，Function Investigation，Industrial-scale Enzyme …… Natural Proteins vs. Recombinant Proteins

Natural Recombinant

Recombinant Limited resources technology

Animal contaminants Animal-free

Low protein yield High protein yield

Batch-to-batch Controlled batch-to- variations batch variations Process for Recombinant Protein Expression

Vector Gene synthesis Transfection construction

Protein Protein QC control purification expression 2

Key Influencing Factors for Protein Expression How to Obtain Recombinant Proteins

• Supernatant / intracellular Key Factors Purification • Lysis method methods • Chromatography purification

• Culture medium • Transfection method Culture • Culture temperature & condition duration

Vector / Host

• Sequence • Vector • Host cells Usage of Expression Host

Bill RM. Front Microbiol. 2014 Expression Hosts Used in Bio- pharmaceutical Area

Yeast, 20%

Mammalian cell, 50%

E. coli, 30%

Bill RM. Front Microbiol. 2014 Expression Systems

Baculovirus-Insect Mammalian E. coli Yeast cells cells

• Low cost • Low cost • Desirable post-translational • Desirable post-translational • Rapid expression • Rapid expression modifications modifications • Easy to scale-up • Easy to scale-up • Easy to scale-up • Soluble proteins • Most widely used • Some post-translational • High cell density • More demanding culture • Rare post-translational modifications • Soluble proteins conditions modifications • Unique glycosylation pattern • More demanding culture • High cost • Inclusion bodies • High mannose content in conditions • Difficult to express higher MW glycan • Lack of partial glycosylation proteins • High cost Expression Hosts - Post-translational Modifications (PTMs)

Protein Activity Low High

Yeast / Insect Stable CHO / E. coli

Yeast Insect

Expression Yield Low High

Yeast E. coli / insect Stable CHO

E. coli Stable CHO Expression Host Choice - Protein Molecule

Protein type Mammalian cell Insect cell Secreted protein ++ +

Extracellular region ++ +

Cytoplasm + ++

Viral proteins + ++

Trans-membrane protein + ++

Kinase + ++

Difficult to express ++ ++ Assisting Protein Expression

 Small-MW tags：His, FLAG, HA, Myc  Large-MW tags：IgG-Fc, GST, SUMO  Tag location：N- or C-terminal  Protease digestion sites：EK, 3C, TEV…

 Tag removal  Serve as peptide linker - Protein Tag

 Facilitate purification Common tags used in Sino Biological  Assist protein folding Expression system Tag

 Improve solubility HEK293 / CHO His, Fc, flag

 Enhance stability Insect His, GST, Fc, flag  Increase yield Yeast His  Prolong protein half-life E. coli His, GST, Trx, Sumo, MBP, NusA, ZZ Expression Vector - Protease Digestion Site Selection

Protease digestion site Protease digestion site

Tag Protein Tag

Enzyme Sequence Protein Tags Impact Activity

Fc tag Fc tag His tag His tag Culture Condition

 Transfection  Culture medium: Ingredient and feed liquid  Vector quality  Culture method:

 Transfection reagents:  Stirring: shear force, foam Liposome, positive ion, etc.  Dissolved oxygen  Cell status  pH  Plasmid-to-cell ratio  Culture temperature & time

Culture Time Optimization

 Culture time affects multiple aspects of protein expression

 Loss and degradation of plasmid during culture  Protein accumulates along with culture

 Track expression status

 Protein aggregation and degradation also increase during the course of culture Purification Technology

Secreted Intracellular Membrane proteins proteins proteins

Solid-liquid Cell lyses Extraction Purification separation

• Dilatometry • High speed centrifugation • Affinity purification: Ni- • Affinity purification • Membrane breakage with • Deep filter sepharose • Ion exchange detergent • Microfiltration • Cation exchange • Hydrophobic chromatography • High pressure crushing • Ultra-filtration • Gel filtration • Ultrasonic crushing Protein Development at Sino Biological

Molecule Sequence Vector Pilot Process Scale-up Protein analysis & host clone expression optimization production QC

4000 3% 12% 3500

3000 6%

2500 8% 52% 2000

1500

Protein number Protein 1000 19% 500

0 HEK293HEK293 cell细胞 Insect昆虫细胞 cell E.大肠杆菌 coli cell Yeast酵母 CHO CHO细胞 cell Human Mouse Rat Monkey Viral Others 3

FAQs & Case Studies Recombinant Protein Expression: Case Studies

Low production

No activity FAQs Degradation

Aggregation Factors Contributing to Low Protein Yield

Causes Solutions

Low expression level Expression vector

Aggregation and degradation Expression host

Toxic protein Culture medium ingredient

Protein misfolding and Culture condition post-translational modifications …… …… Low Protein Yield - Vector Promoter Optimization

