DMD #58347 1 Title Page Biotransformation and In Vivo Stability of

DMD #58347

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Title Page

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate

Selection and Pharmacokinetic Profiling

Michael P. Hall

Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., Thousand Oaks, CA

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on June 19, 2014 as DOI: 10.1124/dmd.114.058347

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Running Title Page

Running Title: Biotransformation and In Vivo Stability of Biotherapeutics

Address correspondence to: Michael Hall, Department of Pharmacokinetics & Drug

Metabolism, Amgen Inc., One Amgen Center Drive, MS 30E-3-C, Thousand Oaks, CA 91320.

Email: [email protected].


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Number of Text Pages: 25

Tables: 0

Figures: 1

References: 78

Abstract: 248 words

Introduction: 377 words

Discussion: 5384 words

Conclusion and Future of Field: 510 words


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Abbreviations: ADME, absorption, distribution, metabolism, and excretion; Ab, antibody;

ADC, antibody-drug conjugate; DPP-IV, dipeptidyl protease IV; DAR, drug-to-antibody ratio;

DAPD, dual-acting peptide for diabetes; ELISA, enzyme-linked immunosorbent assay; FGF21,

fibroblast growth factor 21; Fc, fragment crystallizable region; GLP-1, glucagon-like peptide-1;

GIP, glucose-dependent insulinotropic polypeptide; hc, heavy chain; hPTH, human parathyroid

hormone; IgG, immunoglobin G; ICH, International Conference on Harmonization; ip,

intraperitoneal; iv, intravenous; kDa, kilodaltons; LBA, ligand-binding assay; lc, light chain; LC,

liquid chromatography; MMAE, maleimide-monomethyl auristatin E; MS, mass spectrometry;

MALDI, matrix-assisted laser desorption ionization; MW, molecular weight; mAb, monoclonal

antibody; FcRn, neonatal Fc receptor; NDA, New Drug Application; PEG, polyethylene glycol;

PD, pharmacodynamic; PK, pharmacokinetic; SM, small molecule; sc, subcutaneous; SELDI,

surface-enhanced laser desorption ionization; MS/MS, tandem mass spectrometry; TMP,

thrombopoietin mimetic peptide; TOF, time of flight; FDA, US Food and Drug Administration


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Abstract

Historically, since the metabolism of administered peptide/protein drugs (‘biotherapeutics’) has

been expected to undergo predictable pathways similar to endogenous proteins, comprehensive

biotherapeutic metabolism studies have not been widely reported in the literature. However,

since biotherapeutics have rapidly evolved into an impressive array of eclectic modalities, there

has been a shift towards understanding the impact of metabolism on biotherapeutic development.

For biotherapeutics containing non-native chemical linkers and other moieties besides natural

amino acids, metabolism studies are critical as these moieties may impart undesired toxicology.

For biotherapeutics that are comprised solely of natural amino acids, where end-stage peptide

and amino acid catabolites do not generally pose toxicity concerns, the understanding of

biotherapeutic biotransformation, defined as in vivo modifications such as peripherally generated

intermediate circulating catabolites prior to end-stage degradation or elimination, may impact in

vivo stability and potency/clearance. As of yet, there are no harmonized methodologies for

understanding biotherapeutic biotransformation and its impact on drug development, nor is there

clear guidance from regulatory agencies on how and when these studies should be conducted.

This review provides an update on biotherapeutic biotransformation studies and an overview of

lessons-learned, tools that have been developed, and suggestions of approaches to address issues.

Biotherapeutic biotransformation studies, especially for certain modalities, should be

implemented at an early stage of development to: 1) understand the impact on potency/clearance,

2) select the most stable candidates or direct protein re-engineering efforts, and 3) select best

bioanalytical technique(s) for proper drug quantification and subsequent pharmacokinetic

profiling and exposure/response assessment.


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Introduction

Peptide and protein drugs (‘biotherapeutics’) have become increasingly important modalities for

the treatment of grievous diseases. In 2010, there were 200+ biotherapeutics approved for use

(Walsh, 2010). The types, or modalities, of these biotherapeutics are quite varied. They include

hormones, growth factors and other replacement peptides/proteins; monoclonal antibodies

(mAb); subunit vaccines; fusion proteins; and therapeutic enzymes (Walsh, 2010). Moreover, a

recent report showed that annual revenue for biotherapeutics had grown steadily during the

previous 10 years, and in 2011, biotherapeutics accounted for 15.6% of the total global

pharmaceutical market, which was valued at $138 billion globally. This valuation is expected to

increase to over $320 billion by the year 2020 (GBI Research, 2012).

