Technology Highlights Adam Adler, Ph.D. LakePharma, I nc. October - - PowerPoint PPT Presentation

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Technology Highlights Adam Adler, Ph.D. LakePharma, I nc. October - - PowerPoint PPT Presentation

Oct 27, 2023 161 likes •423 views

Technology Highlights Adam Adler, Ph.D. LakePharma, I nc. October 4, 2013 LakePharma Outline of Technology Highlights Hybridoma cloning Work with complicated hybridoma samples Stable Cell Line Generation Achieve high yield stable cell

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LakePharma

Technology Highlights

October 4, 2013

Adam Adler, Ph.D.

LakePharma, I nc.

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LakePharma

Outline of Technology Highlights

Hybridoma cloning Maxcyte Electroporation Stable Cell Line Generation

  • Improve methods for high viability, high yield transient

protein production (and stable cell generation)

  • Achieve high yield stable cell lines in a short time frame

Recombinant Protein Production

  • Rapid protein production at small-scale
  • DNA sequence to gram scale production in under 6 weeks
  • Work with complicated hybridoma samples

Antibody Humanness Score

  • Developed method to quantify the humanness of

monoclonal antibodies

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LakePharma

Hybridoma Cloning

  • Have successfully cloned I gG hybridoma VH and VL from multiple species
  • Have successfully cloned I gM hybridoma VH and VL from multiple species
  • Have successfully cloned I gG B-cell VH and VL
  • Success rate is > 95% (from more than 400 samples)
  • Turnaround time is < 7 days
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LakePharma

Case Study: Customized Cloning of a Hamster Hybridoma with a Dominant Aberrant Light Chain

Standard cloning method Customized cloning method

QPPLVSVALGQKATITCSGD... QPPLVSVALGQKATITCSGD... QPPLVSVALGQKATITCSGD...

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LakePharma

Case Study: Cloning of a Mouse Hybridoma with Multiple Heavy and Light Chains

Standard cloning method Customized analysis procedure

Relative affinity

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LakePharma

Outline of Technology Highlights

Hybridoma cloning Maxcyte Electroporation Stable Cell Line Generation

  • Improve methods for high viability, high yield transient

protein production (and stable cell generation)

  • Achieve high yield stable cell lines in a short time frame

Recombinant Protein Production

  • Rapid protein production at small-scale
  • DNA sequence to gram scale production in under 6 weeks
  • Work with complicated hybridoma samples

Antibody Humanness Score

  • Developed method to quantify the humanness of

monoclonal antibodies

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LakePharma

CHO Stable Cell Line Generation

  • Antibiotic System Generates Pools in 2 Weeks
  • Serum-free suspension cell culture process
  • Multiple selection markers can co-express multiple genes

– Zeocin, puromycin, and hygromycin B

Pool generation: 2 weeks, yield range is 50-200 mg/L

Transfection Apply selection Cell banking; Ready for production run 1 day 12-14 days 12-14 day production run Transfer to 24 well Transfer to 50 mL tube 100 mL in shake flask Cell banking; Ready for production run 1 day Transfection Apply selection 12-14 days 4 days 9 days 5 days 12-14 day production run

Clone generation: 5 weeks, yield range is 100-500 mg/L

Titer Growth VCD & titer in 96 well plates in shake flasks

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LakePharma

CHO Antibiotic Stable Cells

  • Comparing Pool and High Performing Clone
  • Antibody stable pool generated and

robust clonal lines selected

  • Highest performing clones show

significant titer improvement over pool

Viable cell density Cell viability

Titer

200 1000

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LakePharma

Case Study: I ncrease Expression of a Low Producing Antibody with CHO Antibiotic Stable

  • A mouse IgG2b antibody produced at 4 mg/L in a CHO chemical transient transfection

– 10-fold increase for electroporation or stable pool – 45-fold increase for stable clone

Production yield Conditioned media

4C4 6B5 6C4

Purified sample

Individual Clones Reducing Non-red

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LakePharma

Outline of Technology Highlights

Hybridoma cloning Maxcyte Electroporation Stable Cell Line Generation

  • Improve methods for high viability, high yield transient

protein production (and stable cell generation)

  • Achieve high yield stable cell lines in a short time frame

Recombinant Protein Production

  • Rapid protein production at small-scale
  • DNA sequence to gram scale production in under 6 weeks
  • Work with complicated hybridoma samples

Antibody Humanness Score

  • Developed method to quantify the humanness of

monoclonal antibodies

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LakePharma

MaxCyte STX™ Electroporation

  • Key advantages

– > 95% transfection efficiency in CHO – > 95% viability after transfection – Scalable to 1 liter transfection

  • LakePharma and MaxCyte are partners

– To improve process – To further increase yield

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LakePharma

Case Study: High yield transient production using MaxCyte

Antibody transiently transfected into CHO cells with MaxCyte electroporation – Reached 500 mg/L within 7 days of production

Viable cell density (E6/mL) Cell viability Titer (mg/L)

500

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LakePharma

Case Study: I mprove stable cell line generation process with MaxCyte system

  • Results

– Same DNA, same CHO cells, same selection procedure, different transfection method – Cell viability after transfection is higher with electroporation – 2-3 fold more expression with electroporation

