Cell Culture and Tissue Engineering Biotechnology Progress

DOI 10.1002/btpr.2444

Evaluation of Heavy Chain C-terminal Deletions on Productivity and Product

Quality of Monoclonal Antibodies in Chinese Hamster Ovary (CHO) Cells

Zhilan Hu1#, Danming Tang1#, Shahram Misaghi1, Guoying Jiang2, Christopher Yu3,

Mandy Yim1, David Shaw1, Brad Snedecor1, Michael Laird1, and Amy Shen1*

1
Department of Early Stage Cell Culture; 2 Biological Technologies; 3 Protein Analytical

Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

# These authors contribute equally to this work.

*Correspondence to:

Amy Shen, Electronic mail address: shen.amy@gene.com,

Telephone: (650) 225-6446, Fax: (650) 225-2006

Key words:

CHO cell; Therapeutic monoclonal antibody; Charge variants; C-terminal lysine, Proline

amidation, Productivity

Running title: Lower productivity of C-terminal Lys deleted antibody expressing cell

lines.

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/btpr.2444
© 2017 American Institute of Chemical Engineers Biotechnol Prog
Received: Dec 01, 2016; Revised: Jan 25, 2017; Accepted: Jan 27, 2017

This article is protected by copyright. All rights reserved.

 Biotechnology Progress Page 2 of 27

Abstract

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents

and are used to treat many different diseases. During cell culture production, antibody

charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and

proline amidation. Differences in levels of charge variants during manufacturing process

changes make it challenging to demonstrate process comparability. In order to reduce

heterogeneity and achieve consistent product quality, we generated and expressed

antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK).

Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had

considerably lower specific productivities compared to clones that expressed either wild

type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no

measurable differences in antibody HC and LC mRNA levels, glycosylation and

secretion were observed, our analysis suggests that the lower specific productivity of

clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC

synthesis and faster antibody degradation.

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Introduction

Monoclonal antibodies (mAbs) have become increasingly important as

human therapeutic agents for a wide range of human diseases, including many types of

cancer 1, 2. Process changes in production scale, media, and cell culture process

parameters can result in product quality inconsistencies among different antibody

production batches 3-5. Monoclonal antibody products are heterogeneous and may

undergo different modifications such as glycosylation, deamidation, oxidation, lysine

hydroxylation, N-terminal glutamine cyclization, C-terminal lysine cleavage, and proline

amidation. Variations in levels of these modifications are undesirable and can be

attributed to lack of process control 4, 5. One antibody product quality which requires

monitoring is the relative level of basic and acidic antibody variants 6.

Therapeutic antibodies of IgG1, IgG2 and IgG4 isotypes have a basic lysine residue at

their C terminus (C-terminal lysine). However, the C-terminal lysine is often not detected

or detected at low levels in the final drug products because of peptidase action during

antibody production in mammalian cell cultures 7-9. Antibody C-terminal lysine is

removed by carboxypeptidase D (CpD)10, 11, which results in generation of a mixture of

antibody isoforms bearing zero or one C-terminal lysine residues on each heavy chain

(HC). The HC of the human IgG1 antibody isotype ends with proline-glycine-lysine

residues. Following C-terminal lysine cleavage, peptidylglycine α-amidating

monooxygenase (PAM), utilizing its two enzymatically active domains - peptidylglycine

alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-

amidating lyase (PAL), further catalyzes the hydroxylation of glycine and removal of the

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glyoxylate from the glycine residue, leaving an amidated C-terminal proline (Figure 1A)
12-14
. Both lysine deletion and proline amidation of C-terminus of antibody HC can

contribute to changes in antibody charge variant profiles.

In order to reduce the antibody charge variants heterogeneity and minimize variations in

the level of charge variants among production batches, we decided to generate antibodies

lacking HC C-terminal lysine (-K) or both lysine and glycine (-GK) (Figure 1B). The HC

C-terminal lysine of wild type (WT) antibodies is mostly removed during the mammalian

cell culture production phase and the remaining C-terminal lysine residues are removed

after administration to humans 8. Khawli et al have previously reported that an antibody’s

charge variant levels do not affect its potency, FcRn binding, or PK properties 6.

