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