triple light chain antibodies: factors that influence its formation in cell culture

ARTICLE

Triple Light Chain Antibodies: Factors ThatInfluence Its Formation in Cell Culture

Natalia Gomez,1 Abigail R. Vinson,1 Jun Ouyang,2 Mary D.H. Nguyen,2

Xiaoying-Nancy Chen,2 Vikas K. Sharma,3 Inn H. Yuk1

1Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, MS 32, South San Francisco,

California 94080-4990; e-mail: [email protected] Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco,

California 94080-49903Early Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way,

South San Francisco, California 94080-4990

Received 8 June 2009; revision received 11 August 2009; accepted 12 October 2009

Published online 20 October 2009 in Wiley InterScience (www.interscience.wiley.com
). DOI 10.1002/bit.22580
ABSTRACT: THIOMABs are recombinant antibodies engi-neered with reactive cysteines, which can be covalentlyconjugated to drugs of interest to generate targeted ther-apeutics. During the analysis of THIOMABs secreted bystably transfected Chinese Hamster Ovary (CHO) cells, wediscovered the existence of a new species—Triple LightChain Antibody (3LC). This 3LC species is the product ofa disulfide bond formed between an extra light chain andone of the engineered cysteines on the THIOMAB. Wecharacterized the 3LC by size exclusion chromatography,mass spectrometry, and microchip electrophoresis. We alsoinvestigated the potential causes of 3LC formation duringcell culture, focusing on the effects of free light chain(LC) polypeptide concentration, THIOMAB amino acidsequence, and glutathione (GSH) production. In studiescovering 12 THIOMABs produced by 66 stable cell lines,increased free LC polypeptide expression—evaluated as theratio of mRNA encoding for LC to the mRNA encoding forheavy chain (HC)—correlated with increased 3LC levels.The amino acid sequence of the THIOMAB molecule alsoimpacted its susceptibility to 3LC formation: hydrophilic LCpolypeptides showed elevated 3LC levels. Finally, increasedGSH production—evaluated as the ratio of the cell-specificproduction rate of GSH (qGSH) to the cell-specific produc-tion rate of THIOMAB (qp)—corresponded to decreased3LC levels. In time-lapse studies, changes in extracellular3LC levels during cell culture corresponded to changes inmRNA LC/HC ratio and qGSH/qp ratio. In summary, wefound that cell lines with low mRNA LC/HC ratio and highqGSH/qp ratio yielded the lowest levels of 3LC. These findingsprovide us with factors to consider in selecting a cell lineto produce THIOMABs with minimal levels of the 3LCimpurity.

Biotechnol. Bioeng. 2010;105: 748–760.

� 2009 Wiley Periodicals, Inc.

KEYWORDS: THIOMAB; light chain; glutathione; CHOcells

Correspondence to: N. Gomez

748 Biotechnology and Bioengineering, Vol. 105, No. 4, March 1, 2010

Introduction

THIOMABs are recombinant antibodies with reactivecysteine residues engineered at specific sites (Junutulaet al., 2008b). The thiol groups of these engineered cysteinescan be conjugated to cytotoxic drugs via a reduction-re-oxidation process to form therapeutic antibody–drugconjugates (ADCs), called THIOMAB-drug conjugates(TDCs). Recent preclinical studies suggest that TDCs mayretain the antitumor efficacy of ADCs, while minimizingtheir systemic toxicity and increasing the therapeuticwindow (Junutula et al., 2008b).

In the first step towards the production of TDCs, mam-malian cells stably transfected to express the THIOMAB ofinterest are cultured in suspension. THIOMABs secretedinto the culture medium are purified using a series ofchromatography steps prior to analytical characterization.The engineered cysteines on secreted THIOMABs areusually capped with either a cysteine or a glutathione(GSH) through a disulfide bond (Junutula et al., 2008b). Wereport here a third type of capping: one of the engineeredcysteines forms a disulfide bond with an extra light chain togenerate a Triple Light Chain antibody (3LC) (Fig. 1). Weconsider 3LC to be an undesirable impurity because ofthe potential for free light chain to be conjugated to thecytotoxic drug. Therefore, we remove 3LC to very low levels(<0.5%) during the purification process. However, thesepurification efforts also lower the overall THIOMABrecovery yield. By providing cell culture methods to lower3LC levels when needed, we could minimize the negativeimpact of 3LC on THIOMAB recovery. To accomplish thisgoal, we sought to first understand parameters that influence3LC formation.

Since 3LC is generated by disulfide bond formed betweena free light chain (LC) polypeptide and a full-lengthTHIOMAB (i.e., LCþTHIOMAB$ LC-s-s-THIOMAB),

� 2009 Wiley Periodicals, Inc.

Figure 1. Schematic depicting THIOMABs and Triple Light Chain antibodies

(3LC). A: THIOMAB-HC variant: the engineered cysteine is in the HC sequence. B: 3LC

formed from a THIOMAB-HC variant. C: THIOMAB-LC variant: the engineered cysteine

is in the LC sequence. D: 3LC formed from a THIOMAB-LC variant.

the amount of free LC polypeptide available to react in theER should drive 3LC formation. Free LC polypeptides can betranslated into the ER and remain there as monomers or LCdimers, whereas free HC polypeptides are not released fromthe ER because they associate with immunoglobulin bindingprotein (BiP) (Hendershot, 1990; Vanhove et al., 2001). Inconventional antibodies, these free LC polypeptides canremain unbound to HC and can be secreted with full-lengthantibodies (Shapiro et al., 1966). In THIOMABs, thesefree LC polypeptides can form disulfide bonds with theengineered cysteines. This disulfide bond formation coulddepend on: (1) the amount of LC polypeptide present,(2) the reactivity of the LC polypeptide (e.g., sterichindrance, charge, location of engineered cysteine), and(3) the ER redox state.