Therefore, antibody lacking HC C-terminal lysine should have the same potency as its

WT counterpart. Indeed, our prior evaluations did not reveal any differences in stability,

pharmacokinetic (PK) properties, or potency amongst WT antibodies and antibodies

lacking either HC C-terminal lysine (-K) or both lysine and glycine (-GK)15. In this study,

we have investigated the impact of HC C-terminal amino acids on antibody productivity

and product quality using three different monoclonal antibody molecules (mAbs). We

observed that the antibodies lacking HC C-terminal lysine had increased basic variants

due to C-terminal proline amidation. Additionally, clones that express antibodies lacking

HC C-terminal lysine had lower specific productivities and titers compared to clones that

expressed WT antibodies. Interestingly, the clones that express antibodies lacking both

glycine and lysine had higher productivity and reduced levels of basic variants (by 90%)

compared to those expressing WT antibodies. Our analyses ruled out differences in

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antibody mRNA level, glycosylation and secretion between clones expressing WT

antibody or antibody lacking HC lysine. Our results indicate that the lower specific

productivity of antibodies lacking HC C-terminal lysine is accompanied by lower

intracellular antibody accumulation and higher rates of antibody degradation.

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Results

Clones expressing antibodies lacking HC C-terminal lysine exhibit lower titers and

specific productivities

Plasmids for two different antibody mAb1 and mAb2 bearing HC C-terminal lysine or

glycine-lysine deletions, along with their WT versions, were transfected into CHO cells

and 216 clones from each expression construct were randomly picked and assayed for

antibody titers. Pools of the top 12 highest titer clones from each expression construct

were evaluated in a fed-batch production assay. Pools for both mAbs bearing HC lysine

deletions had lower specific productivities (Figure 1C) and titers (Figure 1D) relative to

WT and glycine-lysine variants. To confirm these findings with another antibody

(mAb3), we transfected CHO cells with plasmids of WT, HC C-terminal lysine deleted

and glycine-lysine deleted versions of mAb3. For each version, we analyzed antibody

expression for a total of 528 clones that were randomly picked per construct (Figure 2A)

and again the lysine deleted version showed lower titers relative to the WT or glycine-

lysine deleted versions. The top 96 clones were further assayed (data not shown) and

subsequently the top 20 individual clones of each expression construct were analyzed in a

14-day fed-batch production assay. The results confirmed that mAb3 clones expressing

lysine-deleted antibody indeed have overall lower titers due to lower specific

productivity, relative to clones expressing WT and glycine-lysine deleted antibodies. The

average values of titers and specific productivities of WT and glycine-lysine deleted

antibodies expressing clones were almost 2-folds higher than the clones expressing lysine

deleted antibodies (Figure 2B & C). We noted that all different versions of the mAb3

allow comparable cell growth as indicated by Day 14 integrated viable cell count (IVCC)

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(Figure 2D). The data collected thus far for all three antibodies shows that, although the

same expression vector was used to express different antibodies, the clones expressing

lysine-deleted versions exhibited an overall lower volumetric productivity, suggesting

that this phenomenon is not a clone or antibody specific effect and is indeed triggered by

deletion of HC C-terminal lysine.

We then used only mAb3 as a model molecule for further analysis. To eliminate clone-

specific variation in antibody production, we pooled the top 20 mAb3 expressing clones

of each version and performed a fed-batch production assay. Again, the pool of clones

expressing antibody lacking HC C-terminal lysine had lower titer throughout the

production compared to the pools expressing WT antibody or antibody lacking glycine

and lysine (Figure 3A). Although Day 14 IVCCs were similar among the three pools

(Figure 3C), the Day 14 specific productivity of the lysine-deleted antibody expressing

pool had decreased 32.3% and 50.2% compared to the WT and glycine-lysine-deleted

antibody expressing pools, respectively (Figure 3B). We then examined whether C-

terminal lysine or glycine-lysine deletion lowers the level of antibody basic variants.

