Optimized Feed Strategies for Increased Titer and Product Quality

Transcription

1 Optimized Feed Strategies for Increased Titer and Product Quality Steve Gorfien, PhD Senior Director, R&D The world leader in serving science

2 Agenda Pain points in fed-batch culture Addressing pain points Nutritional and dilutional gaps Process considerations Importance of product quality Factors that can influence product quality Latest advance in high-throughput glycan testing Conclusions 2

3 Pain Points in Fed-Batch Culture Nutritional gap Engineered cells have high nutritional demands Providing the right nutrients at the right time Matching basal medium and feed Dilutional gap Dilution impact of feed addition How concentrated can you make the feed to be able to feed more? Process High/low ph feeds add to preparation complexity and time Pick clones in a system that reflects ultimate process Desired end point Cell density, product titer, product quality High cell density may not correlate with high titer or with desired critical quality attributes 3

4 Engineered Cells Have High Nutritional Demands Concentration Viable cell density (1 6 cells/ml) EPO titer (mg/l) Run time (days) Run time (days) Arginine Asparagine Cysteine Glutamine 4

5 Balanced Formulations Drive Performance Viable cell density (VC/mL) IgG titer (mg/l) Sample target process Perform spent media analysis Analyze growth/titer Calculate consumption rates Analyze nutritional pathways Optimized balanced formulations Time (days) 5

6 Benefit of Rationally Integrated Optimization Relative IgG titer Cell Line/control process CHO-K1SV high IgG expression Modified catalog base medium Proprietary feed/process Desired outcome Improved titer Proprietary base medium Formulation access Stay with current feed/process 1,6 1,4 1,2 1,,8,6,4,2, Steps Modification of basal medium alone was insufficient to improve titer Jiang et al, BioProcess International 1(3) March, 212 6

7 Process Intensification Addresses Nutritional and Dilutional Gaps Protein titer Theoretical trend Dilutional gap Nutrient feeds Nutritional gap Glucose feed only Integral viable cell density Higher total productivity by maintaining q p 7

8 Sustaining q P Increased Q P in Matched Basal/Feed Media Viable cell density (1 6 cells/ml) IgG titer (mg/l) IgG titer (g) q p = 29 pg/cell/day Culture time (days) IVC (1 12 viable cell days) CD OptiCHO CD OptiCHO w/ EFC CD OptiCHO CD OptiCHO w/ EFC CD FortiCHO CD FortiCHO w/ EFC CD FortiCHO CD FortiCHO w/ EFC Elimination of nutritional gap = ~2-fold titer increase 8

9 Granulated Format Improves Process and Productivity Processing time cut in half vs. DPM Fluid granulation Neutral ph ph range: Super-concentrated feeds Simple to prepare Target: 15 2 g/l Just add water Solution delivery Spray nozzle Product Faster hydrating 9 min Flexible in its use Add less water to concentrate Air preparation unit Spraying Moistening Solidifying Finished granule Trace liquids Powder Liquid bridge Solid bridge AGT granule 9

10 Volume (L) Increased Titer with Highly Concentrated Feeds Antibody titer (mg/l) L 71 L 19 L 1 L 3x 12 L 71 L 88 L 3x Traditional Dilution Maximum working volume Available space Feeding supplement Media working volume ~2% improvement in bioreactor use Culture time (days) mab A, EFC (45%) mab A, 1X EFC+ (45%) mab A, 2X EFC+ (22.5%) mab B, EFC (45%) mab B, 1X EFC+ (45%) mab B, 2X EFC+ (22.5%) 3 4% titer increase by eliminating the dilutional gap 1

11 Clone Ranking Varies with Process Titer (mg/l) 18 Top 1 MabB Clones in SFB & FB Simple Fed-batch Fed-batch 1 Fed-batch F5 54A5 2B7 5B9 5D9 25F1 5C7 6D6 55F2 5A3 Clone ID 11

12 Product Quality is Important Critical quality attribute: A physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range or distribution to ensure the desired product quality * Examples of critical quality attributes of biotherapeutics: Glycosylation Charge variants N- and C- terminal structural features Fragmentation Aggregation * ICH, ICH Harmonized Tripartite Guideline Q8: Pharmaceutical Development, Step 4 version (August 29) 12

13 Glycosylation is a Key Critical Quality Attribute Glycosylation can affect: Protein folding A1 A1F NGA2F Solubility A2F NA2F Man-9 Immunogenicity NA2G1 Man-5 NA2G1F Enzymatic activity Transport Turnover rate Man-6 Man-7 Man-8 Activity and availability of the protein are impacted by glycans 13

