Implementation of Capacitance Probes for Continuous Viable Cell Density Measurements for 2K Manufacturing Fed-Batch Processes at Biogen Idec

Transcription

1 Implementation of Capacitance Probes for Continuous Viable Cell Density Measurements for 2K Manufacturing Fed-Batch Processes at Biogen Idec Jason Wong (Manufacturing Sciences, Cambridge) IFPAC January 24, 2014

2 Outline Introduction Technology Implementation Strategy Example: Process X Commercial Manufacturing Process 2

3 Introduction Viable cell density (VCD) is an important parameter Measured off-line through periodic sampling On-line VCD monitoring benefits to manufacturing: 1) Continuous monitoring of VCDs at bioreactor stages 2) Track growth based on historical performance and troubleshoot growth excursions in real-time manner 3) Reduced sampling 4) Transfer cell culture within seed train bioreactors 3

4 Technology Dielectric spectroscopy (Harris et al., 1987, Kell et al. 1990; and Yardley et al., 2000) Measures the dielectric properties of the medium as a function of frequency Suspension of cells composed of three separate parts: 1. Medium 2. Cytoplasm 3. Plasma Membrane (Non-Conducting) Electrically speaking, Cell Suspension = Suspension of spherical capacitors each containing a conducting matrix and all surrounded by a conducting medium 4

5 Probe Theory Positive Electrode + Medium _ + _ + Plasma Membrane (Poor Conductor) + _ + + _ Negative Electrode Cytoplasm _ Applied electric field results in + ions pushed in direction of field and ions in opposite direction Ions accumulate along this membrane resulting in polarization at poles of cells Magnitude of suspension s field can be measured by its capacitance (pf)

6 Factors Influencing Capacitance Measurements Frequency Setting o # of times electric field changes direction per second o Low frequency vs. high frequency Cell Growth (Increase in cell diameter) o Can increase capacitance by increase of cell volume Non-biomass solids o Dead cells, oil droplets, debris, gas bubbles o Reduce capacitance by reducing cellular volume fraction 6

7 Aber Biomass Monitor 200 Instrument Biomass Monitor 200 Head Amplifier Capacitance Probe Cell Culture Mode Frequency set to 580 Khz (Vendor- Recommended) Standardization done in media immediately prior to inoculation Grounding procedure which included a 6 foot head-amp strap that fastens the head amplifier to foot of bioreactor Note that the probe is suitable for bioreactors only and not for shake flask, spinners, or wavebags 7

8 Implementation Strategy New processes evaluated in pilot & full-scale batches o Technical Development to determine feasibility o Bench-scale experiments using capacitance probe o Pilot-scale, engineering and clinical batches can provide opportunity to assess operational and scalerelated issues Existing processes developed using off-line method o Perform studies to correlate capacitance with offline readings 8

9 Seed Train Transfers for Batch Process Fixed Time and Variable Transfer Volume o Important to have accurate readings at end of culture Variable Time and Full Transfer Volume o Project target transfer time by leveraging growth rate calculation using two time points during the n- 1 process Variable Time and Partial Transfer Volume o Hybrid of the first and second types 9

10 Example: Process X Manufacturing Process Process X was developed using offline CEDEX Transferred into Cambridge 2K Manufacturing Facility for Phase 1 Production Resupply Campaign was initiated for Phase 2 Production and Process Validation Campaign Process Overview: o Shake flask inoculum stage, two seed bioreactors, and final fed-batch production bioreactor stage o High viability through seed and production stage o Short production run with low peak VCD o No complex feeding o Seed Transfer: Fixed Time and Variable Volume 10

11 Example: Process X Manufacturing Process Capacitance data from a total of eight full-scale resupply GMP batches in 2010 showed strong correlation with automated cell counter (CEDEX) 11

12 Example: Process X Manufacturing Process Methods for measuring VCD were not filed Viability measurements were stated in the filing only at the inoculum and production stage Due to high viabilities demonstrated in clinical batches, discontinuation of viability measurements pose minimal risk to product quality and process performance Proposed maintenance of viability measurements throughout the inoculum shake flask stage (using CEDEX) and a final viability measurement at end of production bioreactor (harvest) 12

