© 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

CHOP Ratio (ng/mg): ng CHOP per mg of product

CHOP ELISA Shows Decreasing impurty clearance

Slide 3

© 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.

* *

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

* * *

*

*

*

*

*

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

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

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