recent experiences with host cell protein impurity analysis · host cell protein impurity analysis...
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CaSSS Conference
South San Francisco Conference Center
November 13, 2014
Martin Vanderlaan, Director
Analytical Operations
Genentech
Recent experiences with
Host Cell Protein Impurity Analysis
Increasing interest in Host Cell Protein analysis
• Workshop in Lisbon, associated with BEBPA BioAssay conf., 2012
• Joint BEBPA-USP conference, Washington DC, June 2013
• BEBPA Workshop, Dubrovnik, May 2014
• WCBP Workshop, Washington DC, January 27-29, 2015 (CaSSS.org)
• Training class - Biotherapeutics Analytical Summit, March 2015, Baltimore
(Cambridge Health Inst)
• BEBPA Workshop, May 2015, San Francisco area
USP Guidance Document due out early 2015
(Maura Kibbey, [email protected])
<1132> Residual Host Cell Protein Measurement
in Biopharmaceuticals
EuP Guidance Document expected Q2 2015
Slide 2
Slide 3 FDA emphasis on Quality
The Biopharmaceutical
industry needs to produce
products with low levels of
impurities.
Health Authorities and patients
expect nothing less.
FDA concerns about HCPs
- Immunogenicity
- Biological activity
Frequently asking questions about
the suitability of your HCP control
system.
2-D SDS-PAGE of CHO Protein (CHOP) Standard
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
Slide 4
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”
The issue is one of demonstrating coverage
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
• CHO protein impurity levels depend on the particular mAbs
• “hitchhiker” effect – interaction of product and CHOP
5 Why do some HCPs co-purity with product? Slide 5
Sisodiya, VN, Leguieu, J, Rodriguez, M,
McDonald, P, Lazzareschi, K. 2012. Studying
host cell protein interactions with monoclonal
antibodies using high throughput protein A
chromatography. Biotechnol J 7: 1233-1241.
* *
Antigen Excess Can Result in dilution dependence of the assay
Slide 6
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
* * *
*
*
*
*
*
5 mg/ml
1 mg/ml
0.5 mg/ml
0.3 mg/ml
10 mg/ml
Product
Concentration
Anicetti, VR, Fehskens, EF, Reed, BR, Chen,
AB, Moore, P, Beier, MD, Jones, AJS. 1986.
Immunoassay for the detection of E. Coli
proteins in recombinant DNA-derived human
growth hormone. J Immunol Methods 91:213-
224.
Slide 7
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
Illustration of non-linear dilution of sample
Non-linear response indicates potential host cell protein impurity
in excess of the available antibodies.
Zhu-Shimoni J, Yu C, Nishihara J,
Wong RM, Gunawan F, Lin M,
Krawitz D, Liu P, Sandoval W,
Vanderlaan M. Host cell protein
testing by ELISAs and the use of
orthogonal methods Biotechnol
Bioeng. 2014 Jul 4. doi:
10.1002/bit.25327. [Epub ahead of
print]
Slide 8
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
Chromatography used to Separate HCPs from product
Product
MS used to identify predominant HCP(s)
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) a CHOP impurity in antigen excess
© 2012, Genentech / Proprietary information — Please do not copy, distribute or use without prior written consent
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
Slide 9
A few more details on PLBL2
• PLBL2 gene cloned, expressed in CHO cells, purified, used as immunogen and
analytical standard. PLBL2-specific sandwich ELISAs developed based on rabbit
polyclonal and mouse monoclonal antibodies developed in-house.
• Mammalian PLBL2 is much different from non-mammalian Phopholipases.
• Hamster and human PLBL2 are about 20% different in amino acid sequence, with
many of the differences in surface residues – likely to be immunogenic.
• PLBL2 can be reduced to <1 ng/mg with good purification processes. All
Genentech and Roche antibodies now screened for PLBL2 and purification
improvements implemented where needed.
• Our platform CHOP ELISA detects PLBL2, however other CHOP ELISAs have
been found that do not detect PLBL2.
Page 10
Deuschl, F, Kollmann, K, von Figura, K, Lubke, T. 2006. Molecular Characterization of the hypothetical 66.3 kDa
Protein in mouse: Lysosoma Targeting, glycosylation, processing and tissue distribution. FEBS Letters 580:5747-5752.
Vanderlaan et al. (in preparation) Hamster Phopholipase B-Like 2 (PLBL2), a host cell protein impurity in CHO-derived
therapeutic monoclonal antibodies.
15 kDa
20 kDa
25 kDa
37 kDa
50 kDa
75 kDa
100 kDa
150 kDa
250 kDa
10 kDa
PLBL2 specific Antibodies
ELISA Western Blot
Page 11
Conc
1 10 100
0
0.5
1
1.5
Graph
5-P Fit: y = (A - D)/( (1 + (x/C)^B)^G ) + D: A B C D G R^2
Std CHOP (Std CHOP: Conc vs AvgOD) 0.0972 0.999 1.57e+07 3.61 1.11e+05 1
Std PLB (Std PLB: Concentration vs Values) 0.0886 1.12 5.98 0.698 0.419 0.999__________
Curve Fit Option - Fixed Weight Value
Spiking PLBL2 into CHOP assay shows saturation Slide 12
766
-3 31
1,459
-2 85 9 -1 -1 -4
-200
0
200
400
600
800
1000
1200
1400
1600
1800
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 Slide 14
PLBL2 levels in HCCF differ between cultures
0
2
4
6
8
10
12
14
PL
BL
2 in
HC
CF
(u
g/m
L)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
PL
BL
2 a
s a
%ag
e o
f C
HO
P
Production culture
Slide 15
Conclusions
• Host cell protein (HCP) measurement is a complex science,
but many new resources are becoming available.
• HCPs often co-purify with product by “hitch-hiking along”.
• If one (or a limited number of ) HCP(s) co-purify with a product this may
be manifest in the HCP ELISA as non-linear sample dilution. Non-linear
dilutions should be investigated.
• PLBL2 has been identified as a particular host cell protein that can co-
purify with therapeutic monoclonal antibodies.
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