Buffer Make up Comparison: Continuous Inline versus Cycling Batch. Greg Lepak Craig Sandstrom

Transcription

1 Buffer Make up Comparison: Continuous Inline versus Cycling Batch Greg Lepak Craig Sandstrom

2 Agenda Sample Projects Model Process & Key Assumptions Buffer Estimates Equipment Flow Diagram Comparisons Process Intensification Opportunities Facility comparison Operational comparison WFI comparison

3 Small Scale Project Comparison MAb Drug Product Facility 275 kg/year 300 Production Days/year Batch Facility 2 X 2,500 L Bioreactors (2,000 L WV) 5 g/l Titer 14 Day Cycle 65% Yield 7 day run rate Continuous Facility 2 X 1000 L Bioreactors (780 L WV) 0.5 g/l 2 VVD 60 Day Cycle/50 Day Production 72% Yield

4 Large Scale Project Comparison MAb Drug Product Facility 5,200 kg/year 310 Production Days/year Batch Facility 6 X 15,000 L Bioreactors (12,500 L WV) 5 g/l Titer 14 Day Cycle 65% Yield 2 1/3 day run rate Continuous Facility 4 X 7,000 L Bioreactors (7,000 L WV) 0.5 g/l 2 VVD 60 Day Cycle/50 Day Production 72% Yield

5 Downstream MAb Process

6 Key Assumptions State of the Art Single Use Mix Vessel <2,000 L & < 1/week Single Use Bag <200 L Sodium Hydroxide Distributed from Central Source Tanks & Lines CIPd Separately Buffers in Bags prepared for 30 days Buffers in Tanks s prepared as: Fed Batch: 1/bath Continuous: Match Fed Batch Cycle

7 Buffer Summary Buffer Solution SSF LSF Col 1 Equil Buffer 2,413 14,476 Col 1 Wash Buffer 1,508 9,048 Col 1 Elution Buffer 1,538 9,228 Col 1 Strip Buffer 618 3,709 VI Acid Buffer VI Base Buffer 214 1,283 VI Equil Buffer 506 3,036 Col 2 Equil Buffer 664 3,981 Col 2 Strip Buffer 603 3,619 Buffer Solution SSF LSF Col 3 Equil Buffer 1,096 6,573 Col 3 Wash Buffer 1,056 6,333 Col 3 Elution Buffer 1,508 9,048 Col 3 Strip Buffer 603 3,619 VF Flush Buffer 306 1,836 VF Equilibration Buffer 337 2,020 TFF Equil Buffer 516 3,096 TFF Diafiltration Buffer 792 4,755 Formulation Buffer

8 Buffer Preparation Small Scale Facility Buffer Prep Tank Prep's/week Prep Tanks Buffer Tank - 2,500 L 4 1 Buffer Tank - 1,500 L 3 1 Buffer Tank L 6 1 Large Scale Facility Buffer Prep Tank Prep's/week Prep Tanks Buffer Tank - 15,000 L 18 3 Buffer Tank - 5,000 L 27 3 SUMV 9 1 Buffer Tank L 3 1 SUMV CIP Operations/week 2 CIP Skids 180 CIP Operations/week 4 CIP Skids

9 Small Scale Facility

10 Large Scale Facility Fed Batch

11 Large Scale Facility Continuous

12 Line Size Comparison Small Scale Fed Batch Line Size Re* Continuous Line Size Re Column #1 80 cm 10 cm** Flow Rates 13 lpm 12, lpm lpm 1" 29, lpm 1/4" 2, lpm 38, lpm 2,800 Column #2 80 cm 10 cm** Flow Rates 30 lpm 1" 29, lpm 1/4" 2,300 Column #3 80 cm 10 cm** Flow Rates 8 lpm 11, lpm 460 3/4" 1/4" 25 lpm 34, lpm 1,900 Assume water at 20 o C. ** Scaled on average product mass flow rate.

13 Line Size Comparison Large Scale Fed Batch Line Size Re* Continuous Line Size Re Column #1 200 cm 45 cm** Flow Rates 80 lpm 36,000 4 lpm 5, lpm 2 80, lpm ¾ 13, lpm 110, lpm 16,000 Column #2 200 cm 30 cm** Flow Rates 180 lpm 2 80,000 4 lpm ½ 9,000 Column #3 200 cm 30 cm** Flow Rates 50 lpm 22, lpm 2,700 2 ½ 160 lpm 71,000 4 lpm 9,000 Assume water at 20 o C. ** Scaled on average product mass flow rate.