Understanding the biological absorption, distribution, metabolism, and excretion (ADME)

properties of small molecules (SM) is a critical element in SM drug discovery and development,

spanning early discovery to late development (Baillie, 2008). In contrast, the understanding and

mechanistic details of the ADME properties of biotherapeutics are not as well developed

although there has been recent interest in closing this gap (Prueksaritanont and Tang, 2012). One

reason for the disparity has been attributed to the relatively young age of the era of

biotherapeutics compared to that of SM (Waldmann, 2003). At any rate, as new ADME

characterizations of biotherapeutics become available in the literature, it behooves the scientific

community to assess the state of the science in toto. This review focuses on the current

understanding of metabolism properties of biotherapeutics vs. SM drugs and the concomitant

impact on biotherapeutic discovery and development.


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In order to avoid potential confusion resulting from the common association of the term

“metabolism” with SM drugs or with end-stage protein lysosomal degradation, the term

“biotransformation” will be used to describe the physical alteration of a biotherapeutic due to

peripheral intermediate catabolism and truncation, not including inherent chemical stability.

Other biotransformation events, such as deamidation, oxidation or other amino acid

modifications, will be mentioned briefly but are generally beyond the scope of this review. The

main objectives of this article are to review work that has been done and tools that have been

developed to examine protein (MW > 5 kDa) biotherapeutic biotransformation, clarify the

impact on candidate selection and bioanalysis, and suggest approaches for assessing

biotransformation based upon protein therapeutic modality.


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Metabolism: Small Molecule Drugs vs. Biotherapeutics

Unlike traditional small molecule drugs, biotherapeutics generally exhibit poor oral

bioavailability; therefore, they are dosed in a parenteral manner, usually by intravenous (iv) or

subcutaneous (sc) injection. During the absorption and distribution phases, biotherapeutics

undergo metabolism and clearance through mechanisms that differ substantially from SM drugs.

Metabolism for SM drugs is defined as essential biochemical modifications, which occur

predominantly in the liver, that often render the drugs more susceptible to elimination (for

example, by increasing hydrophilicity). Although detoxification and enhanced clearance are the

predominant outcomes of SM drug metabolism, there is the potential for increased toxification

after metabolism and undesired accumulation of toxic metabolites in tissues or organs. As a

result of this potential for untoward effects, the understanding of metabolism is an absolute

requirement for moving a small molecule candidate forward through the development pipeline.

The analytical tools for understanding small molecule drug metabolism have been well-

established and validated by the pharmaceutical industry. Regulatory agencies also have very

specific guidelines with respect to requirements for characterization of small molecule drug

metabolism that have to be addressed in any New Drug Application (NDA) filing.

In contrast to metabolism of small molecules, metabolism of biotherapeutics is predominantly

defined as the enzymatic hydrolysis (catabolism) of polypeptides to produce smaller peptides and

amino acids for deactivation and increased clearance. Unlike small molecule drugs, the final

small peptide and amino acid breakdown products of all biotherapeutics are produced

predominantly by lysosomal degradation after active or passive cellular uptake and thus are

expected to be similar. Since these end products are not likely to pose clinical toxicity concerns,


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in-depth studies of biotherapeutic metabolism have not been abundant and analytical tools for

probing biotherapeutic metabolism have not been systematically adopted. In addition, guidance

from regulatory agencies with respect to biotherapeutic metabolism is minimal and reflects a

certain indifference. As noted by Hill: “The ICH S6 (R1) guidance [from 1998] suggests that the

consequential metabolism of proteins and peptides is via expected routes and implies only a little

more investigation may be needed…” (Hill, 2010). Newer guidance from the European

Medicines Agency (2007) states that: “metabolites that have pharmacodynamic activity should

preferably be measured, for example, through chromatographic separation, collection and further

in vivo bioassay quantification. However, in cases where measurement of separate active

metabolites or peptide fragments is not technically feasible, pharmacokinetics of the active

moiety could be determined.” This newer guidance implies selective metabolism studies may be

in order, especially in cases where amino acid sequences have been altered. Safety concerns can

arise with these types of biotherapeutics; for example, hybrid biotherapeutics where protein

scaffolds contain unnatural or D-amino acids, polyethylene glycol (PEG), or small molecule

moieties such as those found in antibody-drug conjugates (ADCs) (Hamuro and Kishnani, 2012).

Even with biotherapeutics that contain only natural amino acid sequences, the need for

metabolism studies has to be presently assessed in light of the rapid production of widely varied

protein therapeutic modalities in recent years, ranging from small peptides to large multispecific

chimeric proteins. The catabolic breakdown of biotherapeutics may involve a series of

kinetically disparate proteolytic events as illustrated in Figure 1. In this example, an antibody

(Ab)/peptide fusion construct is administered and during the absorption and distribution phases,

a large intermediate catabolite forms rapidly (kcatabolism) whereby the fused peptides are

proteolytically truncated. This catabolite may be sustained in circulation for a significant amount


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of time after dosing before ultimate lysosomal degradation and/or renal excretion (kelimination)