Sino Biological Leading Brand

293ft cos7 293ft cos7

DG44 HepG2 DG44 HepG2

3-10-fold higher Low Protein Yield

- Vector Promoter Optimization

Protein Production Protein (mg/L)

Leading Leading Plasmid 1 Plasmid 2 Plasmid 3 brand 1 brand 2 from Sino from Sino from Sino Low Protein Yield - Transfection Ratio

MOI adjustment: Improve protein expression

 Low MOI: multiple rounds of transfection  High MOI: most cells are transfected once  Adjust culture time  Focus on protein stability Low Protein Yield - Expression Host Cell Line

Commonly used insect cell lines Protein expression level in different host cells sf9 hi-5 70  Sf9, sf21, high five 60 50  Sf9: virus amplification 40  High five: higher expression level 30 20 10 0 A B C D E F Low Protein Yield - Culture Medium Selection

Leading Brand 1 Leading Brand 2  Cell density Leading Brand 3 Sino Biological  Cell viability

 Post-modification Protein Production Protein (mg/L)

Culture time post-transfection (days) Low Protein Yield - Reduce Aggregation Protein 1 Protein 2 3-fold yield increase 3-fold yield increase

- + - +

Add anti-aggregation reagents to the culture medium to increase protein yield Transient Transfection vs. Stable Expression

Transient transfection Stable cell line

• Short production cycle（within 1 week） • Long production cycle • Sustainable expression within 4-10 days • Plasmids integrate into chromosomes • Plasmid easily lost • High expression level • Batch-to-batch variations • Stable protein expression

 Secreted protein production: CHO stable cell line > 293 transient transfection  Membrane protein and intracellular protein production: little difference Protein Degradation Monitoring and Management

Sequence mutation

Protease Expression inhibitors host

Degradation

Purification Culture condition temperature

Culture duration Reduce Protein Degradation - Change Expression Host

Mk protein has very low degradation in Degradation Comparison High five cell SF9 High five

Kaneda N et al. J Biochem. 1996 Jun;119(6):1150-6 Reduce Protein Degradation - Culture Condition Optimization High Low Low Temperature Reduce expression time +++ + ++ Time

Full length

Fragment

+++ + ++ Extent of degradation Solutions for Protein Aggregation

 Choosing the right cell lysis method

 Control shear force, control foaming

 Choose the right buffer system

 Can only be explored during experiment

 Disulfide bond mediated aggregation: add appropriate reducing agents

 Optimize purification conditions with aggregate removal

 Optimize molecule structure: mutate sites with hydrophobic amino acids Solutions for Aggregation Reduction - Culture and Purification Optimization Culture medium optimization Purification condition optimization

Before Reducing reagent - + - +

After

Non-reducing gel Activity is the Key to Protein Applications

Culture and purification Protein tag optimization

Solubility enhancing tag 1 Solubility enhancing tag 2

activity

enzymatic

Culture A B C C Purification I I I II No activity Active Challenges for Intracellular and Membrane Proteins  Releasing large amounts of protein and nucleic acids after cell disruption  Protease: degrade target protein  Nucleic acids: long-chain DNA and RNA  Like a glue, it forms a sticky substance that can not be removed via centrifugation, filtration, and column purification.  Bind to target protein as a form of contaminant.  Hybrid protein: protein aggregation  Target protein form aggregation with itself  Aggregation between the target protein and hybrid protein  Reduce target protein binding to columns  Difficult to remove and cause reduced purity for target protein Solution for Nucleic Acid Contamination

Add nuclease after cell disruption

 A small amount of nuclease can cleave long- chain nucleic acids into small fragments  High activity, small quantity of enzyme needed

 Low cost Before After SuperNuclease from Sino Biological Membrane

1st 2nd Total proteins Before After Purification purification Protein extraction  Explore of protein extract Separate protein from membrane （detergent , pH, salinity, etc.)

Solid-liquid separation Remove particles

Crude purification  Explore of purification methods Extract target protein （pH, salinity, buffer and purification column)

Purification Remove impurities, improve purity Key Factors

Expression Protein Protein host expression purification

Sequence Cell culture Formulation screening Vector Transfection method

Cleavage enzyme Tag Promoter Culture temperature QC control Signal peptide Culture time Key Factors

Expression Protein Protein host expression purification

Sequence Cell culture Formulation No One-Size Fits screeningAll Vector Transfection method

Cleavage enzyme Tag Promoter Culture temperature QC control Signal peptide Culture time Sino Biological – Accelerates your Research

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