The amount of LC monomer should correlate to the ratioof LC polypeptides copies to HC polypeptide copies sincehigher LC/HC ratios can yield higher levels of free LC dimeror monomer (Schlatter et al., 2005). The reactivity of the LCpolypeptide may depend on the amino acid sequence ofboth the THIOMAB and the LC polypeptide. Finally, thedisulfide bond reaction requires redox enzymes and anoxidative environment (Frand et al., 2000; Raina andMissiakas, 1997). GSH is known to be a fundamentalregulator and redox buffer in the ER for the formation ofdisulfide bonds (Chakravarthi et al., 2006). In particular,GSH has been shown to introduce reducing equivalents tothe ER that prevent hyperoxidation by Ero1, the majoroxidative pathway (Cuozzo and Kaiser, 1999). GSHinfluences the ER redox state, controls disulfide bondformation, and reduces non-native bonds and oxidoreduc-tases (Chakravarthi and Bulleid, 2004; Jessop and Bulleid,2004; Molteni et al., 2004).

In this work, we investigated the effects of mRNA LC/HCratio and GSH production on 3LC formation for 66 stable

cell lines that secreted 12 different THIOMABs. We analyzedthe LC amino acid sequences in these THIOMABs toidentify properties that increased antibody susceptibility to3LC formation. In addition, we considered the impact of thelocation of the engineered cysteines—which can be on theLC or HC—on 3LC formation (Junutula et al., 2008a,b).By determining key attributes of cell lines and THIOMABsthat impact 3LC formation, we may identify methods tominimize 3LC formation in cell culture, and thereforemaximize recovery of the desired THIOMABs for sub-sequent conjugation into TDCs.

Materials and Methods

CHO Cell Lines

Stable cell lines secreting IgG1-based THIOMABs werederived from a CHO dihydrofolate reductase-negative(DHFR-) host. To generate these cell lines, CHO cells weretransfected with a DNA plasmid encoding genes for DHFR,light chain (LC) and heavy chain (HC) and cultured with aproprietary medium containing methotrexate (MTX). Foreach of the 12 THIOMABs discussed in this work, four tonine cell lines with the highest titers (Table I)—correspond-ing to a total of 66 stably transfected cell lines—wereevaluated.

Shake Flask Production Cell Culture

For production experiments, cell lines were cultured intriplicate shake flasks (125 or 250 mL, Corning, Corning,NY) for 14 days. After seeding the cultures (1� 106 cells/mL) in a proprietary basal medium, additional nutrientswere supplied in the form of a batch feed on day 3. Shakeflasks were cultured at 378C for 48 h, and subsequently at338C for the rest of the experiment. On days 3, 7, 10, and 14,the cultures were analyzed for viable cell concentration(VCC) and viability (Vi-Cell XR, Beckman Coulter,Fullerton, CA), for pH, glucose and lactate (Bioprofile400, Nova Biomedical, Waltham, MA), and for titer (proteinA HPLC). Day 14 supernatants were analyzed for free LCpolypeptides, amino acid concentration and 3LC and totalGSH content. Day 7 cell pellets were used for mRNA analysisand Western blotting.

Integrated viable cell concentration (IVCC) was calcu-lated as follows:

IVCCi ¼ IVCCi�1 þVCCi þ VCCi�1

2

� �� ðti � ti�1Þ

i is the sample number (samples 1, 2, 3, 4, and 5 werecollected on days 0, 3, 7, 10, and 14, respectively) and t is theday of sample collection. Cell-specific productivity (qp) foreach cell line was determined from the slope of a linear fit oftiter versus IVCC.

Gomez et al.: 3LC in THIOMABs 749

Biotechnology and Bioengineering

Table I. Characteristics of stably transfected cell lines and THIOMABs evaluated.

THIOMABa Symbol No. of cell linesb Cell line number Final titerc (g/L) qp (pg cell�1 day�1) Final cell viabilityc (%) IVCCc (106 cell day L�1)

A-HC ^ 4 1–4 1–3 17–37 74–91 3–12

B-HC � 4 5–8 1–1.8 9–20 77–84 8–12

C-HC þ 4 9–12 1–1.9 8–15 94–98 10–13

D-HC D 4 13–16 1.4–2 12–16 88–97 10–17

E-LC && 4 17–20 0.5–2.2 5–35 70–94 5–9

E-HC 4 21–24 1.1–2.1 20–29 87–92 5–9

F-LC * 6 25–30 0.1–0.7 2–10 20–70 5–7

F-HC * 8 31–38 0.4–2.2 3–48 70–98 3–16

G-HC Z 8 39–46 0.4–2.1 5–34 50–76 3–11

H-HC X 3 47–49 0.002–0.2 0.01–2 55–78 6–9

I-LC & 9 50–58 0.8–1.2 4–20 89–95 7–13

I-HC & 8 59–66 0.7–1.9 9–20 86–96 6–14

aTwo versions of THIOMABs were evaluated: HC¼ engineered cysteine in HC, LC¼ engineered cysteine in LC.bSixty-six stably transfected cell lines were evaluated in shake flasks for a total of 12 different THIOMABs (4–9 cell lines/THIOMAB).cValues corresponding to day 14 measurements.