(Hall et al., 2010; Hager et al., 2013). This peripheral, non-lysosomal catabolism of

biotherapeutics is referred to as ‘biotransformation’ and is systemic, but the major sites of

enzymatic breakdown are blood, liver, and kidney (Werle and Bernkop-Schnürch, 2006; Lin,

2009). Proteases with exo- or endopeptidase activity have been identified and characterized in

these tissues/organs (Werle and Bernkop-Schnürch, 2006). Although clinical safety may not be

generally affected, the potential impact of circulating catabolites on potency and clearance may

require consideration or assessment. Knowledge of specific sites of proteolytic cleavage is

critical for engineering more stable biotherapeutic candidates by mutation of susceptible residues

or application of other types of stabilization. In addition, the traditional bioanalytical tools for

quantification of biotherapeutics and determining pharmacokinetic (PK) exposure such as ligand-

binding assays (LBA) may not take catabolism into account, and the resulting concentration

measures may therefore either underestimate or overestimate true exposure. The impact on

bioanalysis is fully addressed in the next section.


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Impact of Protein Biotherapeutic Biotransformation on Bioanalysis

LBA is the standard method by which biotherapeutics, especially larger protein biotherapeutics,

are quantified in preclinical and clinical samples for PK and pharmacodynamic (PD) profiling.

Non-competitive “sandwich” enzyme-linked immunosorbent assay (ELISA) is the standard LBA

for bioanalysis. In this assay format, capture and detection reagents track distinct epitopes of the

analyte of interest. Often, the capture and detection reagents are chosen based upon assay

optimization using only the purified, intact biotherapeutic analyte spiked into serum or plasma.

For this reason, biotransformation has generally not been considered during LBA development.

For the purposes of correct PK/PD profiling, the optimal LBA will track only those entities

(parent and bioactive catabolites) that contribute to true exposure. For instance, glucagon-like

peptide-1 (GLP-1) is a 37 amino acid peptide that has been investigated for treatment of type II

diabetes. In order to increase its half-life and bioavailability, it has been conjugated to the N-

terminus of a carrier mAb (Murphy et al., 2010). After iv administration of this construct to

mice, quantification of the therapeutic was achieved by two different LBA formats that both

utilized anti-idiotypic capture of the Ab portion of the construct; however, two different

detection reagents were utilized: 1) anti-human immunoglobulin G (IgG), for detection of the

mAb only (“total” assay), or 2) anti-GLP-1 (N-terminal specificity), for detection of intact GLP-

1 (“N-terminal” assay). The N-terminal assay produced drug concentration results that were

significantly lower than the total assay throughout the PK time course. These results indicated

that while the concentration of the mAb portion of the construct was sustained, the N-terminal

region of the GLP-1 portion was quickly degraded. More importantly, the pharmacological

action of GLP-1 is contingent upon having an intact N-terminus (Deacon, 2004). Therefore, if


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the total assay were to be used for PK profiling, it would grossly overestimate the true exposure,

as the reported concentration of the biotherapeutic is predominantly derived by inactive

catabolites that contain degraded GLP-1. Thus, the N-terminal assay would be the appropriate

LBA for PK/PD profiling. Additionally, since the discrepancy between the total and N-terminal

LBAs was significant, subsequent mass spectrometry (MS) studies were undertaken to determine

the exact vulnerable proteolytic loci of the GLP-1. This information was used to stabilize the

GLP-1 N-terminal region with the goal that the results of the total and N-terminal assays would

ultimately approach convergence. The indispensable utility of MS for analysis of biotherapeutic

biotransformation will be addressed in the next section of this review.

This GLP-1/mAb fusion example demonstrates the possibility for LBA to significantly

overestimate exposure if biotransformation is not considered. Alternatively, the following

example demonstrates how exposure can be underestimated. Romiplostim is a novel protein

biotherapeutic used for the treatment of idiopathic thrombocytopenia, consisting of tandem

repeats of thrombopoietin mimetic peptides (TMPs) fused to the C-terminus of the fragment

crystallizable region (Fc) of human IgG1 (Molineux and Newland, 2010). In the intact construct,

there are a total of 4 TMPs. Two LBA formats were developed to quantify romiplostim in

plasma samples obtained from preclinical in vivo experiments (Hall et al., 2010). For capturing

romiplostim, both assays utilized a rabbit polyclonal reagent raised against TMP. In the first

assay (bridging assay), detection of the captured romiplostim was achieved by using the same

polyclonal reagent. In the second assay (TMP/Fc assay), the detection reagent was an anti-human

Fc mAb. In an in vivo rat PK study, there was a large discrepancy between the two assays with

the bridging assay producing markedly lower drug concentration values than the TMP/Fc assay.