13 Implementation Strategy for Process X Track VCD (vc/ml) of culture using a simple linear regression model that relates capacitance to VCD o One equation for 100L & 750L o Different equation for 2K Created report justifying use of the capacitance probe: 1. State probe as equivalent VCD measuring device 2. Analysis of Fit and Summary of Equations 3. Automation & Engineering Decisions 4. Details of Process Monitoring and Risk Mitigation 13

14 Example: Process X Manufacturing Process Automation & Engineering Decisions: o Capacitance converted to VCD, displayed on Delta V o Zeroing procedure completed via Delta V o No automation of transfer calculations 14

15 Example: Process X Manufacturing Process Process Monitoring & Risk Mitigation: o Lack of redundant probe o Probe failure is supplemented by ability to measure offline VCD using CEDEX o Instructions provided in batch record to use offline option 15

16 Example: Process X Manufacturing Process Normal VCD Trend 100 FVIII_2000L_T1_COMv1 - Unitgroup., Unit -, Sub-Batch _, M1 VCD (vc/ml)_t1 +3 std. dev. Average Batch -3 std. dev. RECD VCD (vc/ml)_t M Days2000L (smoothed and shifted) SIMCA-Batch On-Line Client :53:11 (UTC-5) Uniform, Stable, Consistent Within 3SDs of Historical Average (Clinical & Resupply Batches) 16

17 Example: Process X Manufacturing Process Atypical Probe Behavior (Obvious) FVIII_2000L_T1_COMv1 - Unitgroup., Unit -, Sub-Batch _, M1 VCD (vc/ml)_t1 +3 std. dev. Average Batch -3 std. dev. RECD RECD VCD (vc/ml)_t M Days2000L (smoothed and shifted) SIMCA-Batch On-Line Client :12:03 (UTC-5) Erratic readings due to probe issues Significant offset, operator did not zero the probe prior to inoculation Switch to CEDEX as primary instrument for monitoring 17

18 Example: Process X Manufacturing Process Atypical Probe Behavior (Non-Obvious) FVIII_2000L_T1_COMv1 - Unitgroup., Unit -, Sub-Batch _, M1 VCD (vc/ml)_t1 +3 std. dev. Average Batch -3 std. dev. RECD VCD (vc/ml)_t M Days2000L (smoothed and shifted) SIMCA-Batch On-Line Client :13:12 (UTC-5) Non-Routine Offline CEDEX Sample on Day 0 and 1 to confirm results Seed density confirmed low, probe results comparable to CEDEX Continued to use probe as primary instrument for monitoring 18

19 Summary of Abnormal Trendings Three Common Behaviors of VCD Abnormal Trending: Probe Behavior Type Description and Recommended Course of Action #1 Obvious Erratic or flat-line signal. No fixed course. The trend displays lack of uniformity, regularity or consistency. The VCD probe profile can be within or outside of action limits. Regardless of these results, the CEDEX will be used as a primary instrument since the probe is deemed malfunctioned or problematic. #2 Obvious Significant offset noticed. The VCD probe profile is shifted due to zeroing, calibration or stabilizing issues. The VCD readings typically lie outside the action limit. Use CEDEX as the primary instrument. #3 Non-Obvious Profile is uniform and stable but is inconsistent with previous batches. Non-routine sample required for investigation. 19

20 20 Process X: Comparison Probe and CEDEX

21 Summary: Process X Manufacturing Process Capacitance showed consistency and comparability with offline CEDEX Probe performance during PVR and commercial batches were good Created a risk mitigation strategy and identified common atypical VCD trends and proposed course of action 21

22 Acknowledgements Mark Byers (Manufacturing Sciences) Jeff Simeone (Manufacturing Sciences) Sriram Ramakrishnan (Manufacturing Sciences) John Jewett (Manufacturing) Matt Leprohon (Manufacturing) James Sur (Manufacturing) David Winters (Automation) Barbara Woppmann (Technical Development) Valerie Tsang (Technical Development) Mitch Bennett (Quality) Elijah Tan (Regulatory) 22