14 Buffer Concentrates & Inline Dilution Sodium Hydroxide (NaOH) Commonly supplied at 1M Inline dilution w/ WFI at chrom skids to M Batch dilution at UF/DF to M common Process Buffers Typical 1x 10X concentrations used for chromatography Accuracy critical for chromatography elution buffers Typical 1x, use strength Not all buffers are good candidates for inline dilution Titrated buffers with tight ph or conductivity tolerances High viscosities (e.g. polysorbates) Buffer concentrations near saturation (e.g. 6M Urea, 3M NaCl) Exothermic reactions and off gasing Chrom Eluate Buffer Conditioning Blending buffer with product stream during chromatography load Precipitates can be an issue, buffer/product dependent

15 Batch vs Continuous In-line What Equipment is needed for Buffer prep? What will be the size of the equipment? How will the Buffers be made? How simple or complex will be operations? How will the Choice influence WFI system?

16 Small Scale Batch Small Scale MAb, batch process Two 2000 L batch Bioreactors Cycle 14 days, one batch each week 5 gm/l Titer and 65% efficiency Processing 300 days/year

17 Chromatography Buffer Requirements Protein A - 80 cm Diameter Column Operation Step Buffer Volumn (CV) Velocity (cm/h) Flow Rate (lpm) Duration (hr) Equilibration 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Load Product Chase 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Wash 2 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Elution 25 mm Acetate, ph 3.7 ± Strip 250 mm Acetic Acid, 250 mm NaCl Total Time (hr) 2.55

18 Chromatography Buffer Requirements Step Equilibration Protein A - 80 cm Diameter Column Operation Firsrt Run Volume (L) Second Run Volume (L) Buffer 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Total Volume (L) Load Product mm Phosphate, 1 M Sodium Chase Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Wash 2 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Elution 25 mm Acetate, ph 3.7 ± Strip 250 mm Acetic Acid, 250 mm NaCl

19 Chromatography Buffer Requirements Protein A - 80 cm Diameter Column Operation Step Buffer Firsrt Run Volume (L) Second Run Volume (L) Required Buffer Volume (L) Equil/Chase/Wash2 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Elution 25 mm Acetate, ph 3.7 ± Strip 250 mm Acetic Acid, 250 mm NaCl

20 Chromatography Buffer Requirements Protein A - 80 cm Diameter Column Operation Step Buffer Total Volume (L) Tank Working Volume (L) Tank Volume (L) Equil/Chase/Wash2 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Elution 25 mm Acetate, ph 3.7 ± Strip 250 mm Acetic Acid, 250 mm NaCl

21 Chromatography Buffer Requirements Protein A - 80 cm Diameter Column Cleaning Operation Step Cleaning Buffers Total Volume (L) Tank Working Volume (L) Tank Volume (L) Equilibration WFI 905 N/A N/A Cleaning 1 0.1M NAOH Storage 2% Benzyl alcohol, 100 mm Na Acetate Skid Sanitize 1.0 M NAOH

22 Protein A - Batch Buffer Summary 7 tanks and WFI feeding a LHM Line size 3/4" Small tanks can have close proximately to LHM Buffer is used to complete 2 chrom cycles of 3 hr each Buffer made up once per week and used within 6 hours Buffer volumes L Make up tank 1500 L can make 1200 L for larger buffer volumes 1500 L can make 400L 1200 L, double batch the 2000L Total 8 batches per week, 7 buffers

23 Chromatography Buffer Requirements Bind and Elute Ion Exchange - 80 cm Diameter Column Operation Step Buffer Volumn (CV) Velocity (cm/h) Flow Rate (lpm) Duration (hr) PreEquilibration Equilibration 50 mm Tris-HCl, ph 7.4, 1M NaCL mm Tris-HCl, ph Load Product Elute Strip/Regeneration 50 mm Tris-HCl, ph mm Tris-HCl, ph 7.4, 1M NaCL Total Time (hr) 1.12