The TMP/Fc assay values were similar to radioassay results where 125I was incorporated into the


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Fc region of romiplostim; thus, this LBA appeared to track all Fc-containing catabolites and

could be considered a total assay with the caveat that at least one TMP was present in the

construct. Exploration of what the bridging assay measured revealed that the entire construct had

to be essentially intact in order to produce any measurable assay signal; a romiplostim analog

containing only one TMP on each monomer of Fc failed to produce a signal in this assay. Further

mass spectrometric studies showed extensive catabolism of the terminal TMPs with the internal

TMPs remaining largely intact after 24 h post dose. Although relative bioactivities were not

available, it could be reasonably hypothesized that all catabolites of romiplostim that contained

at least one viable TMP would be bioactive and should be monitored for accurate PK profiling.

Thus, in this case, the TMP/Fc assay would be most appropriate for reporting romiplostim

concentrations as it reflected both the intact construct plus its bioactive metabolites. Using the

bridging assay was too specific, providing concentration information for intact romiplostim

alone, thereby leading to underestimation of true exposures.

As was the case with the GLP-1/mAb construct, MS studies were crucial to assess: 1) the

exact molecular details of romiplostim biotransformation, 2) which catabolites any given LBA

was detecting, and 3) the most appropriate LBA for PK assessment. In addition, the extensive

biotransformation of romiplostim led to the design of a newly stabilized construct where the

TMPs were inserted between the CH2 and CH3 loops of Fc, thus rendering the TMPs less

susceptible to catabolism (Hall et al., 2010).

To circumvent these issues of the effects of biotransformation on LBA development and

specificity, one natural inclination is to bypass LBA entirely and move towards use of

quantitative MS assays where specificity concerns are ameliorated. Biotherapeutics or associated

surrogate peptides as well as any important catabolites could be specifically quantified by MS. In


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fact, several researchers have published studies with this ultimate goal in mind (Dubois et al.,

2007; Dubois et al. 2008; Heudi et al., 2008; Ezan et al., 2009; Liu H et al., 2011; Bronsema et

al., 2012; Li et al., 2012). Historically, there have been several drawbacks associated with

quantitative MS methods compared to LBA, including: 1) MS has been generally less sensitive;

2) large protein analytes have suffered from low throughput; and 3) quantitative MS methods for

macromolecules are much more nascent compared to established antibody-based methodologies.

Presently, however, detection sensitivity has improved to nearly match that of LBA due to

advances in MS instrumentation and work flows such as the utilization of nanoflow liquid

chromatography (LC) coupled to nanospray MS (Duan et al., 2012) and use of pre-enrichment

techniques such as affinity-MS, and throughput has increased substantially by the

implementation of automation. We are at an exciting juncture where PK profiling achieved by

quantitative MS is becoming more commonplace and may be eventually used with similar

frequency as LBA. Until then, LBA remains the gold standard for biotherapeutic bioanalysis, and

as such, critical information about biotransformation, whether it is obtained from MS or other

techniques, can be used to develop the correct LBA for the most accurate PK profiling.


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Workflows for Studying Protein Biotherapeutic Biotransformation

*Mass Spectrometry (MS)

MS is becoming the analytical tool of choice for assessing biotransformation of protein

biotherapeutics. Although differential LBA results, as demonstrated in the examples in the

previous section, offer an indication that gross metabolism is occurring by utilizing multiple

formats that assess different regions of the analyte (e.g., total vs. intact), LBA results can only

serve as an important yet inexact screen for probing biotransformation. Moreover, these results

cannot easily provide exact molecular details about the specific locations of proteolytically

sensitive residues, or “hotspots.” Due to the high mass resolution of current mass spectrometers,

however, this molecular understanding is readily obtainable; the observance of the catabolic loss

of just one amino acid is easily achieved. In addition, other biotransformation events such as

oxidation of vulnerable amino acids such as methionine and tryptophan, can be easily tracked by

high-resolution MS; these events would be nearly impossible to probe by LBA.

*Sample Generation and Collection

Biotransformation studies require the generation of appropriate samples for analysis. Since in

vitro test systems have been successfully implemented for the analysis of the metabolism of SM

drugs, researchers have also attempted to use in vitro test systems to understand biotherapeutic

biotransformation. For example, studies have been conducted in whole blood/serum/plasma, and

liver and kidney homogenates (Boulanger et al., 1992; Powell et al., 1992; Fredholt et al., 2000;

Sofianos et al., 2008). A couple of cautionary issues for using in vitro test systems need to be

addressed. First, the level of catabolism, especially for tissue homogenates, is generally much


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greater than that observed in in vivo models (Werle and Bernkop-Schnürch, 2006). Second, the

in vitro catabolic profile may not adequately describe that which actually occurs in vivo. An

example of this is given by the study of catabolism of stromal-derived factor-1, CXCL12, an 8

kDa chemokine (Antonsson et al., 2010). CXCL12 catabolism was studied by both in vitro and

in vivo mouse models. In vitro, truncation of the N-terminus of CXCL12 up to five amino acids

was observed; however, the protein was further truncated in vivo by two additional amino acids

(Antonsson et al., 2010). Additionally, when a methionine residue was added to the N-terminus,

it completely protected the N-terminus from degradation in vitro but not in vivo (Antonsson et

al., 2010). Therefore, although in vitro test systems are attractive due to ready availability and

low cost, at present, in vivo studies produce the most reliably accurate descriptions of

physiologically relevant biotherapeutic biotransformation. Furthermore, since in vivo studies are

preferable, analysis of biotherapeutic biotransformation and in vivo stability has been nearly

exclusively monitored in collected blood/plasma/serum.