Cell-specific GSH production rate (qGSH) and cell-specificcysteine/cystine consumption rates (qcys) were calculated asfollows:

qGSH ¼ GSHextracellular½ �tIVCCt

;

qCys ¼Cys½ �medium� Cysextracellular½ �t

IVCCt

t is the day of sample collection.For time-lapse studies, day 3, 7, 10, and 14 supernatants

were analyzed for 3LC and GSH content. Day 3, 7, 10, and14 cell pellets were used for mRNA and intracellular GSHanalysis.

Real-Time RT-PCR

Day 7 shake flask culture samples were centrifuged (4� 106

cells, 5,000 rpm, 2 min) and cell pellets were stored (�708C).mRNA extraction and purification were performed usinga RNeasy kit (Qiagen, Valencia, CA) according to themanufacturer’s instructions. For real-time RT-PCR, mRNA(5mg) was added to a reaction mixture (20.5mL) preparedwith RT-PCR kit (Applied Biosystems Inc., Foster City, CA)containing forward and reverse primers (0.3mM each) andprobe (0.2mM). Primers were prepared in-house for LC,HC, and beta-2-microglobulin (used as the housekeepinggene). TAMRA and FAM were used as quencher andreporter dyes, respectively. An ABI Prism 7700 SequenceDetection System (Applied Biosystems Inc.) was used fordetection (40 cycles at 958C, 15 s and 608C, 1 min). mRNAconcentrations for LC, HC, and beta-2-microglobulin werequantified with the calibration curve method using the Ct

value derived from the detection system. The mRNA LC/HCratio was calculated by dividing the amount of LC mRNA bythe amount of HC mRNA.


Western Blotting of Cell Lysates

Day 7 shake flask culture samples were centrifuged (1� 106

cells, 5,000 rpm, 2 min). The resulting cell pellets storedat �708C were lysed with a lysis buffer (Cell Signaling,Boston, MA) containing protease inhibitor cocktail (Roche,Manheim Germany). Total protein content was determinedwith a Bradford assay (Bradford Assay Kit, Pierce, Rockford,IL). Tris–glycine SDS sample buffer was added to cell lysatesand heated (908C, 3 min). Cell lysate (10mg) was loadedinto a 1 mm, Nu-PAGE 4–12% Bis-Tris gel (Invitrogen,Carlsbad, CA) and run for 50 min in a Nu-PAGE electro-phoresis apparatus (Invitrogen) using 1� MOPS buffer(Invitrogen). Proteins were transferred to a nitrocellulosemembrane (BioRad, Hercules, CA) with the XCell IITM

Blot Module (Invitrogen) using 1X CAPS buffer with 3%methanol for 90 min. Membranes were blocked (1 h) in 1�NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl (pH7.4), 0.05% Triton X-100) with 5% gelatin and incubatedovernight with a mouse anti-human LC antibody con-jugated to HRP (SouthernBiotech, Birmingham, AL) oranti-b-actin antibody (Sigma–Aldrich, St. Louis, MO). Forb-actin staining, additional secondary antibody incubationusing an anti-mouse IgG antibody conjugated to HRP (GEHealthcare Biosciences, Piscataway, NJ) was performed(1 h). After four washes (15 min each), the membraneswere incubated (1 min) with an ECL kit (Amersham-GEHealthcare Biosciences, Piscataway, NJ), prior to filmexposure and development.

Measurement of Total Glutathione Concentration

Total GSH (including both reduced GSH and oxidizedGSSG) was measured using the enzyme recycling methoddeveloped by Tiezle and modified by Baker et al., (Bakeret al., 1990; Tietze, 1969). To prepare samples, 3� 106 cellswere centrifuged (2,000g, 10 min, 4oC). The supernatant wasused to determine extracellular GSH concentration. The cell

pellet was rinsed and resuspended in MES buffer, followedby three consecutive cycles of freeze-thaw and centrifugation(10,000g, 15 min). Both supernatant and cell lysate weredeproteinated by adding an equal volume of 1 M metapho-sphoric acid (Sigma–Aldrich). This mixture was vortexed,left at room temperature (5 min), and then centrifuged(5,000g, 3 min). The resulting supernatant was removed andstored (�20oC). To complete the assay, thawed sampleswere treated with 4 M triethanolamine (Sigma–Aldrich) at100mL per 1 mL of sample to increase sample pH from�1 to �7. The Cayman Chemical Glutathione Assay Kit(Cayman Chemical Co., Ann Arbor, MI) was used accordingto the manufacturer’s kinetic method instructions.

Measurement of 3LC With Bioanalyzer (MicrochipElectrophoresis)

3LC was quantified using the Agilent 2100 Bioanalyzer(Agilent Inc., Santa Clara, CA). Sample preparation andprotein separation were conducted according to manufac-turer’s instructions. Briefly, 4mL of protein A purifiedsamples (�1 mg/mL) was mixed with 24mL of denaturingsolution (50 mM iodoacetamide and 0.5% SDS) and 2mL ofsample buffer, and incubated (708C for 5 min). This mixturewas diluted (60mL water) prior to loading 6mL onto theprotein chip for electrophoresis. Results were analyzed usingAgilent 2100 Expert software. The percentage of 3LC in eachsample was determined as follows:

%3LC ¼ 3LC peak area

total area� 100

3LC peak¼ peak at 175 kDaTotal area¼ area for all peaks (except markers) in

electropherogram.