14 Productivity Increase While Maintaining Quality Profile Protein titer Protein titer 25% 25% 2% 15% CD OptiCHO medium +3% EFA +1% FMax 8% Percent glycosylation at harvest 2% 1% 5% 7% 15% Culture duration (days) 6% GF G1F G2F G M5 5% 1% 5% CD OptiCHO medium + 3% EFA 4% 3% 2% % Integral viable cell density EFA = Gibco CHO CD EfficientFeed A Supplement and Fmax = Gibco FunctionMAX Titer Enhancer % CD OptiCHO medium + 3% EFA CD OptiCHO medium + 3% EFA + 1% FMax No change in key glycan structures 14

15 Addressing Media and Process Impact on Glycosylation* ph DO, pco 2 Temperature Low glucose/glutamine Mn, NH 3, glycerol, nucleotide sugar content Cell viability at harvest Shear stress Perfusion vs. fed batch * From review by Hossler P, Khattak SF and Li KJ, Glycobiology, 19(9): , 29 15

16 Preventing Ammonia Accumulation Galactosylation Percent (%) GF G1F G2F G M5 Condition 1 Condition 2 Condition 3 Day 14: NH 4 +: 9 mm Day 14: NH 4 +: 6 mm Day 14: NH 4 +: 4.5 mm 16

17 Controlled Conditions in Bioreactors Galactosylation Percent (%) GF G1F G2F G G1 M5 1 Bioreactor Shake flask 17

18 Lower Harvest Viability GF, G1F Percent (%) GF G1F 5 G2F G G1 M5 Day 14 Day 19 95% Viability 45% Viability Harvest day 18

19 CHO Transcriptome Comparison CHO-S DG44 CHO 9% 8% 8% 7% 6% 5% 4% 3% 2% 1% GF G1F G2F G G1 M5 7% 6% 5% 4% 3% 2% 1% GF G1F G2F G G1 M5 % Day 5 Day 7 Day 11 Day 14 % Day 5 Day 7 Day 11 Day 14 Higher terminal galactose in DG44 Increased transcripts in DG44: ALG2, ALG3, ALG9 and ALG12, ALG8 and ALG1, ALG14, and B4GALT5 Glycosylation differences between cell lines linked with transcriptome profiles 19

20 Media/Feed Composition Influences Glycosylation Percent (%) GF G1F G2F G G1 M5 2

21 Rational Glycan Modulation % Glycosylation 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% % GF G1F G2F G M5 21

22 Additives Only Provided Partial Modulation of Glycans % Relative quantity control.3x.17x.36x.68x.87x 1.X 1 A1F/MAN5 GF G1F_1 G1F_2 G2F An additive approach alone did not provide the ability to target a desired glycan profile 22

23 Another Approach Was Tried Using Gibco GlycanTune C+ Total Feed VCD (x1 6 cells/ml) IgG titer (% of control) A B 18 12% % 12 8% 1 8 6% 6 4% Culture time (days) 2% Culture time (days) Glucose control 15% 3X GTC+ Transition day 15 15% 3X GTC+ Transition day 11 15% 3X GTC+ Transition day 7 15% 3X GTC+ Transition day 4 2% 2X EFC+ Control 15% 3X GTC+ Transition day 13 15% 3X GTC+ Transition day 9 15% 3X GTC+ Transition day 5 23

24 Another Approach Was Tried Using GlycanTune C+ Total Feed % Relative quantity GF G1F_ G1F_2 G2F Mann5/A1F EFC+ Control Day 15 Day 13 Day 11 Day 9 Day 7 Day 5 Day 4 Day of transition from EFC+ to GTC+ The timing of transition from Gibco EfficientFeed C+ (EFC+) to GlycanTune C+ (GTC+) makes it possible to target specific glycosylation profiles. 24

25 Case Study: Modulating Glycan Profile Problem Solutions Results Starting point: Customer was satisfied with yield but required product quality modulation to match the innovator molecule (4/12 glycan attributes within an acceptable range) Optimization Analyze product quality data In-house program DoE using in-house IP/literature SA2 SA2F SA1 SA1F M5 G GF Client analyzes PQ Evaluate in ambr 15 HT system G1 G1Fa G1Fb G2 G2f PQ improvement Outcome: 9 of 12 attributes within range, remaining 3 within 8% of target. 25

26 Current Glycan Analysis Solutions Single channel (CE & LC) low-throughput (days/96 samples) separation Poor data quality of high-throughput methods Labor intensive workflow (pipetting, centrifugation, etc.) Use of toxic sodium cyanoborohydride chemistry Kit supplier Instrument supplier Generic software Use of vacuum centrifugation steps Generic software taking long analysis time Non integrated solution from multiple vendors Commercial kits with high cost per sample No validation support for result/solution Non integrated solution 26