To begin an in vivo biotransformation study, an animal species has to be selected. Preclinical

studies of catabolism are dominant in the literature, demonstrating that issues of

biotransformation should be addressed early in a research program so that potential candidates

can be stabilized by re-engineering if liabilities are found. One could theorize that

biotransformation studies in nonhuman primates are most likely to be extrapolatable to humans

although there is little to no literature to support this. One study reports that biotherapeutic

catabolism profiles between rodent and monkey studies were identical except for the temporal

appearance of any given catabolite (Hager et al., 2013). This is an important linkage given the

higher expense of conducting monkey studies. Additional studies are needed to examine cross-


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species differences in biotherapeutic biotransformation, and more importantly, the correlation of

preclinical to clinical data.

Once the animal species has been chosen, the biotherapeutic is administered by the desired

mode of introduction, usually iv, sc, or intraperitoneally (ip). There are no strict guidelines as to

the dosing level as there have been no studies to date showing any effect of dose on catabolic

profiles; however, the dose should be high enough to track catabolism for the desired period of

time post-dosing. Furthermore, due to the generally lower sensitivity of MS methods compared

to LBA, a dosing level higher than that used for LBA is usually implemented. The route of

administration may potentially impact the resultant catabolic profile. The appearance of

catabolites may be delayed following sc vs. iv dosing, but the identities of the catabolites

generated may be independent of the route of administration. After sc administration, it is

generally believed that the lymphatic system is the primary route of absorption for protein

biotherapeutics with molecular weights >16 kDa (McLennan et al., 2005). At the injection site

and during lymphatic transport, it can be hypothesized that a biotherapeutic may be exposed to

unique proteases that could result in biotransformation prior to systemic exposure. Although

there are reports that biotherapeutics are stable after in vitro incubation in lymph (Charnan et al.,

2000; Wang et al., 2012), catabolic degradation of biotherapeutics has been observed in

subcutaneous skin tissue homogenate and lymph node suspensions (Wang et al., 2012). It is not

known, however, if this catabolic activity results in complete destruction of the administered

biotherapeutic or can produce larger catabolic fragments that appear in systemic circulation.

More studies are clearly needed to assess the impact of route of administration upon

biotherapeutic biotransformation.


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Blood collection from dosed animals needs to be carefully considered in order to maintain

biotherapeutic integrity. The guidelines for sample collection for maintaining integrity of protein

biomarkers and plasma proteomes can serve as a useful template for biotherapeutic

biotransformation studies (Omenn et al., 2005; Rai et al., 2005; Rai and Vitzthum, 2006;

Tammen, 2008). Any catabolites discovered during a study need to be attributed to

biotransformation and not to artifactual proteolysis occurring during sample collection.

Collection of plasma is generally preferential to that of serum to avoid any degradation due to

activated proteolysis that could occur during blood clotting. In addition, ex vivo stability should

be monitored by adding the analyte of interest to control plasma/serum and carrying this sample

through the same ensuing processing and analysis steps as the actual in vivo samples.

Furthermore, collection of very early time points (<5 min) can serve as a positive control where

in vivo catabolism is expected to be minimal; the presence of proteolyzed analyte in this case

would suggest artifactual ex vivo degradation. Although not routine, the addition of protease

inhibitors during collection could also inhibit any ex vivo proteolysis.

*Sample Preparation

Once plasma/serum samples have been collected properly, the next step is preparing the samples

for MS analysis. Plasma/serum is a very complex proteinaceous mixture with endogenous

proteins that span an enormous dynamic range of concentrations – at least 9 orders of magnitude

(Adkins et al., 2002). Therefore, direct analysis of biotherapeutic catabolites from plasma/serum

directly is nearly impossible. Extraction methods are thus needed to remove endogenous proteins

and concentrate the biotherapeutic and cognate catabolites of interest. Methods include protein

precipitation, solid-phase extraction, and affinity purification (Ji et al., 2003; Dai et al., 2005;


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Ackermann and Berna, 2007; Heudi et al., 2008; Ezan et al., 2009; Lu et al., 2009; Liu H et al.,

2011; Li et al., 2012). The first two methods are more generally applied to peptides and low

molecular weight proteins and will not be addressed in detail here. Affinity purification,

however, provides a highly selective way in which to enrich a protein biotherapeutic and its

associated catabolites. Selection of an appropriate affinity matrix depends upon the

biotherapeutic of interest. Polyclonal Abs against the therapeutic can serve as an appropriate

matrix with multi-epitope capture. In addition, polyclonal Abs are much easier and less

expensive to generate than monoclonal Ab reagents. Alternatively, if human Ab or Ab fragments

(e.g., Fc) are part of the biotherapeutic, these can serve as useful catabolically stable “handles”

for enrichment. Commercially available Protein A or Abs specific for human Ab or Ab

fragments can be used in these cases.