Interestingly, we observed that the antibodies bearing lysine deletion had 18.5% basic

variants, which was even higher than that for the WT antibodies (15.6%) (Figure 3E).

While levels of uncleaved C-terminal lysine in the lysine deleted version of antibody

were reduced to 0%, an increase in levels of proline amidation (16.9%) accounts for an

overall increase in levels of basic variants compared to WT (Figure 3D). In contrast,

glycine-lysine double deleted antibodies with only 3.5% basic variants displayed the most

uniform HC C-terminus (-SLSLSP) (Figures 3D&E). Similar trends were observed when

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%basic variants were compared for individual clones of each expression construct

(Figure 3F).

Clones expressing C-terminal lysine deleted antibody have higher rates of HC

degradation

To determine the root cause of lower specific productivity of clones expressing antibody

lacking HC C-terminal lysine, we used the pool of the top 20 mAb3 clones for each of the

WT, lysine deleted, and glycine-lysine deleted versions of antibody and analyzed the

intracellular antibody levels by Western blot analysis. We observed that the intracellular

level of HC molecules in the cells expressing lysine deleted antibody was lower than that

of the cells expressing WT or glycine-lysine deleted antibody, while LC levels were

comparable across all antibody versions (Figure 4A). Measuring HC and LC mRNA

levels revealed that the lysine deleted version had slightly higher HC mRNA levels

relative to the WT version, with the glycine-lysine deleted version having the highest HC

mRNA level among all three versions (Figure 4B, top). Levels of antibody LC mRNA

were comparable across all versions (Figure 4B, bottom). Therefore, altered mRNA

levels do not account for the observed low levels of intracellular HC bearing C-terminal

lysine deletion. On the other hand, inhibition of proteasomal degradation resulted in

accumulation of similar levels of ubiquitinated antibody species among WT, lysine

deleted, and glycine-lysine deleted versions. Considering that the lysine-deleted version

has the lowest levels of intracellular HC (Figure 4C), quantification of ubiquitin:HC

Western blot signal ratios revealed that HC bearing lysine deletion has ~2-fold higher

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ubiquitination rate and so perhaps higher degradation rate relative to WT HC or HC

bearing glycine-lysine deletion (Figure 4C, compare lane 4 to lanes 2 and 6).

We also decided to analyze the antibody glycosylation pattern as a way to examine HC

localization within the ER/Golgi pathway. As long as antibody HC molecules are

residing in the ER, their N-linked glycans are sensitive to EndoH digestion but once in

the Golgi, due to glycan modifications they become resistant to EndoH digestion.

However, all HC glycans remain sensitive to PNGase-F digestion, irrespective of their

localization. We did not observe any differences in intracellular glycosylation and

trafficking patterns among the three versions of antibody, since comparable banding

patterns among all antibody versions were obtained upon EndoH or PNGaseF treatment

(Figure 4D). Lack of EndoH resistant antibody species shows that antibody molecules are

not retained in the Golgi.

Clones expressing C-terminal lysine deleted antibody have lower antibody synthesis

rates

We also investigated rates of antibody synthesis for WT antibody and antibody lacking

C-terminal lysine or glycine-lysine. Cells were treated with cyclohexamide (CHX) to stop

protein synthesis and within 6 hours almost all the intracellular antibody molecules were

secreted from the cells into the media (Figure 5A). Removal of CHX restored protein

synthesis and levels of intracellular and secreted antibodies were monitored at 0 and 2

hours (Figure 5B). For the cells expressing antibody lacking glycine-lysine, maybe

because the intracellular HC levels were higher and overwhelmed secretion capacity,

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there was some HC molecules left in the cells even after 6 hours of CHX treatment

(Figure 5A). Hence, there was also higher levels of HC molecules at the beginning of the

