Hamster Phospholipase B-Like 2 (PLBL2), a host cell protein...
Transcript of Hamster Phospholipase B-Like 2 (PLBL2), a host cell protein...
© 2009, Genentech / Proprietary information – Please do not copy, distribute or use without prior written consent.
Martin Vanderlaan, Ph.D., M.B.A
Director, Analytical Operations
Genentech – a member of the Roche Group
Hamster Phospholipase
B-Like 2 (PLBL2), a host cell
protein impurity in
CHO-derived therapeutic
monoclonal antibodies
CaSSS CMC strategy Forum
Washington DC
January 25, 2015
2-D SDS-PAGE & WB of CHOP Standard
pH 3 pH 10
Sypro Ruby-stained gel Anti-CHOP immunoblot
50
80
70
100
10
40
30
25
15
20
90
60
120
kDa
160
pH 3 pH 10
~24,383 predicted gene products
Broad range of proteins recognized by anti-CHOP antibodies •Proteins not equally recognized
•Abundant vs rare/absent antibodies
•Single number “result” for HCP assay is “immunologically weighted”
Platform HCP Immunoassays allow cross product comparisons
Krawitz DC, Forrest W, Moreno GT, Kittleson J,
Champion KM. 2006. Proteomic studies support
the use of multi-product immunoassays to
monitor host cell protein impurities. Proteomics
6(1):94–110
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
Atypical case of non-linear dilution of sample
Non-linear response indicates potential host cell protein impurity
in excess of the available antibodies.
Zhu-Shimoni et al. 2014 Host cell
protein testing by ELISAs and the use
of orthogonal methods Biotechnol
Bioeng. 111: 2367-2379.
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Antigen Excess: one cause of assay dilution dependence
CHOP-A CHOP-B
0
10
20
30
40
50
0.01 0.1 1 10 100
CH
OP
Con
c.
[ng
/mL
]
Product Conc. [mg/mL]
CHOPinmAb2Pool
CHOP-A CHOP-B
* *
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
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*
*
*
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5 mg/ml
1 mg/ml
0.5 mg/ml
0.3 mg/ml
10 mg/ml
Product
Concentration
Anicetti, VR, et al. 1986. Immunoassay for the
detection of E. Coli proteins in recombinant
DNA-derived human growth hormone. J
Immunol Methods 91:213-224.
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
Chromatography used to Separate HCPs from product
Product
Wang, X, Hunter, AK, Mozier NM. 2009. Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk
Assessment Biotechnol. Bioeng 103: 446-458.
A280
88% CHOP step yield
CHOP
CHOP ELISA of fxns
Separation of product from CHOP using ceramic hydroxyapetite (CHT)
Identification of Phospholipase B-like 2 (PLBL2) by LC-MS/MS of tryptic digests of gel bands
Procedure:
1) SDS-PAGE of pooled ELISA positive fractions from HPLC
2) Excise and digest bands resolved on gel
3) Analyzed with nano LC-MS/MS
4) Searched mammalian UniProt sequence database
5) Confirmed the sequences by MS and MS2
Coverage Report for Phospholipase B-like 2 (PLBL2) protein
IgG
IgG
PLBL2
G3I6T1_CRIGR Putative phospholipase B-like 2
IgG
ctr
l
CH
T F
xn
Phospholipase B-like 2 (PLBL2) protein chemistry
pI = ~ 6.0
mannose-6-phosphate carbohydrates, indicative of Lysosomal organelle location
Synthesized as a pre-pro-enzyme, activated by acidic pH by cleavage into two parts that remain
associated non-covalently. This clip improves accessibility of the enzyme active site.
Most likely an Acyl amidase, not digesting phospholipid bilayer membranes but true biologic
substrate not yet identified.
Sometimes abbreviated PLB2; Hamster 80% homologous to human
Deuschl F, Kollmann K, von Figura K, Lubke T.
2006. Molecular characterization of the
hypothetical 66.3-kDa protein in mouse:
lysosomal targeting, glycosylation, processing
and tissue distribution. FEBS Lett
580(24):5747-52.
15 kDa
20 kDa
25 kDa
37 kDa
50 kDa
75 kDa
100 kDa
150 kDa
250 kDa
10 kDa
PLBL2 specific Immunoassay
Clone gene
Express in CHO, purify
Immunize mice, rabbits
ELISA for PLBL2
Rabbit-anti-PLBL2 WB shows expected
staining of PLBL2 fragments
Vanderlaan, M, et al (in press) Hamster PLBL2: a host cell protein impurity in CHO-derived
therapeutic monoclonal antibodies. BioProcess International
Recovery of PLBL2 in the CHOP ELISA
Accurate spike-recovery can be obtained only in dilute samples
Further support for “antigen excess” interpretation of results.
Positive sample mixed with excess Rabbit-anti-PLBL2 in solution
Results in linear dilution in CHOP ELISA, with low CHOP value
Showing 100% of CHOP assay non-linearity can be blocked with anti-PLBL2
CHOP Std Curve
PLBL2 spike
% Recovery
PLBL2
Conc
(ng/mL)
Analyst 1 Analyst 2
20 53 53
10 74 75
7.5 82 80
5 92 91
2.5 99 107 ng/mL CHOP Std or PLBL2
Op
tica
l D
en
sity
Quantitative LC-MS/MS assay using an analytical standard
10 ppm 20 ppm
50 ppm
30 ppm
100 ppm
Linear quantification of PLBL2 peptide by LC-MS/MS assay
Confirms CHOP ELISA – PLBL2 present at ~300 ng/mg in Run 3
Comparable PLBL2 level estimates by all methods
Sample PLBL2
ELISA
CHOP
ELISA*
PLBL2 by
LC-MS/MS
Mab 1 Run 1 83 104 87
Mab 1 Run 2 122 139 90
Mab 1 Run 3 34 25 32
Mab 1 Run 4 137 155 103
Mab 1 Phase IIb Run 2 242 179 141
Mab 1 Phase IIb Run 3 328 310 241
Mab 1 Phase IIb Run 4 273 189 154
Mab 2 run 1 41 59
Mab 2 run 2 56 66
Mab 2 run 3 39 59
*value from “dilute to LOQ” approach to CHOP testing
What about other platform anti-CHOP antibodies?