Besides enrichment, other sample preparation steps may be appropriate. MS analysis can be

greatly helped by reducing the molecular complexity and size of the analyte of interest. For

dimeric or higher-order structure biotherapeutics that are held together by disulfide bonds,

reduction and alkylation can be beneficial either before or after enrichment. If N-linked

glycosylation is present, treatment with glycosidases such as PNGaseF can greatly help to reduce

the complexity. In other cases, enzymatic digestion with site-specific proteases such as trypsin,

chymotrypsin, Lys-C, and Asp-N can release smaller polypeptide fragments where MS analysis

is greatly facilitated. For example, the N-terminal catabolism of glucose-dependent

insulinotropic polypeptide (GIP) was monitored by tracking the smaller N-terminal tryptic

peptide as opposed to the intact molecule (a difference of 26 amino acids), resulting in a

sensitivity improvement of 250-fold (Siskos et al., 2009).


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In many cases, enrichment is sufficient for direct analysis by MS. There are cases, however,

where orthogonal separations such as LC may be necessary for additional purification.

Generally, reverse-phase LC is the method of choice for peptide/protein separation. The

chromatography can be done offline, or in-line with the mass spectrometer.

*Choice of mass spectrometer

Protein biotherapeutic catabolites can be investigated by a number of different types of ion

sources [e.g., electrospray ionization, matrix-assisted laser desorption ionization (MALDI),

surface-enhanced laser desorption ionization (SELDI)] and mass analyzers [e.g., quadrupole,

time of flight (TOF), ion trap/orbitrap]. Since the parameters for mass spectrometer choice are

essentially the same as for general proteomic studies and protein analysis, the details will not be

described here as there are already excellent review articles covering this topic (Mann et al.,

2001; Ens and Standing, 2005; Domon and Aebersold, 2006; Ahmed 2008; Tipton et al., 2011).

Catabolites and other biotransformed entities can be confirmed by mass differences in the

resultant mass spectra and corroborated by gas-phase fragmentation data if necessary.


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Criticality of Biotransformation Studies based on Biotherapeutic Modality

*Cytokines, growth factors, and replacement proteins

This broad therapeutic class is characterized by biotherapeutics that have a large range of

molecular weights and have endogenous counterparts. Marketed examples include epoetin alfa

for renal failure, filgrastim for neutropenia, and interferon-β for multiple sclerosis. The

circulatory half-lives can vary widely which can be the result of a combination of both catabolic

deactivation and/or inherent clearance via other pathways. For example, if a biotherapeutic has a

very short half-life mainly due to rapid clearance of the intact molecule by the kidney or by

tissue-mediated drug disposition, then extensive studies regarding possible circulating catabolites

may not be necessary. However, it may be difficult to know this a priori, and thus it is

challenging to suggest general guidance for the need of biotransformation studies. Several

enlightening examples of biotransformation studies in this biotherapeutic class do however merit

discussion.

RANTES/CCL5 is chemokine that plays a role in leukocyte trafficking and homing (Schall et

al., 1990). A 68 amino acid mutant of RANTES/CCL5, [44AANA47]-RANTES, has been shown

to inhibit the recruitment of native RANTES and reduce the severity of a murine model of

multiple sclerosis (Johnson et al., 2004). Chemokines have been shown to be susceptible to

deactivation by proteases in vivo; therefore, studies were performed to analyze the catabolism of

[44AANA47]-RANTES (Favre-Kontula et al., 2006). Using immobilized anti-RANTES

polyclonal Abs for enrichment followed by interrogation by SELDI TOF-MS, it was shown that

[44AANA47]-RANTES quickly formed two major catabolites, the 3-68 and 4-68 forms where the

first 2 or 3 N-terminal amino acids were lost, respectively. These catabolites are important in that


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loss of the initial N-terminal residues can significantly alter the biology of this chemokine

(Proost et al., 1998). Using the SELDI TOF-MS results, a quantitative MS approach that would

be able to easily track the parent and catabolites was developed. An alternative approach would

have been to use LBA with reagents that have specificity for the N-terminus of the molecule.

