4/28/2004

Implementing Process Improvements After IND

Submission

James Rebbeor, Ph.D., Pete Vandeberg, Ph.D.

Bayer Technology, Bioanalytics

Critical Questions
• How do we define “comparability”?

• Have we sufficiently demonstrated

comparability? Does Product with process
change have comparable characteristics?

• How do we decide/when can we move

forward with process change?

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Plasminogen Process
Toxicology and Phase I/II Process

Freeze
Hold
Diafiltration

Caprylate Cake Depth Filter 1st Affinity Affinity Eluate

Extraction & Lipid pptn Chromatography
PEG Dowex Process

Dowex cation exchange

PEG/Cuno Filtrate Dowex Flow-Through

•Toxicology and Ph I/II Clinical Lots: Lysine

removed by UF/DF
•Dowex Lots: Lysine removed by cation-exchange
resin
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Benefit of Process Change:

simpler, faster, more efficient

Fewer people required,

less throughput time from start to finish,
fewer buffers used,
fewer BPRs needed,
less PEG waste generated

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Plasmin Process
Significant purification occurs downstream of proposed process change
(lysine and benzamidine affinity column purification steps)

1 2 3 4 5 6 7 8

Freeze
Hold

Streptokinase Sterile Freeze Dry

Caprylate 2nd Affinity Hydrophobic Nanofilter UF/DF
Activation Filter
Virus Chromatography Chromatography Virus Removal Formulate
Inactivation

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Experimental Plan for Dowex

• Produce Two Plasmin lots at Tox/Clinical scale under protocol

(Dowex ETP): use same equipment, BPRs, feed stock
• Characterize intermediates and final product with same assays
• If product is comparable to UF/DF process, present to FDA
• If no concerns raised by FDA, incorporate Dowex into Ph III
Clinical Production and engineering for commercial facility
• If product not comparable, or if FDA requests significant follow-
up studies, continue with UF/DF
• Process Definition declared by Sept. 30, 2003

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Dowex Comparability Strategy

• Extensive Characterization
– Same testing performed on Tox and Ph I/II clinical Lots
– Key test points:
• Affinity Column-1 Load (just after Dowex)
• Affinity Column-1 Eluate
• Final Container
– Test Parameters
• potency (chromogenic, fibrinolytic, A2AP inhibition, etc.)
Impurities - process related (proteins, lysine, etc.)
• Impurities - product related (degradation)
• glycosylation, isoform identity
• Process Parameters Throughout Process
– pH, conductivity, volumes, A280, turbidity,conversion,
yield, etc.
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Summary of Dowex Comparability Results

• Affinity Column-1 Load (Dowex Flow-Through)
– total protein loaded is the same (about 0.5x protein conc.)
– [Caprylate] ~ 7 mM; no added NaCl
– Impurity proteins remain same on normalized basis
• Affinity Column-1 Eluate
– Higher Purity Plasminogen
• lower alpha2-macroglobulin (0.08 mg/mL to 0.04 mg/mL)
• lower aggregates (4% vs. 8%)
• specific activity is up (about 1.00 vs. 0.85)
– Converted to Lys-Plasminogen
• 60% has loss of pre-activation peptide, compared to 40% in Tox/Ph I/II
clinical lots - impact not expected to be significant
• Pre-activation peptide normally lost in SK-activation
• May not be related to Dowex
– Conversion to plasmin is comparable
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Summary of Dowex Comparability:

Final Container Results
• Final Containers Comparable in all test parameters
– potency, specific activity, a2AP Inhibition, fibrinolytic
activity, lysine binding, etc.
– Similar purity profile by non-reduced methods
• Size Exclusion HPLC, SDS-PAGE, MALDI
– Similar purity profile by reduced methods
• Reduced SDS-PAGE, MALDI
– Slight Differences by Reduced Bioanalyzer
• Capillary Gel Electrophoresis Method
• Profile is the same, with slightly more heavy chain damage
– Difference is expected to be within range of normal final
containers and may not be related to Dowex

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Summary: Dowex vs.Tox and Ph I/II Clinical

• Products from Dowex Process are comparable to

Tox/ Ph I/II Clinical
– Affinity Column-1 Eluate: Subtle differences in Affinity
column-1 eluate (lower A2M levels, higher Lys-Pmg levels)
– Final Container: Slightly higher level of clipping in heavy
chain by reduced bioanalyzer
– Differences may not be related to Dowex (normal process
variation).
• Overall Pm yields were slightly lower (n=2)
• Recommendation: Proceed with Dowex

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Path Forward: Present to FDA

• Authored Technical Registration Documents (TRD)

detailing process materials (Dowex resin) and
product characterization
– Revised existing Narrative description, flow charts, outlines
– produced new documents
– Submit to allow 30 days for review, then follow-up
• Proceeded with Batch Record document revisions to
implement Process change
• Responded to FDA queries as they arrived
– Resin qualifications were only questions

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Conclusion: Change, A Very “Do-able” Process

• Time and Resources required were significant

– Pilot scale production is costly and requires many qualified,
highly trained personnel, and facility resourcing
– cost of goods is high, and final container material is only useful
for stability studies
– Technology, Bioanalytical, QA, and RA resources required
• Long-term benefit still to be realized
• Implementation for Phase III clinical lot production has
begun
– Time savings already apparent (3-4 runs/wk)
• Training and experience enables more changes to be
considered, and implemented before commercial scale

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