  • Hypothesis

– More copies of DNA are introduced to the cells by electroporation

Stable Pool Comparison # 1 [Antibody] Stable Pool Comparison # 2 [Fc-fusion]

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LakePharma

Outline of Technology Highlights

Hybridoma cloning Maxcyte Electroporation Stable Cell Line Generation

  • Improve methods for high viability, high yield transient

protein production (and stable cell generation)

  • Achieve high yield stable cell lines in a short time frame

Recombinant Protein Production

  • Rapid protein production at small-scale
  • DNA sequence to gram scale production in under 6 weeks
  • Work with complicated hybridoma samples

Antibody Humanness Score

  • Developed method to quantify the humanness of

monoclonal antibodies

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LakePharma

High-Throughput Small Scale Antibody Production

Constructs complete; DNA scale-up; transfection Purification SDS-PAGE, QC 7 days Start DNA construction 5 days 2 days

Rush order [3-5 antibodies]: ~ 2 weeks

Example: – Produced 4 antibodies for a client in 12 days

  • Started from DNA sequence: synthesis through purification
  • Included an international holiday

– Obtained ~ 1 mg of each antibody

Constructs complete; DNA scale-up Purification SDS-PAGE, QC 10-14 days Start DNA construction 2 days 2 days 5 days Transfection [50-100 mL]

Standard order [10-100 antibodies per week]: 3-4 weeks

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LakePharma

[Low-Throughput Large Scale]

Gene Synthesis to Gram Scale Antibodies I n 6 Weeks

  • Gene synthesis and cloning are integrated; can be completed in 1 week
  • Multiple production routes

Route Yield range (mg/ L) Gene synthesis + cloning Pilot test run Stable pool generation Scale up production/ purification Total time 293 transient

50 – 500 1 week 1 week

N/A

2 weeks 4 weeks

CHO transient (MaxCyte EP)

50 – 500 1 week 1 week

N/A

3 weeks 5 weeks

CHO stable

50 – 500 1 week

N/A

2 weeks 3 weeks 6 weeks

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LakePharma

Case Study: High yield 293 transient production

Transient transfection of antibody into 293 cells:

0.0 1.0 2.0 3.0 4.0 5.0 d0 d1 d2 d3 d4 d5 d6 d7

1E6/mL

20 40 60 80 100 120 d0 d1 d2 d3 d4 d5 d6 d7

%

100 200 300 400 500 600 700 d0 d1 d2 d3 d4 d5 d6 d7

mg/L

Viable cell density Cell viability Titer

kDa 75 20 15 10 150 25 100 37 50

At 600 mg/ L, a 2 liter production would be sufficient for gram scale

DNA complete; transfect cells QC’d purified protein is ready Start DNA construction 10 days 9 days

Timeline: < 3 weeks

600

QC’d purified protein is ready New transfection 9 days

After second production run: 4 weeks total

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LakePharma

Case Study: Using a Stable Pool For Rapid Production

Viable cell density Cell viability Titer

Start production run QC’d purified protein is ready Start DNA construction for stable 7 days 14 days

Timeline: 5 weeks

DNA complete; electroporate; apply selection 14 days

300

Make CHO stable pool immediately followed by production run: At 300 mg/ L, 4 liter production would be sufficient for gram scale [or 10 liter WAVE would produce 3 grams]

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LakePharma

Case Study: Purify 1 Gram of a Low Producing Ab

Viable cell density Cell viability Titer

  • Antibody was low producing in transient: ~ 1.5 mg/L
  • CHO stable clone was developed: ~ 50 mg/L
  • Two 10 L production runs (WAVE) were performed:

1,126 mg antibody was purified  I f speed was priority, same goal could be achieved with stable pool and lager volume production

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LakePharma

Outline of Technology Highlights

Hybridoma cloning Maxcyte Electroporation Stable Cell Line Generation

  • Improve methods for high viability, high yield transient

protein production (and stable cell generation)

  • Achieve high yield stable cell lines in a short time frame

Recombinant Protein Production

  • Rapid protein production at small-scale
  • DNA sequence to gram scale production in under 6 weeks
  • Work with complicated hybridoma samples

Antibody Humanness Score

  • Developed method to quantify the humanness of

monoclonal antibodies

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LakePharma

T20 Score Analyzer to Quantify Ab Humanness

  • We developed a tool to quantify the humanness of the variable regions of monoclonal

antibodies – Based on sequence alignment to 1000s of sequenced human antibodies

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LakePharma

T20 Score Analyzer to Quantify Ab Humanness

Heavy (variable) Light (variable) T20 Score Analyzer

T20 score  From 0-100;  Higher score = More human-like

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LakePharma

T20 Score Analyzer to Aid Antibody Humanization

  • T20 scores increase following

humanization of therapeutic antibodies – Though not all technically look “human-like” – These humanized antibodies have little to no immunogenicity

  • Important to check humanness score

throughout humanization process – Humanized v2 and v3 have increasing # of “back mutations” to the rodent framework

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LakePharma

T20 Score Analyzer Available Online

Tool is freely available for use online: http:/ / abanalyzer.lakepharma.com/

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LakePharma

I nformation Online

www.lakepharma.com