Size-Exclusion Chromatography of THIOMAB

Size-exclusion chromatography (SEC) was conducted usingan Agilent 1100 liquid chromatography system equippedwith a binary pump (Agilent Inc.). Briefly, THIOMAB(�50mg in 50mL) was injected onto a size-exclusioncolumn from Tosoh Bioscience (Montgomeryville, PA):TSK-G3000SWXL, 5mm particle size, 7.8 mm ID, and 30 cmlength. Protein was eluted isocratically for 30 min using amobile phase (0.2 M potassium phosphate, 0.25 M potas-sium chloride, pH 6.2) and a flow rate of 0.5 mL/min.Protein elution was monitored by absorbance at 280 nm. Toreduce the sample prior to SEC, �10 mM of dithiothreitol(DTT) was added to the protein solution (in pH 7.5 Trisbuffer) and the mixture was incubated (378C, 30 min).

Liquid Chromatography–Mass SpectrometryAnalysis of THIOMAB

THIOMAB was deglycosylated prior to liquid chro-matography–mass spectrometry (LC–MS) analysis. Onemicroliter of N-glycosidase (New England Biolabs, Ipswich,

MA) was added to 100mL of 1 mg/mL THIOMAB (inpH 7.5 Tris buffer) and incubated overnight (378C). Liquidchromatography was conducted on an Agilent 1100 LC systemwith a binary pump. Approximately 10mg of THIOMABwas injected onto the Agilent Poroshell 300SB C3 column(1 mm� 75 mm, 5mm, 300 A) equilibrated in 72% buffer A(0.1% formic acid in water) and 18% buffer B (0.1% formicacid in ACN), and held for 10 min at 18% buffer B. Proteinwas eluted from the column (maintained at 758C) with aflow rate of 0.2 mL/min by a linear gradient to 95% buffer Bover 8 min with detection at 280 nm. The mass spectro-metric analysis was carried out in positive ion mode on anABI Q-Star Pulsar electrospray ionization (ESI)-qTof massspectrometer equipped with a Turbo Spray ionizationsource (Applied Biosystems Inc.). Liquid chromatographyflow was introduced directly to the ESI source with sprayvoltage of 5300 V and source temperature of 4008C.The declustering and focusing potentials were 100 and300, respectively. Other parameters were optimized formaximal signal intensity and resolution. The instrument wascalibrated externally in an m/z range of 780–3,500 amu usingcesium tridecafluoroheptanoate. Molecular masses werederived from multiply charged ions and deconvoluted usingthe BioAnalyst 1.1 software package (Applied BiosystemsInc.).

High Performance Liquid Chromatography(HPLC) Assays

Extracellular THIOMAB titer was determined usingconventional protein A affinity chromatography with UVdetection (Fahrner et al., 1999). Free extracellular LCmonomer and dimer polypeptides in cell culture super-natants were measured using an affinity chromatographycolumn (anti-kappa light chain) coupled with reverse phaseHPLC (Battersby et al., 2001). The percentage of total freeextracellular LC polypeptide (monomer and dimer) contentwas determined as follows:

%Lþ LL ¼ LCmonomer peak areaþ LC dimer peak area

total area

� 100

Cystine concentrations were determined by free aminoacid analysis of cell culture supernatants with pre-columnderivatization and reversed-phase HPLC (Cohen andMichaud, 1993).

Statistical Analysis

Statistical tests were performed using JMP 7.0 software (SASInstitute Inc., Cary, NC). Statistical significance was definedby P values <0.05.

Results and Discussion

This is the first report to describe the existence of the 3LCspecies and to investigate factors that influence its formation



in cell culture. 3LC is unique to THIOMABs because theengineered cysteine in the THIOMAB amino acid is reactiveand can disulfide bond to a free cysteine, glutathione, orLC polypeptide. Scientists selected the location of thiscysteine to be on the surface of the antibody to facilitate easyaccess of the drug to the cysteine during the conjugationprocess (Junutula et al., 2008a). This accessibility shouldalso facilitate 3LC formation through disulfide linkagebetween the engineered cysteine and a free LC polypeptide.We hypothesized that the quantity of 3LC formed in thecell culture process is a function of multiple parametersincluding: (1) free LC polypeptide concentration, (2)THIOMAB amino acid sequence, and (3) GSHconcentration.

3LC Characterization

We first detected the 3LC species during a routine size-exclusion chromatography (SEC) analysis of a THIOMAB:

Figure 2. Characterization of THIOMAB and 3LC. A: A representative SEC chromatogra

DTT-reduced (in black) THIOMAB. Upon reduction, the shoulder diminishes while a later-elu

be observed because its oxidized form absorbs at 280 nm. C: A representative SEC chromato

280 nm. Monomer (HHLL) is present regardless of DTT treatment. D: Deconvoluted mass spe

of a THIOMAB comprising two heavy chains and two light chains (HHLL), while mass 168802

Bioanalyzer instrument used to detect 3LC. The 3LC species is identified as a peak at app

antibody; [HHLL]2¼ dimer.


we identified a shoulder peak before the monomer peak(Fig. 2A). The location of this peak indicated that the newspecies was intermediate in size between the monomeric(HHLL) and dimeric ([HHLL]2) forms of the antibody.Upon reduction with DTT (Fig. 2B), the shoulder peakdisappeared, whereas a peak eluting later than the mainpeak—corresponding to a lower molecular weight species—emerged. DTT reduces antibody inter-chain disulfides,while retaining the antibody native monomer structurethrough non-covalent interactions during a non-denaturingSEC analysis (Fig. 2C). This observation indicated thatthe species responsible for the shoulder peak consisted ofthe THIOMAB linked to an extra component by a reduciblebond. The extra component detached from the intactTHIOMAB upon DTT treatment and could not re-associatedue to its non-native nature. Since this shoulder peak is notdetected in non-THIOMAB versions of the same antibody(Fig. 2C), the engineered cysteine on the THIOMABprobably mediated the reducible bond formed with the extracomponent.

