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, MCK@USP.org)

<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

Mab2 Mab1

SPR shows differential binding of PLBL2 to Mabs Slide 13

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.

Page 16