24 Chromatography Buffer Requirements Bind and Elute Ion Exchange - 80 cm Diameter Column Operation Step PreEquilibration Equilibration Buffer Firsrt Run Volume (L) Total Volume (L) 50 mm Tris-HCl, ph 7.4, 1M NaCL mm Tris-HCl, ph Load Product Elute Strip/Regeneration 50 mm Tris-HCl, ph mm Tris-HCl, ph 7.4, 1M NaCL

25 Chromatography Buffer Requirements Bind and Elute Ion Exchange - 80 cm Diameter Column Operation Step Buffer Total Volume (L) PreEquilibration/Strip/Regenerati on 50 mm Tris-HCl, ph 7.4, 1M NaCL 604 Equilibration/Elute 50 mm Tris-HCl, ph

26 Chromatography Buffer Requirements Bind and Elute Ion Exchange - 80 cm Diameter Column Operation Step PreEquilibration/Strip/ Regeneration Equilibration/Elute Buffer Total Volume (L) Tank Working Volume (L) Tank Volume (L) 50 mm Tris-HCl, ph 7.4, 1M NaCL mm Tris-HCl, ph Bind and Elute Ion Exchange - 80 cm Diameter Column Operation Total Volume (L) Tank Working Volume (L) Step Buffer Column Storage/ Skid Store 0.1 M NaOH Column Cleaning/Skid Sanitize 1.0 M NaOH Tank Volume (L)

27 Bind and Elute - Batch Buffer Summary 4 tanks feeding a LHM Line size 3/4" Small tanks can have close proximately to LHM Buffer is used to complete 1 chrom cycles of 2 hr Buffer made up once per week and used within 3 hours Buffer volumes L Make up tank 1000 L can make 700 L for buffer volumes Total 4 buffer batches per week, 4 buffers

28 Chromatography Buffer Requirements Flow Through Ion Exchange - 80 cm Diameter Column Operation Step Buffer Volumn (CV) Velocity (cm/h) Flow Rate (lpm) Duration (hr) Equilibration BN Load Product Wash BN Elution BN Regeneration BN Total Time (hr) 2.46

29 Chromatography Buffer Requirements Step Flow Through Ion Exchange - 80 cm Diameter Column Operation Buffer Firsrt Run Volume (L) Second Run Volume (L) Total Volume (L) Equilibration BN Load Product Wash BN Elution BN Regeneration BN

30 Chromatography Buffer Requirements Flow Through Ion Exchange - 80 cm Diameter Column Operation Step Buffer Firsrt Run Volume (L) Second Run Volume (L) Required Buffer Volume (L) Equilibration BN Wash BN Elution BN Regeneration BN

31 Chromatography Buffer Requirements Step Flow Through Ion Exchange - 80 cm Diameter Column Operation Buffer Total Volume (L) Tank Working Volume (L) Tank Volume (L) Equilibration BN Wash BN Elution BN Regeneration BN Step Flow Through Ion Exchange - 80 cm Diameter Column Operation Buffer Total Volume (L) Tank Working Volume (L) Tank Volume (L) Column Storage/ Skid Store BN Column Cleaning/Skid Sanitize BN

32 Flow Through Ion Exchange Buffer Summary 6 tanks feeding a LHM Line size 3/4" Small tanks can have close proximately to LHM Buffer is used to complete 2 chrom cycles of 3 hr each Buffer made up once per week and used within 6 hours Buffer volumes L Make up tank 1500 L can make 1200 L for larger buffer volumes 1500 L can make 400L 1200 L Total 6 batches per week, 6 buffers

33 Batch Buffer Summary Total Batch Buffers for Chromatography Operations Total Tank Working Buffer Volume (L) Volume (L) Tank Volume (L) Step 50 mm Phosphate, 1 M Equil/Chase/Wash2 Sodium Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Elution 25 mm Acetate, ph 3.7 ± mm Acetic Acid, 250 mm NaCl Strip PreEquilibration/Strip/ 50 mm Tris-HCl, ph 7.4, 1M Regeneration NaCL Equilibration/Elute 50 mm Tris-HCl, ph Equilibration BN Wash BN Elution BN Regeneration BN Equilibration WFI 905 N/A N/A Cleaning 1 0.1M NAOH % Benzyl alcohol, 100 mm Storage Na Acetate Skid Sanitize 1.0 M NAOH