m of THIOMAB at 280 nm. B: SEC chromatograms at 280 nm of non-treated (in gray) and

ting peak representing a smaller size species (labeled as fragment) emerges. DTT can

gram of a non-treated (in gray) and DTT-reduced (in black) non-THIOMAB antibody at

ctra of collected fractions from SEC (main peak and shoulder peak): 145480 is the mass

is the 3LC variant of the THIOMAB. E: Representative electropherogram obtained from

roximately 175 kDa. HHL¼ Two heavy chains and one light chain; HHLL¼ full-length

LC–MS analyses of the main and shoulder peaks fractionscollected from SEC confirmed the presence of a larger massin addition to the expected THIOMAB mass (Fig. 2D).The difference between the two masses was approximately23,000 Da, matching the expected mass of an LC polypep-tide. When we characterized and quantified the 3LC speciesusing an electrophoretic method, we found the correspond-ing 3LC peak at �175 kDa (Fig. 2E). During analysis of 12different IgG1-based THIOMABs secreted by 66 differentcell lines (Table I), we detected varying amounts of 3LCspecies in the cell culture harvest fluid (Fig. 3). Althougheach THIOMAB used in this work contains two engineeredcysteines and therefore has the potential to form an antibodywith four light chains, we either do not detect these four lightchain species or detect them at very low levels (<0.5%).

Free LC Polypeptide Concentration

Since 3LC results from the disulfide bond formed betweenan engineered cysteine in the HC or LC of a THIOMAB andan additional LC polypeptide, we hypothesize that theamount of free LC polypeptide expressed is a criticalparameter in determining %3LC generated by cell linessecreting THIOMABs. To test this hypothesis, we used realtime RT-PCR to quantify mRNA encoding for LC and HCpolypeptides. The percentage of free LC and LC dimerpolypeptides (Lþ LL) secreted into the cell culture mediumincreased with the mRNA LC/HC ratio (Fig. 3A). Theamount of intracellular free LC polypeptides in cell lysatesalso increased with the mRNA LC/HC ratio (Fig. 3B).Schlatter et al., (2005) reported similar results when theyadjusted the proportion of plasmids coding for LC and HCin transient transfections—the amount of extracellular LCdimer polypeptides analyzed in Western blots increased withlower HC to LC gene ratios.

The linear correlation between the amount of secreted3LC and mRNA LC/HC ratio or Lþ LL is statisticallysignificant (P< 0.0001 for both cases). These trends wereapproximately linear up to an mRNA LC/HC ratio of �1.5and a corresponding Lþ LL of �30% (Fig. 3C and D).THIOMAB D-HC cultures showed significantly higher%3LC than any other THIOMAB cultures, suggesting thatcertain THIOMABs are more susceptible to 3LC formationthan others. For most of the THIOMAB cultures, percentageof 3LC rarely exceeded 10%, even when the mRNA LC/HCratio was high (>1.5). We speculate that 3LC formation in

Figure 3. Effect of free LC polypeptide concentration on 3LC levels. A: Secreted

Lþ LL as a function of mRNA LC/HC ratio. Lþ LL was measured in day 14 cell culture

supernatant using an affinity column coupled to an HPLC. mRNA LC/HC ratio was

measured from cell pellets on day 7 using real time RT-PCR. B: Non-reducing Western

blots of cell lysates for nine representative cell lines expressing three THIOMABs. Cell

lysates were collected on day 7 and analyzed by PAGE and subsequent blotting with

anti-LC antibody. C and D: %3LC as a function of mRNA LC/HC ratio and Lþ LL,

respectively. 3LC was measured from day 14 supernatants using Bioanalyzer. All data

points represent an average of three biological replicates (n¼ 3). SEMs ranged from

2% to 12%, but are not shown to improve clarity for the symbols.

the ER may become limited when the amount of free LCpolypeptide is similar to or greater than the amount of full-length antibody because the free LC polypeptides are morelikely to react with each other to form dimers than to reactwith the full-length antibody to form 3LC.



Unlike other THIOMAB cell lines, cell lines secretingH-HC did not generate any detectable %3LC regardlessof the mRNA LC/HC ratio (Fig. 3C). The only notabledifference between the H-HC cell lines and the other 11THIOMAB cell lines pertained to cell-specific productivity(Table I): the H-HC cell lines had the lowest qp values(<2 pg cell�1 day�1). To explain these results, we proposethat qp needs to exceed a certain threshold for 3LC to beformed. Since 3LC formation should be facilitated byproximity between the THIOMAB antibody and free LCpolypeptide in the ER, 3LC formation would be lessprobable when qp is low. In addition, the extra non-nativedisulfide bond resulting from 3LC formation should bemore readily reduced when qp is low, because more GSHshould be available per antibody (i.e., high qGSH/qp) in theER. This postulated effect of high qGSH/qp in the ER on 3LCreduction is discussed in detail later in this section.