CHX washout step (Figure 5B). Within 2 hours, antibodies started to accumulate in the

cell and small amounts were secreted into the cell culture media. While levels of antibody

secretion for both WT and HC C-terminal lysine deleted versions were minimal (Figure

5B, lane 2 vs lane 4 and Figure 5C), intracellular accumulation of newly synthesized

lysine deleted antibody HC was only 42% of that of WT and was much lower than

glycine-lysine deleted antibody HC (Figure 5B and 5C). Interestingly, our findings

suggest that for antibody bearing glycine-lysine double deletion, the HC translation,

translocation and folding is the fastest (Figure 5C). This may be partially due to its higher

mRNA level (Figure 4B). The faster production of folded antibody could explain the

higher titer and specific productivity of clones expressing the glycine-lysine deleted

version of mAb3. Our findings suggest that the HC translation/translocation (and folding)

rate is slower for HC lacking C-terminal lysine relative to WT, and HC lacking both

glycine and lysine. In sum, clones expressing antibody lacking HC C-terminal lysine

have slower intracellular accumulation of antibody HC and faster intracellular antibody

degradation, resulting in lower overall titer and specific productivity.

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Discussion

In this study, we have shown that clones expressing antibodies with C-terminal lysine

deletion consistently have lower titers, a phenomenon that was observed in three different

test cases. After ruling out altered mRNA levels accounting for the observed protein level

differences, we found that the low productivity of these clones seems to be due to lower

antibody peptide synthesis, translocation and/or folding rates combined with a higher rate

of antibody degradation. It remains to be determined whether the identity of the C-

terminal amino acid plays a role in regulating the release of a newly synthesized HC

molecule from the ribosome, or its translocation into the ER lumen and subsequent

folding. Perhaps a nascent HC polypeptide with a charged C-terminal lysine (WT) or a

proline (glycine-lysine deleted version) residue, can more easily leave the ribosome/ER

membrane interface and enter the aqueous ER lumen than a nascent HC polypeptide with

a glycine C-terminus (lysine deleted version). Once it folds and exits the ER, the lysine

deleted version of antibody is readily processed and transported out of the cell, as

indicated by EndoH treatment (Figure 4D) and the CHX washout experiment (Figure 5).

We don’t think the low specific productivity in –K construct is due to its poor binding to

the protein A/G column, because it has been shown via crystallographic refinement that

the primary binding site for protein A is on the Fc region, between the CH2 and CH3

domains 16.

We also observed that deletion of C-terminal lysine increased basic variant levels due to

more prevalent proline amidation (Figure 3E). In WT HC, proline amidation requires

removal of the C-terminal lysine by endogenous carboxypeptidase D11. However, for the

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HC bearing a C-terminal lysine deletion this step has already been bypassed, leaving it

more prone to proline amidation. On the other hand, clones expressing antibody lacking

both HC C-terminal lysine and glycine have the lowest levels of C-terminal amino acid

heterogeneity and similar or higher productivity compared to the WT antibody. It is

noteworthy to keep in mind that all the antibodies tested here are of IgG1 isotype and for

now our findings with regards to –K and –GK versions of antibodies only apply to this

isotype.

Considering that each of the mAbs 1, 2, and 3 have different targets and hence different

CDR sequences, we are fairly confident that the observed higher titers of –GK mAbs is

not antibody specific. Based on this, we suggest that -GK version of an antibody could

be a feasible option for reducing antibody C-terminal processing, therefore decreasing

heterogeneity and the need for monitoring this variable during manufacturing. Since

complement dependent cytotoxicity (CDC) occurs only when antibody is injected into the

bloodstream and the C-terminal lysine is removed, both WT (due to function of carboxyl

peptidases) and –GK versions of an antibody can equally accommodate this purpose. If

however an effector-less antibody is desired, an aglycosylated –GK antibody may be

engineered 17. While our initial investigations suggested that deletion of C-terminal lysine

or glycine-lysine residues from HC does not affect antibody glycosylation, potency, PK

and bioavailability relative to the WT antibody, 6, 15 nevertheless, the immunogenicity of

the glycine-lysine deletion in antibodies will need to be tested in order to ensure patient

safety prior to its utilization for commercial production.