34 Small Scale Batch Buffer Summary 13 tanks and WFI feeding three LHMs Buffer made up once per week and used within 48 hours Buffer volumes L Make up tanks two 1500 L 1500 L can double batch the 1700 and 2000L, 6 batches/week 1500L can batch , 10 batches/week Batch 3 buffers in each tank per day, six buffers per day for three days

35 Small Scale Continuous Buffer Prep Small Scale MaB, batch process Two 2000 L batch Bioreactors Cycle 14 days, one batch each week 5 gm/l Titer and 65% efficiency Processing 300 days/year

36 Continuous Buffer Requirements Total Batch Buffers for Chromatography Operations Total Volume (L) 10:1 Concentrate Volume (L) Bag Concentrate Volume (L) Step Buffer 50 mm Phosphate, 1 M Sodium Equil/Chase/Wash2 Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Elution 25 mm Acetate, ph 3.7 ± mm Acetic Acid, 250 mm Strip NaCl PreEquilibration/Strip/R 50 mm Tris-HCl, ph 7.4, 1M egeneration NaCL Equilibration/Elute 50 mm Tris-HCl, ph Equilibration BN Wash BN Elution BN Regeneration BN Equilibration WFI 905 N/A N/A Cleaning 1 0.1M NAOH / 60/ / 65 / 65 2% Benzyl alcohol, 100 mm Na Storage Acetate Skid Sanitize 1.0 M NAOH / 50/ / 55 / 55

37 Continuous Buffer Requirements 21 bags feeding three LHMs Disposable tubing connects to LHM Buffer made up once per week and used within 48 hours Buffer volumes L Make up tanks two 300 L, High aspect ratio tanks 300 L can batch the 165, 195, 200L, 3 batches/week, fill seven bags 300L can batch L, 10 batches/week Batch 4 buffers in each tank per day, eight buffers per day for two days

38 Small Scale Batch Purification Buffer Prep Comparison Batch Small Stainless Tankage Batch make-up slower Buffers made in 3 days Minimal issues with MOC Much CIP piping Limited Expansion Continuous In-line Dilution Plastic Single use Bags Batch make-up is faster Buffers made in 2 days Slight concern with Skid MOC Much CIP avoided Greater Expansion Possible

39 Small Scale WFI Comparison Step Buffer WFI Requirements Batch Volume (L) In-line Volume (L) Equil/Chase/Wash2 50 mm Phosphate, 1 M Sodium Chloride, ph 7.0 ± Wash 1 25 mm Acetate, ph 5.5 ± Elution 25 mm Acetate, ph 3.7 ± Strip 250 mm Acetic Acid, 250 mm NaCl PreEquilibration/Strip/Rege neration 50 mm Tris-HCl, ph 7.4, 1M NaCL Equilibration/Elute 50 mm Tris-HCl, ph Equilibration BN Wash BN Elution BN Regeneration BN Equilibration WFI N/A N/A Cleaning 1 0.1M NAOH / 65 / 65 Storage 2% Benzyl alcohol, 100 mm Na Acetate Skid Sanitize 1.0 M NAOH / 55 / 55 total

40 WFI Requirements WFI Requirements - Buffer make-up and Tankage CIP WFI Demand (L) days for activity Daily Make-up Water (L) Daily WFI for CIP (L) Peak WFI (day) Weekly WFI Demand (L) Batch Making 12, Batch Making CIP 7, In-Line Buffer Making 1, In-line Dilution 10,

41 WFI Requirements WFI Requirements Still Capacity Time for Make-up Minimum WFI WFI Still Weekly WFI Demand (L) (days) Capacity (L/day) Capacity (L/day) Batch 28, In-Line 14, WFI Requirements Storage Capacity Peak WFI Demand (L) WFI Still Capacity (L/Day) WFI Working Volume (L) WFI Tank Volume (L) Batch 7, In-Line 10,

42 Small Scale WFI Comparison Continuous Buffer Make up Dilution allows for Bag Technology Reduced WFI for CIP Batch requires twice the WFI capacity Peak Load greater for Continuous WFI Storage Volume similar

43 Questions Craig Sandstrom, PhD Director Process Engineering 100 Fluor Daniel Drive Greenville, SC Greg Lepak Principal Process Engineering 100 Fluor Daniel Drive Greenville, SC