THIOMAB Amino Acid Sequence

To explore the impact of the amino acid sequence on 3LCformation, we analyzed the net hydrophilicity of the LCpolypeptide for each THIOMAB. By summing the water-to-octanol transfer free energy values of the whole residue(DGwoct) for all amino acids in the sequence, we obtainedthe net hydrophilicity value for each LC polypeptide (Whiteand Wimley, 1998). Since the transfer free energy value istypically negative for hydrophobic amino acids and positivefor hydrophilic amino acids, a higher positive value reflectsan enhanced hydrophilicity.

We generated slopes specific to the 11 differentTHIOMABs by graphing the linear portion of %3LC versusmRNA LC/HC ratio for each THIOMAB (Fig. 4A). Whenwe plotted these slopes against the hydrophilicity values foreach THIOMAB LC polypeptide (Fig. 4B), we found a linearrelationship (P¼ 0.0058). The most hydrophilic THIOMAB,D-HC, showed an increase of 12.4 units in %3LC per eachunit increase in mRNA LC/HC ratio, whereas the mosthydrophobic THIOMAB, A-HC, only showed an increase of1.1 units in %3LC per each unit increase in mRNA LC/HCratio. In addition, D-HC had the lowest LC polypeptideisoelectric point (�4 compared with 8–9 for the otherTHIOMABs). These findings indicate that the amino acidsequence in the THIOMAB light chain affects 3LCformation. We speculate that the more hydrophobic LCpolypeptides have a higher propensity to participate inreactions such as aggregation or misfolding, and thereforehave a lower probability of reacting with the engineeredcysteine to form 3LC.

The engineered point mutation to replace an amino acidwith a cysteine residue can be located in the LC or HC aminoacid sequence of the THIOMAB (Junutula et al., 2008b).This difference in the location of the engineered cysteinecould influence 3LC formation. For instance, in addition tothe cysteine that normally forms an interchain disulfidebond to the heavy chain, LC polypeptides from the LC


variant of these IgG1-based THIOMABs also contain anengineered reactive cysteine. Therefore, LC polypeptidesoriginating from the LC variant of THIOMABs have an extracysteine for disulfide linkage to the full-length antibody.However, this parameter may have minimal effect on 3LCformation (Fig. 4A). We found comparable slopes—%3LCversus mRNA LC/HC ratio—for THIOMAB E (E-HCslope¼ 6.8, E-LC slope¼ 6.5) and F (F-HC slope¼ 8.0,F-LC slope¼ 8.7), but a more obvious increase forTHIOMAB I (I-HC slope¼ 6.0, I-LC slope¼ 11.4).

GSH Concentration

Since GSH can reduce disulfide bonds in nascent proteinsin the ER directly or by reducing oxidoreductases(Chakravarthi and Bulleid, 2004; Cuozzo and Kaiser,1999; Jessop and Bulleid, 2004; Molteni et al., 2004), higherlevels of GSH may promote reduction of the non-nativebond between the third LC polypeptide and the antibodymonomer. Additionally, GSH can react with the engineeredcysteines on THIOMABs; liquid chromatography–massspectrometry analyses confirmed that the engineeredcysteine residues formed mixed disulfides with cysteine orGSH (Junutula et al., 2008b). High concentrations of GSHin the ER (�10 mM) may cause glutathionylation of allsurface-exposed protein thiols in the ER (Bass et al., 2004).Therefore, engineered cysteines in THIOMABs may beinitially capped with GSH or another LC polypeptide whilein the ER, and some of these cappings may subsequentlybe exchanged for cysteine cappings after the THIOMABsare secreted.

We speculate that GSH influences 3LC formation byregulating disulfide bond formation or by directly reactingwith the engineered cysteines, or both. To test thishypothesis, we quantified total intracellular and extracellularGSH using an enzyme recycling assay. Since cytoplasmicGSH levels correlated with reduction rates in the ER (Jessopand Bulleid, 2004; Molteni et al., 2004), measurements ofintracellular GSH should represent the reducing activity inthe ER.

To generate the data in Figures 6 and 7, we usedextracellular and intracellular GSH levels at the end ofproduction (day 14). From the extracellular GSH value, wecalculated a cell-specific production rate of GSH (qGSH). Wepropose that qGSH—which could correlate to intracellularGSH levels, and therefore also correlate to GSH levels in theER—is an appropriate estimate of GSH production because%3LC¼ f(GSHintracellular)/ f(GSHextracellular/# cells)� f(qGSH).For convenience, we used a single measurement ofextracellular GSH at day 14 to calculate qGSH at a fixedtime in our analyses of all 66 cell lines.

To corroborate this approach, we performed time-courseanalyses (days 3, 7, 10, and 14) of GSH production onfour model cell lines (A-HC cell line # 1, D-HC cell line# 15, F-HC cell line # 33, I-HC cell line # 64) (Fig. 5).Although all four cell lines showed different extracellular

Figure 4. Effect of THIOMAB amino acid sequence on 3LC levels. A: Slopes (m) of %3LC versus mRNA LC/HC ratio for each individual THIOMAB (using only the linear portions

of the graphs). Slopes were obtained by a linear fit of the data. B: Net hydrophilicity value of the THIOMAB light chain, obtained from the linear amino acid sequence, compared to

the slopes of %3LC versus mRNA LC/HC ratio. A net hydrophilicity value was obtained from the primary sequence of each light chain by summation of theDGwoct values for all amino

acids in the primary sequence.

GSH concentrations on day 14, three of the cell lines hadindistinguishable intracellular GSH concentrations on day14. The cell line with the highest day 14 qGSH value alsoyielded the highest intracellular and extracellular GSHvalues throughout all 14 days of culture, and vice versa.

