Using Selective Washes to Solve a Purification Issue Involving a Host Cell Protein
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Transcript of Using Selective Washes to Solve a Purification Issue Involving a Host Cell Protein
ISSUE: Particulates were discovered during stability testing of
BDS
MASS SPEC ANALYSIS identified the particulates as “cathepsin-
A like” host cell protein
PURIFICATION CHALLENGE: Cathepsin A is likely bound to
desired protein and is being partially co-purified in the process
STRATEGY: Investigate Capto Adhere and Mimetic Blue
chromatography by adding wash steps to disrupt the association
between the molecules
TIMELINE: Perform experiments within a 3 week timeframe and
demonstrate effectiveness in week 4
1
Identify column wash conditions that disrupt the product-HCP
interaction without severely reducing overall yield
Primary
Screening
Secondary
Screening
Identify promising
candidates
Refinement
Refine successful
runs
Test second set of
conditions
Confirmation
Further refinement
of top 2-3
conditions
Test column steps
in series to
demonstrate
additive effects
Perform entire
purification of
Product at bench
scale
Perform entire
purification of
product at
intermediate scale
Note: Screen study monitored by HCP ELISA and Western blot analysis
2
3
Conclusion:
0.1M NaCl, 0.2M L-Arg, 5% glycerol pH 8.0 wash
provides the highest HCP clearance
Does not negatively impact yield
Note: Runs 2-18 used the 1x15L B002 CCH as the load; Runs 19-26 used the 200L PD Production CCH as the load
MW
(kDa)
191
64
51
39
28
Cathepsin A
Anti cat A western blot
0
500
1000
1500
2000
2500
0 20 40 60 80 100
HC
P (
pp
m)
recAP yield (%)
Mimetic Blue Screening [HCP] vs. yield %
ID Load material Capto condition Test Wash Condition Elution conditions recAP
Yield (%)
HCP
(ppm)
Run 1 15L B02 Baseline none (baseline) 103 2047
Run 4 15L B02 Baseline 1M Urea , pH 8.0 25mM Phos + 130mM NaCl 90 1066
Run 5 15L B02 Baseline 5% glycerol, pH 8.0 25mM Phos + 130mM NaCl 100 1981
Run 8 15L B02 Baseline 10% ethylene glycol, pH 8.0 25mM Phos + 130mM NaCl 88 1878
Run 10 15L B02 Baseline none 25 mM phos 99 862
Run 14 200L harvest 200mM L-arginine wash baseline 25 mM phos 88 667
Run 18 200L harvest 200mM L-arginine wash 20, 40, 60 mM NaCl, pH 7 25 mM phos 84 1042
Run 20 200L harvest 200mM L-arginine wash 10% eth glycol + 20, 40, 60
mM NaCl, pH 8 25 mM phos 77 128
Run 21 200L harvest 200mM L-arginine wash 1M urea 25 mM phos 83 59
Run 22 200L harvest 200mM L-arginine wash 2M urea 25 mM phos 85 299
Run 23 200L harvest 200mM L-arginine wash 40mM arginine 25 mM phos 83 54
Run 24 200L harvest 200mM L-arginine wash baseline 25 mM phos 93 1145
Run 25 200L harvest 200mM L-arginine wash 10% ethylene glycol + 1M
urea 25 mM phos 86 777
Run 26 200L harvest 200mM L-arginine wash 40mM arginine + 1M urea 25 mM phos 71 46
Run 27 200L harvest 200mM L-arginine wash 1M urea (3g/L load) 25 mM phos 94 371
Run 28 200L harvest 200mM L-arginine wash 40mM arginine (3g/L load) 25 mM phos 88 59
Run 29 200L harvest 200mM L-arginine wash 40mM arginine + 1M urea
(3g/L load) 25 mM phos 66 66
Conclusion: 40mM L-Arg pH 8.0 wash + phosphate only elution buffer provide the highest HCP clearance Does not negatively impact yield
MW
(kDa)
191
64
51
39
28
Cathepsin A
Anti cat A western blot
4