A Chromatography Process from Lab to Production Scale: A Column Packing Study

Transcription

1 A Chromatography Process from Lab to Production Scale: A Column Packing Study Wei Guo, B Thiyagarajan and Nandu Deorkar Mallinckrodt Baker Inc. / Tyco Healthcare November 2006 Trademarks are owned by Mallinckrodt Baker, Inc. unless otherwise noted.

2 Dimensions of Chromatographic Processes Resolution Speed Recovery Capacity

3 Key Attributes of Ideal Chromatographic Media Physical and mechanical properties o o Simple column packing procedure Stable column bed under various conditions Chemical stability o o Cleaning in place and sterilization using strong reagents Column regeneration Backbone and functional groups o o Minimum irreversible adsorption Selectivity and consistent performance

4 Novel BAKERBOND TM Polymeric Chromatographic Media Name PolyPEI Functionality Weak anion exchanger Ion Exchange Capacity, meq/ml (anion) PolyABx PolyCSx PolyQUAT Mixed mode- Primarily weak cation exchanger with weak anion exchange sites Mixed mode- Primarily Strong cation exchanger with weak cation and anion exchange sites Mixed mode- Primarily strong anion exchanger with anion exchange sites (cation) (anion) (strong cation) (weak cation) (anion) 0.15 (Weak anion) 0.25 (strong anion)

5 Schematic Ligand Structure of BAKERBOND PolyABx Group Carboxyl -COOH Secondary amine NH- Tertiary amine N= Functionality Weak cation exchange Weak anion exchanger

6 Objectives Investigate lab to process scale column packing and performance Develop robust column packing protocol Correlate column integrity and separation performance

7 Characteristics of a Well Packed Column Constant bed volume under different flow rates Reproducible separation performance Optimal flow distribution across the column Acceptable efficiency and asymmetry for chosen application

8 Common Packing Methods Flow packing Pressure packing Suction packing Axial compression packing In situ packing (slurry transfer) Combination of some methods above Dry packing

9 BAKERBOND PolyABx Pressure Flow Profile Pressure Drop (psi) Linear Velocity (cm/hr) Column size: 165 X 90 mm ID and 155 X 450 mm ID

10 Column Packing Instrumentation

11 Column Packing Parameter Slurry and packing solution o o o Slurry and pack in 50 mm sodium chloride 2 M sodium chloride / pack with 1 M NaCl Slurry in sterilant buffer (sodium acetate and benzyl alcohol)/ pack with 1 M NaCl Slurry ratio (20-60%) Packing flow rate

12 Slurry Transfer and Flow Packing Procedure for BAKERBOND PolyABx Column Measure amount of BAKERBOND PolyABx-35 Desired packed column volume = 1.05 X settled BAKERBOND PolyABx volume Make a 50% slurry in packing buffer 2 M sodium chloride Transfer the slurry to the empty column with diaphragm pump at 100 to 700 cm/hr Pack with 1 M NaCl at 100 to 700 cm/hr for 2 to 3 column volume

13 Slurry Transfer and Flow Packing Procedure for BAKERBOND PolyABx Column (Continued) Lower down the upper adapter near to the top of bed Pack again with 1 M NaCl at 100 to 700 cm/hr for 2 to 3 column volume or till bed height is constant Lower down the upper flow adaptor to the top of bed and lock it in place

14 Particle Size Distribution over Repeated Use 24 Frequence (%) Comparison at 80 Cycles Initial polymer Ratio 1:2 Ratio 1: Diameter (micron) Particle Size Distribution BAKERBOND PolyABx After Slurry Transfer of BAKERBOND PolyABx to Column (50% and 25% slurry)

15 Particle Size Distribution over Repeated Use Initial BAKERBOND PolyABx BAKERBOND PolyABx after 6 cycles of packing and unpacking cycles.

16 Column Integrity Testing Protocol Equilibrate column with 2 to 5 column volumes of 1.0 M NaCl Load 0.5% to 2.0% column volumes of 2.0 M NaCl / 0.4% acetone solution Elute column with 2 column volumes of 1.0 M NaCl at 20 to 100 cm/hr Determine asymmetry and efficiency

17 Lab Scale Packed Column Performance c:\unicorn\local\fil\reason06\result\xk column packing\!efficiency01009\efficiency01001.res efficiency01001:1_cond efficiency01( )001:1_cond2 efficiency01( )001:1_cond efficiency01( )001:1_cond ms/cm ms/cm Pack Efficiency Asymmetry Efficiency (Ave.): (1s 675) Asymmetry (Ave.): 1.11 (1s 0.07) ml 4.0 XK16 column (120X16 mm ID)

18 Process Scale Column Packing Slurry height: 30 cm Bed height: 15.5 cm Uniformity in slurry and ease processing Left- Slurry in the column before packing Right: After 2-3 column volume packing buffer flow at 100 cm/hour

19 Packed Column Integrity Test 125 X 450 mm ID; Flow rate: 22 cm/hr Detection Efficiency Asymmetry Conductivity

20 Evaluation of Process Column Column Performance by Conductivity Efficiency (plate/m) Efficiency 1.5 Asymmetry Assymetry Test Number Efficiency (Ave.): (1s 585) Asymmetry (Ave.): 1.28 (1s 0.09) Eastern River column (120 X450 mm ID), column packed using 2 M NaCl slurry. Sample: 1% CV 2 M NaCl

21 Packing BAKERBOND PolyABx from Different Slurry Solutions Eastern River Column 125 X 450 mm ID % Slurry in 2 M NaCl % Slurry in initial sterilant buffer

22 Comparison of Process and Lab Scale Columns Lab Column (100 X 10 mm ID) Process Column (140 X 90 mm ID) Sample efficiency Sample efficiency IgG 22,589 IgG 9,888 Lysozyme 18,544 Cytochrome C 23,023 Lysozyme 15,677

23 Conclusion Properties of BAKERBOND PolyABx-35 allow easy transfer from lab to process scale Developed column packing procedure can be used for packing lab scale to process scale column High mechanical strength permit easy and fast column packing No defining or buffer exchange required Slurry transfer at high flow rate Packing at high flow rate/pressure Linear pressure flow profile The packed column provide reproducible and consistent protein separation performance from lab scale to process scale

24 Charles R Deily Bob Buss Audrey Parker Joe Mladosich Charles Keller Acknowledgements