Therefore, these results support our use of day 14extracellular GSH concentration to generate qGSH valuesfor representing the time-course trend of GSH productionin each cell line. By contrast, day 14 intracellular GSH valueswould not capture the history of GSH production over the



Figure 5. GSH levels as a function of cell culture time. A: Cell growth versus time. B: Cell viability versus time. C–E: Intracellular GSH, extracellular GSH, and qGSH versus time,

respectively. Four model cell lines were analyzed: (^) A-HC # 1; (D) D-HC # 15; (*) F-HC # 33; (&) I-HC # 64. Intracellular and extracellular GSH were measured from cell culture

supernatants and cell pellets, respectively, on days 3, 7, 10, and 14. All values represent averages of three biological replicates (n¼ 3). Error bars correspond to SEM.

culture duration because they only provide a snapshot ofGSH production specific to that limited time window. Theseresults also confirm our earlier speculation that higherintracellular GSH values would lead to higher extracellularGSH values.

In addition to qGSH, we also quantified the cell-specificproduction rate of THIOMAB (qp) because the amount ofTHIOMAB translated and folded in the ER lumen mayimpact the reducing activity of GSH in the ER. Whenthe amount of protein translated in the ER increases,the oxidative equivalents generated by Ero1 shouldbe more rapidly consumed. Since GSH competes for theseoxidative equivalents (Molteni et al., 2004), we speculatethat qp will impact the GSH activity in the ER. Therefore,we investigated the ratio of qGSH to qp (qGSH/qp)—whichtranslates to amount of GSH per unit of THIOMAB—as apotential parameter affecting 3LC formation.

When we plotted %3LC as a function of the ratio betweenqGSH and qp (moles of GSH produced per gram ofTHIOMAB), we obtained a nonlinear relationship between%3LC and qGSH/qp (Fig. 6A). We further analyzed thisrelationship by transforming the abscissa into ln(qGSH/qp),and found a statistically significant linear correlationbetween %3LC and ln(qGSH/qp) (P¼ 0.001) (Fig. 6B). Wedid not find significant correlation (P¼ 0.17) between%3LC and day 14 intracellular GSH concentration (Fig. 6C).

To differentiate the effect of mRNA LC/HC ratio fromGSH production, we plotted %3LC as a function of ln(qGSH/qp) for stratified mRNA LC/HC values (Fig. 7). The trendof decreasing %3LC with increasing values of ln(qGSH/qp)is evident within each mRNA LC/HC ratio range. Inparticular, these trends suggest that the effect of GSH


production was most pronounced for mRNA LC/HC ratiosin the 0.4–1.0 range.

Since GSH formation depends on cysteine availability(Griffith, 1999), we investigated cysteine/cystine consump-tion (Fig. 6D). Although there appeared to be somecorrelation between qcys and qGSH for qcys values higher than4mmol/108 cell day, 86% of the data lay in the lower leftquadrant (<4mmol/108 cell day), where the correlation waspoor (P¼ 0.443). Additionally, multiple linear regressionanalysis revealed no statistically significant correlationbetween qcys and %3LC (Table II). These results indicatethat cysteine/cystine availability was not critical for theformation of GSH or 3LC, or both. Alternatively,these results suggest that cysteine/cystine levels were notlow enough to cause an impact on 3LC formation.

Effect of Other Cell Culture Parameters

We also investigated the impact of five additional cell cultureparameters—titer, qp, IVCC, % cell viability, and pH—on3LC formation (Table II). We analyzed these parametersboth individually (univariate regression) and also simulta-neously, with all other parameters as part of a multiple linearregression (multivariate least square regression). In addi-tion, we performed a stepwise regression and found thatmRNA LC/HC ratio, ln(qGSH/qp), and final pH had thelargest impact on 3LC formation, although the effect offinal pH was not statistically significant (P¼ 0.08). Thestatistical analysis showed no significant impact on %3LCfrom any of the additional parameters as depicted by theP values, and confirmed the impact of both mRNA LC/HC

Figure 6. Effect of GSH on 3LC levels. A–C:: %3LC as a function of qGSH/qp,

ln(qGSH/qp), and intracellular GSH, respectively. qGSH was calculated from the ratio of

extracellular GSH to IVCC on day 14. Extracellular and intracellular GSH were

calculated from cell culture supernatant and cell pellets on day 14, respectively.

D: qGSH versus qcys. qcys was calculated from the ratio of (cys in medium� cys on day

14) divided by IVCC on day 14. GSH measurements were done in a representative

sample of three biological triplicates. For all other measurements, data points

represent an average of three biological replicates (n¼ 3). SEMs ranged from 2%

to 12%, but are not shown here to improve clarity for symbols.

ratio and ln (qGSH/qp) (P< 0.0001 and P¼ 0.0002,respectively). Bioreactor experiments exploring the effectsand interactions of other cell culture parameters such astemperature, pH, dissolved oxygen, and medium composi-tions are in progress using bioreactors and the results will bereported elsewhere. The multivariate step-wise correlationgenerated the following linear model to predict %3LC valuesas a function of mRNA LC/HC ratio and ln(qGSH/qp). Thissimplified model partially explains the variability in %3LC;it does not take into account parameters beyond what weexplored in this work

%3LC ¼ �18þ 3:56 mRNALC

HC

� �� 1:42 ln

qGSH

qp

� �

ðR2 ¼ 0:57;P < 0:0001Þ

Time-Lapse Studies

To investigate time-dependent changes in 3LC formation,we analyzed the profiles of %3LC, mRNA LC/HC ratio, andqGSH/qp ratio over time in four different cell lines (Fig. 8).We selected these cell lines to span a %3LC range from 1% to18% at the time of harvest.