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Materials and Methods

Cell Lines and Cell Culture

Antibody-producing cell lines were generated at Genentech and derived from the

CHOK1 host. CHO cells were cultured in a proprietary DMEM/F12-based medium in

125 mL shake flask vessels shaking at 150 rpm, 37oC and 5% CO2. Cells were passaged

with a seeding density of 3x105 cells/mL every 3 - 4 days 18

. DNA sequences of C-

terminal deletion mutants of antibody heavy chain were designed similarly as WT only

lacking the nucleotides encoding C-terminal lysine or glycine-lysine.

Real-Time Quantitative RT-PCR Analysis (TaqMan assay)

Total RNA from cells was isolated using the RNeasy 96 kit following the manufacturer’s

protocol (Cat# 74181, Qiagen) and was treated with DNase (Cat#79254, RNase free

DNase kit, Qiagen) to remove any residual DNA that may be present in the isolated RNA

sample. qRT-PCR was performed using a universal qRT-PCR master mix according to

the manufacturer’s instruction (Cat#4309169, Applied Biosystems) and mRNA levels of

antibody heavy and light chains were normalized to mRNA levels for the housekeeping

gene β-microglobulin.

Primer and probe sequences used for RT-PCR were as follows:

Heavy chain forward primer: TCA AGG ACT ACT TCC CCG AAC

Heavy chain reserve primer: TAG AGT CCT GAG GAC TGT AGG ACA GC

Heavy chain probe: FAM- ACG GTG TCG TGG AAC TCA GGC GC- TAMRA

Light chain forward primer: TGA CGC TGA GCA AAG CAG AC

Light chain reserve primer: CAG GCC CTG ATG GGT GAC

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Light chain probe: FAM -ACG AGA AAC ACA AAG TCT ACG CCT GCG A-

TAMRA

CHO β-microglobulin forward primer: TCC TCT CAG TGG TCT GCT TGG

CHO β-microglobulin reserve primer: TGG CGT GTG TAG ACT TGC ACT T

CHO β-microglobulin probe: FAM - TGC CAT CCA GCG TCC CCC A - TAMRA

Western Blot Analysis

Western blot analysis was performed in order to determine the antibody heavy and light

chain expression levels. β-actin was used as the endogenous control protein. Cell pellets

were collected and lysed in lysis buffer (Cat# 9803, Cell Signaling Technology, Danvers,

MA). Total protein content of the lysed supernatants was quantified by Bradford assay

(Cat#1856210, Thermo Scientific, Rockford, IL). The samples were heated at 95°C for

3 min, and loaded onto SDS–PAGE gel and transferred to nitrocellulose membranes.

Membranes were then incubated with the indicated antibodies (anti-HC antibody,

Cat#A80-104P, Bethyl Laboratories; anti-LC antibody, Cat#ICN55223, Fisher Scientific,

and β-actin, Cat#A2228, Sigma).

Proteasome inhibition experiment

Twenty-clone pool seed train samples from each construct were used for MG132

treatment studies. Seed train cells were seeded at 2 x 106 cells/mL for a total of 5 mL (10

x 106 cells) and treated with or without MG132 (Cat#474791, Sigma) at 10 µg/mL for 6

hours. At the end of the incubation, cells were spun down and analyzed by Western blot

analysis as described above.

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Endo H and PNGase F treatment assay

Cells were lysed in lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5%

NP-40, protease inhibitor). Monoclonal antibodies were pulled down by protein A/G

agarose beads and boiled in 150 µL 1X glycoprotein denaturing buffer as instructed by

the Endo H (Cat# P0702S) and PNGase F (Cat# P0704L) manufacturer (Bio Labs) for 10

min. 30 µL of the denatured antibody was kept as non-treated sample, the rest was

divided and transferred to two new tubes, incubated with either PNGase F or EndoH as

instructed at 37 oC for 1 hr. The samples were then analyzed by SDS-PAGE and Western

blot as explained above.