27 Key Features Desired for a High-Throughput Glycan Analysis Platform Easy sample prep Magnetic bead-based sample prep Hands-on time <3 hrs for 96 samples Fewer pipetting steps No sodium cyanoborohydride use No vacuum centrifugation steps Dye labeling Multiple dyes with superior sensitivity High sensitivity Analyze as little as 1 µg of IgG High throughput Low cost Sample prep and data of 96 samples in 7 9 hrs Robust instrument and capillaries with low running cost Resolution Sialylated glycans Structural isomers Fucose species High-mannose species Fully integrated Full sample prep, hardware, and software solution 27

28 Novel CE-based HT Glycan Analysis System Glycan release Glycan purification Dye labeling Data collection Data analysis Fast <1 hr IgG inputs (1.6 5 µg) Reproducible & quantitative (Magnetic beads) Multiple dyes Reactivity Sensitivity Resolution Multi-capillary Run-to-run reproducibility Cap-to-cap concordance Fit for use software Robust alignment algorithms No dry down Complete sample prep and data of 96 samples in 7 9 hours 28

29 Different Dyes for Better Specificity APTS Turquoise Teal Separation of major glycans was possible 29

30 Conclusions Improved volumetric productivity (Q p ) can be achieved by use of rationally designed, matched basal and feed media to sustain specific productivity (q p ) A granulated feed format enables highly concentrated feeds which can further improve productivity and ease of use Product quality can be impacted by choice of media, feed, and process conditions High-throughput glycan analysis by capillary electrophoresis simplifies and improves the workflow Future challenges: Methods for custom optimization of other critical quality attributes, including charge variant and aggregation profiles Rapid, high-throughput, and sensitive methods for analysis of other critical quality attributes 3

31 Acknowledgments Ryan Boniface Michael Gillmeister Jaime Goldfuss Shawn Barrett Mark Stramaglia Serena Smith Doan Tran Baburaj Kunnummal Shaheer Khan JenKuei Liu 31

32 Caution: For use as a raw material in further manufacturing applications 216 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. For more information on glycosylation, visit thermofisher.com/glycantune The world leader in serving science

33 Appendix The world leader in serving science

34 IgG titer (% of control) VCD (x1 6 cells/ml) % Relative quantity Another Approach Was Tried Using Gibco GlycanTune A+ Total Feed A % B Culture time (days) 1% 8% 6% 4% 2% Culture time (days) Glucose control 2% 2X EFC+ control 15% 3X GTC+ transition day 15 15% 3X GTC+ transition day 13 15% 3X GTC+ transition day 11 15% 3X GTC+ transition day 9 15% 3X GTC+ transition day 7 15% 3X GTC+ transition day 5 15% 3X GTC+ transition day EFA+ Control Day 15 Day 13 Day 11 Day 9 Day 7 Day 5 Day 4 Day of transition from EFA+ to GTA+ A. The growth profiles for the DG44 cell in Gibco CD OptiCHO medium were similar with both EFA+ and with the use of or when transitioning to GTA+. B. DG44 titer comparison between feeding conditions. 1% titer is the titer of 15% 3X EFA+ on day 16. Titer results indicate that the use of and transition to GTA+ does not negatively affect protein production. GF G1F_1 G1F_2 G2F Mann5/A1F The timing of transition from Gibco EfficientFeed A+ (EFA+) to GlycanTune A+ (GTA+) makes it possible to target specific glycosylation profiles. Modulating GF from 72% down to 3%, while increasing G1F (1 and 2) and increasing G2F. 34

35 IgG titer (% of control) VCD (x1 6 cells/ml) Another Approach Tried with Gibco GlycanTune B+ Total Feed % Relative quantity A Culture time (days) 12% 1% 8% 6% B Glucose control 2% 2X EFC+ control 15% 3X GTC+ transition day 15 15% 3X GTC+ transition day 13 15% 3X GTC+ transition day 11 15% 3X GTC+ transition day 9 15% 3X GTC+ transition day 7 15% 3X GTC+ transition day 5 15% 3X GTC+ transition day EFA+ Control Day 15 Day 13 Day 11 Day 9 Day 7 Day 5 Day 4 Day of transition from EFA+ to GTA+ The timing of transition from Gibco EfficientFeed B+ (EFB+) to GlycanTune B+ (GTB+) makes it possible to target specific glycosylation profiles. Modulating GF from 8% down to 41%, while increasing G1F (1 and 2) and increasing G2F. GF G1F_1 G1F_2 G2F Mann5/A1F 4% 2% A. The growth profiles for the DG44 cell in CD CHO medium were similar with both EFB+ and when transitioning to GTB+. The large error bar at day 1 was due to clumped cells Culture time (days) B. DG44 titer comparison between feeding conditions. 1% titer is the titer of 15% 3X EFB+ on day 16. Titer results indicate that the use of and transition to GTB+ does not negatively affect protein production. Also works with other matched basal/feed media 35