Native human parathyroid, hPTH (1-84), has a very complex endogenous variant and

catabolic pool with substantial amounts of both N-terminal and C-terminal truncated species

(D’amour and Brossard, 2005; Lopez et al., 2010). HPTH (1-84) and a truncated variant hPTH

(1-34) both have shown to induce bone formation, and hPTH (1-34) has been approved for the

treatment of osteoporosis (Neer et al., 2001; Quattrocchi and Kourlas, 2004). One reason for the

prevalent use of the shorter analog may be the avoidance of the catabolic production of C-

terminal fragments of PTH, which have been shown to antagonize the bone growth effects of

hPTH (1-34) (D’amour and Brossard, 2005). Furthermore, C-terminal fragments of hPTH (1-84)

can accumulate in renal failure patients causing PTH resistance and may potentially cause other

bone disease (D’amour and Brossard, 2005). With respect to hPTH (1-34), it has been shown that

this short PTH analog is catabolized readily in vitro using rat kidney, liver, and lung

homogenates, but these results have not been confirmed in vivo (Liao et al., 2010). Interestingly,

no formal in vivo catabolism studies of hPTH (1-34) have been reported. In fact, an LC-tandem

MS (MS/MS) method for quantification of this analyte has recently been developed even though

the authors assert that there has been no indication of catabolites of hPTH (1-34) that could

interfere with the already developed LBA methods (MacNeill et al., 2013).

*Monoclonal Antibodies (mAb)


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More than thirty mAbs, including denosumab for the treatment of osteoporosis and bevacizumab

for the treatment of various cancers, have been approved as drugs by the FDA (Riechert, 2012).

Of all of the biotherapeutic modalities, mAbs arguably have the richest wealth of knowledge

concerning their PK and in vivo disposition properties (Lobo et al., 2004; Tabrizi et al., 2006;

Kuang et al., 2010; Deng et al., 2012). This class of biotherapeutic is composed of human or

humanized IgG molecules. An IgG molecule consists of two identical light chains ( lc, ~25 kDa)

and two identical heavy chains (hc, ~50 kDa). Each lc is linked to a hc by a disulfide bond, and

the two hc are covalently linked to each other by two or more disulfide bonds. Human IgG hc

has four subclasses (IgG1-1gG4) although most biotherapeutic mAbs are of the IgG1 or IgG2

subclass. The human IgG lc has two subclasses (κ and λ).

MAbs are attractive biotherapeutics due to their intrinsically long circulatory half-lives. The

long half-life is predominantly dictated by neonatal Fc receptor (FcRn) recycling, which also

protects the molecule from lysosomal catabolism (Roopenian and Akilesh, 2007; Suzuki et al.,

2010). Due to this protection as well as the inherent stability of the molecule, intermediate

catabolism and the presence of mAb fragments is not expected. Therefore, for mAbs, extensive

biotransformation studies are not generally warranted. Indeed, there are several studies in the

literature that have revealed other types of mAb biotransformation events, such as oxidation,

deamidation, glutamate/pyroglutamate conversion, and C-terminal lysine processing (Liu YD et

al., 2009; Cai et al., 2011; Liu YD et al., 2011), but very little has been published about mAb

biotherapeutic catabolic fragments. One study has reported the presence of mAb fragments by

incubation of a fluorescently labeled mAb in whole blood followed by analysis by capillary

electrophoresis (Correia, 2010). The exact molecular nature of the fragments was not, however,


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identified (i.e., if the fragments were actually proteolytic fragments), and the relevance to

potential in vivo catabolism is not clear.

*Peptide/protein Fusions with Half-Life Extenders

A relatively new class of biotherapeutic involves attaching a peptide or small protein with

intrinsically high clearance (short half-life) to a large scaffold with a long half-life. The goal is to

produce a biotherapeutic with desired pharmacological activity that has sustained circulatory

concentration that is dictated by the half-life extender. Examples of half-life extenders are mAbs,

fragments of mAbs such as Fc, albumin, transferrin, and PEG (Kontermann, 2011). The half-life

extenders are chosen due to their longevity and in vivo stability with respect to

biotransformation; however, this stability is not necessarily conferred to the fused pharmaco-

active peptide or protein. Examples of this disparity and the impact on protein engineering and

bioanalysis have already been discussed for GLP-1/mAb and Fc/TMP (romiplostim) constructs

in a previous section of this review. Another recent example of the criticality of understanding

biotransformation for this type of biotherapeutic is that of fibroblast growth factor 21 (FGF21)

fusions to human Fc (Hager et al., 2013). FGF21 is a promising biotherapeutic for the treatment

of type II diabetes. Native FGF21 has a very short half-life and is cleared predominantly by renal

excretion after administration (Hager et al., 2013). In order to attempt to create a longer-acting

therapeutic, FGF21 was initially recombinantly fused to the C-terminus of human Fc (Hecht et

al., 2012). However, by using differential ELISA coupled with ligand-binding MS, it was found

that the C-terminus underwent very fast peripheral catabolism at a proline residue 10 amino acids

upstream of the terminus. Since the C-terminus of the construct had to remain intact to retain

potency, protein engineering efforts were undertaken to stabilize this catabolic liability. After


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numerous constructs were generated, the construct with this proline mutated to a glycine residue

showed retained potency with complete stability against biotransformation in this region (Hecht

et al., 2012; Hager et al., 2013). These efforts demonstrated the necessity of understanding

biotransformation, using this information to stabilize the molecule for retained potency and

decreased clearance, and selection of the proper LBA to track only the bioactive entities during

PK profiling.