Product Quality Analysis

Charge variants were measured via capillary iso-electric focusing (cIEF). Liquid

chromatography followed by mass spectrometry (LC-MS) was used to estimate the

percentage of C-terminal lysine and amidated proline residues.

Shake Flask Fed-Batch Production Assay

Fed-batch production cultures were performed in shake flasks with proprietary

chemically defined medium together with bolus feeds on days 3, 7, and 10. A

temperature shift from 37°C to 35°C was carried out on day 3. Day 14 titers were

determined using protein A affinity chromatography with UV detection. Percent viability

and viable cell count was determined using Vi-Cell XR instrument (Beckman Coulter) 18.

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Protein Synthesis Inhibition and Resumption

Cells (20 x 106) from a pool of the top-20 clones expressing either WT, -K or –GK

antibody were washed with PBS, pelleted and resuspended into 30 mL seed train media

containing 20 µg/mL cyclohexamide (Cat# C7698, Sigma) and grown in shake flasks. At

0 hr, 3 hr and 6 hr time points, 2 x 106 cells (3 mL) were sampled and supernatants were

separated from the cells by centrifugation. After 6 hours, the remaining cells were

pelleted, washed with PBS and resuspended into 21 mL of seed train media in the

absence of cyclohexamide. At 0 hr and 2 hr time points, 2 x 106 cells (3 mL) were

sampled and cell pellets and supernatants were separated. All collected cell pellets were

washed with PBS and lysed in 1 mL lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl,

1% Triton X-100, with protease inhibitors). Lysates were cleared by centrifugation at

14,000 rpm (20,000 × g) for 10 min. Antibodies from both cell lysates and supernatants

were immunoprecipitated by protein A/G beads for further analysis including SDS-PAGE

and Western blot.

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Acknowledgements:

We would like to thank the Analytical Operations group at Genentech for their excellent

service and support, and Mike Laird and Steve Lang for their critical review of the

manuscript. The authors have no conflict of interest to declare.

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Figure legends

Figure 1. C-terminal lysine deletion of antibody HC results in lower specific

productivity and titer.
A. The process of C-terminal amidation of antibody heavy chain. In cell culture, the C-
terminal lysine residue is cleaved by carboxypeptidase D (CpD). In the presence of
copper, peptidylglycine α-amidating monooxygenase (PAM), utilizing its two
enzymatically active domains - peptidylglycine alpha-hydroxylating monooxygenase
(PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), further
catalyzes the hydroxylation of glycine and removal of the glyoxylate from the glycine
residue, leaving an amidated C-terminal proline.
B. Types of basic variants generated from wild type (WT) antibody or antibody with HC
C-terminal lysine (-K) and glycine-lysine (-GK) deletions.
C. Specific productivity (Qp) of pools of clones expressing indicated versions of
antibodies for two different mAbs. Each bar represents the average from two replicate
production experiments. Each error bar denotes 1 standard deviation.
D. Day-14 (D14) titer of pools of clones expressing indicated versions of antibodies for
two different mAbs. Each bar represents the average from two replicate production
experiments. Each error bar denotes 1 standard deviation.

Figure 2. Individual clones expressing antibody mAb3 bearing HC C-terminal lysine

deletion have relatively lower productivities.
A. Titers of the 528 clones expressing indicated versions of antibody mAb3 were
analyzed by ELISA.
B-D. Titer (B), specific productivity (C) and integrated viable cell count (IVCC) (D)
from a 14-day production assay of the top 20 clones producing indicated versions of
mAb3. Blue line represents the average value of the top 20 clones producing each
version of mAb3.

Figure 3. Pool of clones expressing antibody mAb3 bearing HC C-terminal lysine

deletion has lower productivity and higher basic variant levels.