The %3LC time profiles differed amongst the cell linesinvestigated (Fig. 8A). The mRNA LC/HC ratio and qGSH/qp

ratio also changed over culture time (Fig. 8B and C). Thesetrends were unique to each cell line and generally consistentwith the changes in 3LC values: higher mRNA LC/HC ratiosand lower qGSH/qp ratios increased %3LC, and vice versa.Cell line I-HC # 64 exhibited a constant mRNA LC/HC ratioof 0.6 with a decrease in qGSH/qp ratio from days 3 to 7.Overall, this cell line showed the lowest mRNA LC/HC ratioand the highest qGSH/qp ratio, and these factors shouldaccount for its 3LC profile: cell line I-HC # 64 showed thelowest overall %3LC amongst the four cell lines analyzed.

Conclusion

3LC is an impurity unique to THIOMABs. Our analyticalcharacterization of this impurity indicates that it is formedby a non-native disulfide bond between an LC polypeptideand a full-length THIOMAB. In our studies covering 12IgG1-based THIOMABs produced by 66 stably transfectedcell lines, 3LC formation correlated to the mRNA LC/HCratio and the ln(qGSH/qp) value. While 3LC formationseemed to depend primarily on free LC polypeptideconcentration, increased hydrophilicity of the LC polypep-tide also enhanced 3LC formation. In addition, GSH inthe ER may modulate 3LC formation since higher GSHconcentrations could promote reduction of the extradisulfide bond. Time-course studies showed differences in3LC profiles for different cell lines. These 3LC trends weregenerally analogous to the changes in mRNA LC/HC ratioand qGSH/qp ratio over time. In summary, our results suggest



Table II. Results of least-square linear regressions for %3LC as a function of cell culture parameters.

VariableaUnivariate linear regressionb

(P-values)

Multivariate linear regressionc

(P-values)

Stepwise linear regressiond

(P-values)

Final titer (mg/L) 0.046� 0.475

qp (mg 10�8 cell�1 day�1) 0.194 0.150

Final cell viability (%) 0.732 0.190

IVCC (108 cell day L�1) 0.843 0.816

Final pH 0.587 0.147 0.080

qcys (mmol 10�8 cell�1 day�1) 0.801 0.215

mRNA LC/HC <0.0001� <0.0001� <0.0001�

ln(qGSH/qp) 0.001� 0.0006� 0.0002�

aCell culture parameters evaluated from shake flask cultures for 66 stably transfected cell lines.bRegression for %3LC as a function of each individual variable.cRegression for %3LC as a function of all eight variables.dRegression using a step-wise algorithm.�Statistically significant parameter.

Figure 7. Effect of GSH on 3LC levels according to mRNA LC/HC ratio. A–F: %3LC versus ln(qGSH/qp) for a mRNA LC/HC ratio of 0.2–0.4, 0.4–0.6, 0.6–0.8, 0.8–1.3, and 1.3–2.5,

respectively. Data correspond to Figure 6B but are grouped according to the mRNA LC/HC ratio for each cell line.


Figure 8. %3LC as a function of cell culture time. A–C:: %3LC, mRNA LC/HC ratio, and qGSH/qp versus time, respectively. Four model cell lines were analyzed: (^) A-HC # 1; (D)

D-HC # 15; (*) F-HC # 33; (&) I-HC # 64. 3LC, mRNA LC/HC ratio, and qGSH/qp were measured from cell culture supernatants or cell pellets on days 3, 7, 10, and 14. All values

represent averages of three biological replicates (n¼ 3). Error bars correspond to SEM.

that cell lines with low mRNA LC/HC ratio and high qGSH/qp

ratio would produce THIOMABs with lower levels of 3LC.

Nomenclature

3LC
triple light chain antibody
ADC
antibody drug conjugate
CHO
Chinese hamster ovary
Cys
cysteine/cystine
ER
endoplasmic reticulum
Ero1
endoplasmic reticulum oxidoreductin 1
GSH
glutathione
HC
heavy chain
HPLC
high-performance liquid chromatography
IVCC
integrated viable cell concentration
LC
light chain
Lþ LL
light chain monomer polypeptideþ light chain dimer
polypeptide

mRNA
messenger ribonucleic acid
PDI
protein disulfide isomerase
qcys
cell-specific cys production rate
qGSH
cell-specific GSH production rate
qp
cell-specific productivity
SEM
standard error of the mean
t
time
TDC
THIOMAB drug conjugate
THIOMAB
engineered monoclonal antibody with additional cysteines
RT-PCR
reverse transcriptase-polymerase chain reaction
VCC
viable cell concentration
The authors thank the cell line development groups for all the stable

cell lines evaluated; analytical operations for performing assays for

titer, amino acid analysis, protein A purification and free LC; media

preparation group for all the media and solutions used during cell

culture; cell banking group for supplying ampules for all the cell lines

evaluated; Genentech oligo synthesis; Christina Lee for support with

RT-PCR; Kate Moiseff, Adrian Nava, Bryan Mclaughlin, and James

Giulianotti for support with Western blots; Tom Stapp for discussion

on GSH detection; Dan Coleman for support with statistics; John Joly

for data discussion and support.

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triple light chain antibodies: factors that influence its formation in cell culture

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