1
10
100
1000
0.01 0.1 1 10 100 1000
CH
OP
Rat
io [
ng/
mg]
[mg/mL]
CHOP Results comparing two anti-CHOP antibody sources
Conclude: Results differ depending on anti-CHOP reagent
Otherwise acceptable anti-CHOP reagents do not see non-linear dilution
Original anti-CHOP antibodies
Alternate anti-CHOP antibodies
Different source of
platform anti-
CHOP antibodiers
Sample:
Mab lot containing
PLBL2
• CHO protein impurity levels depend on the particular mAbs
• “hitchhiker” effect – interaction of product and CHOP
14 Why do some HCPs co-purity with product?
Sisodiya, VN et al. 2012. Studying
host cell protein interactions with
monoclonal antibodies using high
throughput protein A
chromatography. Biotechnol J 7:
1233-1241.
Levy NE, Valente KN, Choe LH,
Lee KH, Lenhoff AM. 2014.
Identification and characterization
of host cell protein product-
associated impurities in monoclonal
antibody bioprocessing. Biotechnol
Bioeng 111(5):904-12.
Co-purifying HCPs may bind to product
Red line –CHOP assay Blue line – IgG assay Black line – UV trace
Most CHOP migrate with either the aggregate peak or the leading edge
of the main peak.
0 2 4 6 8 10 12 14
0,0
0,1
0,2
0,3
0,4
0,5
volume (ml)
A2
80
nm (
AU
)
0
1
2
3
4
5
6
7
8
CH
OP
concentr
ation (
ng/ m
l)
DL
0
1
2
3
4
5
IgG
concentr
ation (
mg/m
l)SEC of Mab
Mab with co-purifying PLBL2 Control Mab
SPR shows differential binding of PLBL2 to Mabs
SPR sensogram of 1 mm PLBL2 binding to immobilized Mab
When biding is observed, it is readily reversible binding (reverts to baseline in 2 min)
Control antibody shows no binding
766
-3 31
1,459
-2 85 9 -1 -1 -4
-200
0
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400
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800
1000
1200
1400
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Mab 2 Mab 1 Mab 6 Mab 2 Mab 1 Mab 6 Mab 2 Mab 1 Mab 6 buffer
Binding of Mab antibodies and fragments to PLBL2 (1 mM)
PLBL2 binds preferentially to Fab’2 portion
PLBL2 levels in HCCF differ between cultures
Variable PLBL2 levels:
0.76-7.75 ug/mL
0.16-1.6% of total CHOP
Multiple independent HCCF
samples were measured for
many Mab products.
High values (solid bars)
low values (stippled bars). 0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1 2 3 4 5 6 7 8 9 10 11 12 13
PL
BL
2 a
s %
ag
e o
f to
tal C
HO
P
Monoclonal Antibody ID #
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
1 2 3 4 5 6 7 8 9 10 11 12 13
Monoclonal Antibody ID #
PL
BL
2 in
HC
CF
(m
g/m
L)
Some thoughts on PLBL2
• PLBL2 is a widely expressed CHO cell protein
• Tested All Roche antibody products
• PLBL2 found at varying levels in several Mabs in clinical development
• Purification processes revised to reduce PLBL2 to <1 ng/mg
• All HCP testing now involves testing samples at multiple dilutions
near the LOQ of the assay to assess “antigen excess”
• Not all platform CHOP assays detect PLBL2
• Recommended: LC-MS/MS is a valuable orthogonal method to HCP
ELISAs – allow ID and potential quantification of PLBL2.
• CusioBio’s “Hamster PLBL2 ELISA” did not recognize our PLBL2
Lessons Learned for Avoiding HCP Impurities
Use a broadly reactive immunoassay using antigen-affinity
purified antibodies. Recognize that the test result is
“immunologically weighted” and the assay will not detect all
HCPs.
Test for sample dilution linearity, and investigate non-linear
dilutions.
Use orthogonal methods to detect HCPs that might be missed
in the ELISA
- Silver, Fluorescent, or Western stained gels
- Positive ID of all gel bands using LC-MS/MS or WB
- LC-MS/MS of protease-digested material
- Product capture/dissociation of HCPs
Use these tests in your purification development process.
Points to consider in setting limits on HCP impurities
Dose of impurity
Duration of treatment
Route of Administration
Therapeutic MOA
Clinical indication
Patient Population
Potential for immunogenicity
Potential for biological activity
Potential for “adjuvant” to increase ATAs
Factor
Thank you to the team
Wendy Sandoval
Peter Liu
Julie Nishihara
George Tsui
Margaret Lin
Feny Gunawan
Sara Parker,
Robert Ming Wong
Justin Low
Xiangdan Wang
Jihong Yang
Karthik Veeravalli
Patrick McKay
Benjamin Tran
Rajesh Vij
Chris Fong,
Chris Yu
Lori O’Connell
Kathleen Francissen
Judith Zhu-Shimoni
Valerie Quarmby
Denise Krawitz
John Matthews