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A-C. Titer (A), specific productivity (B), and IVCC (C) of the pools of the top 20 clones
expressing indicated versions of the antibody mAb3. Each bar represents the average
from two replicate production experiments. Each error bar denotes 1 standard deviation.
D. Percentage and type of basic variants of the pools of the top 20 clones characterized
by mass spectrometry (ND = Not Detectable).
E. Percent of basic variants of the pools of the top 20 clones expressing indicated
versions of the antibody mAb3.
F. Percent of basic variants of the single clones expressing indicated versions of the
antibody mAb3.

Figure 4. Pool of clones expressing antibody mAb3 lacking HC C-terminal lysine

has similar antibody mRNA level and trafficking pattern compared to WT antibody
and antibody lacking glycine-lysine, but higher degradation rates.
A. Intracellular protein level of indicated versions of antibody mAb3 in pools of top 20
clones. Cells were lysed, analyzed by SDS-PAGE and immunoblotted for indicated
proteins.
B. Heavy chain and light chain mRNA levels of indicated versions of antibody mAb3 in
pools of top 20 clones. Each bar represents the average from the triplicate Taqman
samples. Each error bar denotes 1 standard deviation.
C. Ubiquitination of antibody mAb3. Cells expressing indicated antibodies were treated
with or without MG132 for 6 h. Cell lysates were analyzed by Western blot for
intracellular HC levels. Immunoprecipitation and Western blot analysis were used to
visualize the ubiquitinated HC species. Band intensities were quantified by ImageJ.
The ratio of band intensities of ubiquitinated HC to non-ubiquitinated HC of each
MG132 treated sample has been calculated and stated.
D. Glycosylation analysis of antibody mAb3. Lysate of cells expressing indicated
versions of antibodies were treated with EndoH or PNGaseF, and analyzed by SDS-
PAGE and Western blot. The band shift indicates the removal of glycans from heavy
chain by the enzymes. Note that lack of EndoH-resistant bands indicates that the
intracellular HC molecules mainly localize in the ER and are not retained in the Golgi
complex.

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Figure 5. Low productivity of mAb3 bearing HC C-terminal lysine deletion is

accompanied by lower intracellular HC expression/accumulation.
A. Cells of top 20 clone pools producing the indicated versions of mAb3 were pre-
washed and resuspended into culture medium containing cycloheximide (CHX) and
incubated for 6 hours. Cells and medium at indicated time points were collected.
Antibodies in the cell lysate or the medium were precipitated by Protein-A agarose
and analyzed by SDS-PAGE and Western blot.
B. After 6 hours of translation blockage, CHX was washed out (CHX washout) from the
culture medium. HC levels in the cells and in the medium at indicated time points
were analyzed as in (A).
C. Quantification of (B), the amount of newly synthesized antibodies in the cell (left
panel) and antibodies secreted into the culture medium (right panel), at indicated time
points.

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Page 23 of 27 Biotechnology Progress

Figure 1. C-terminal lysine deletion of antibody HC results in lower specific productivity and titer.
Figure 1
508x677mm (72 x 72 DPI)

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Figure 2. Individual clones expressing antibody mAb3 bearing HC C-terminal lysine deletion have relatively
lower productivities.
Figure 2
508x677mm (72 x 72 DPI)

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Figure 3. Pool of clones expressing antibody mAb3 bearing HC C-terminal lysine deletion has lower
productivity and higher basic variant levels.
Figure 3
508x677mm (72 x 72 DPI)

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Figure 4. Pool of clones expressing antibody mAb3 lacking HC C-terminal lysine has similar antibody mRNA
level and trafficking pattern compared to WT antibody and antibody lacking glycine-lysine, but higher
degradation rates.
Figure 4
508x677mm (72 x 72 DPI)

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Page 27 of 27 Biotechnology Progress

Figure 5. Low productivity of mAb3 bearing HC C-terminal lysine deletion is accompanied by lower
intracellular HC expression/accumulation.
Figure 5
508x677mm (72 x 72 DPI)

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