In contrast, there have been reported studies where proteolytically labile peptides have

become protected by fusion to half-life extenders. For example, a 31-amino acid peptide coined

DAPD (“dual-acting peptide for diabetes”) is a hybrid peptide that has both GLP-1 agonist and

glucagon antagonist activity (Pan et al., 2006). DAPD is vulnerable to deactivation by proteases

including dipeptidyl protease IV (DPP-IV). Due to the fast clearance of DAPD, the half-life was

extended by conjugation to a high mass branched PEG (43 kDa) via maleimide conjugation

through the C-terminal cysteine residue (Claus et al., 2007). By introducing the branched PEG,

the in vivo half-life was significantly increased by protecting the DAPD from both catabolism

and renal filtration.

In short, since the use of half-life extenders such as Fc, mAb, albumin or PEG is invoked in

order to increase the in vivo persistence of quickly clearing pharmaco-active peptides and small

proteins, it is paramount to confirm that biotransformation does not inadvertently derail this

extended half-life strategy.

*Antibody-drug Conjugates (ADCs)

ADCs represent a novel modality where the understanding of in vivo stability and

biotransformation is especially crucial. ADCs are comprised of mAbs that have been conjugated


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with small molecule toxins or chemotherapeutic agents. The attachment of the small molecule

moieties to the Ab is generally through different types of linkers and can be directed

nonspecifically (e.g., through lysine ε-amino or endogenous cysteine groups) or specifically

through engineered sites (e.g., free thiols or other reactive groups) (Nolting 2013; Perez et al.,

2013; Behrens and Liu, 2014; Tian et al., 2014). The stoichiometry of small molecule drugs

conjugated to each carrier antibody is referred to as the drug-to-antibody ratio (DAR), and this

ratio is desirably preserved until the ADC reaches its target. Overall, the intended pharmacology

of ADCs is to utilize the Ab portion of the conjugates to deliver the conjugated toxins directly

and specifically to drug targets differentially expressed on tumors and reduce toxicity issues of

systemic administration of high doses of the chemotherapeutic agent alone. For example,

calicheamicins are highly potent cytotoxic agents that have been conjugated to mAbs that target

surface targets, such as CD22, CD33, and LeY that are highly expressed on various types of

tumors (Bross et al., 2001; Boghaert et al., 2004). Clearly, it is undesirable if the

chemotherapeutic agent deconjugates from the Ab carrier in circulation before it engages the

intended antigen target, as this will impact overall potency and more importantly increase

potential toxicity due to free toxin.

Numerous groups have studied the in vivo stability of ADCs with respect to toxin attachment.

Differential LBA has been suggested as a way in which to assess ADC stability. In this case,

assays are generated with differential specificity to measure conjugated and unconjugated forms

of carrier mAbs. For example, in one report, two assays were developed to track total

calicheamicin concentration (free or conjugated to an ADC) vs. concentration of the mAb carrier

only (Hussain et al., 2014). Using these assays, the concentration ratio of total calicheamicin to

that of the mAb carrier did not change for the first 6 hours after dosing but declined in a log-


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linear manner such that ~50% of the conjugated calicheamicin was deconjugated over the 336-

hour PK time course.

In addition, affinity MS methods have been used to understand the loss of toxins from ADCs in

vitro and in vivo. This is likely due to the aforementioned argument that MS methods provide

molecular level details more readily than differential LBA methods do, and the MS methods

have the added benefit of not requiring procurement or generation of LBA reagents. More

explicitly, changes of the DAR of any ADC due to in vivo or in vitro loss of the conjugated

toxins from the carrier Ab can be easily tracked by MS whereas this information is difficult to

obtain by alternative methods. For example, Shen et al. (2012) described the impact of

conjugation site on in vivo stability and potency of an ADC comprised of trastuzumab

conjugated to maleimide-monomethyl auristatin E (MMAE) through engineered cysteine sites in

various places in the Ab. They utilized affinity LC-MS to monitor the loss of MMAE after

incubation of the various ADCs in human plasma and found that certain sites of attachment led

to more stable ADCs. They ultimately concluded that stability was conferred to those sites with

less solvent accessibility whereby the mechanism of MMAE deconjugation was abated.

Significantly, these in vitro results were also observed in vivo using a mouse model.


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Conclusion and Future of Field

Unlike small molecules, the need for studies of biotherapeutic metabolism (i.e.,

catabolism/biotransformation) has only recently been addressed. Although there has been

minimal guidance from regulatory bodies with respect to biotherapeutic biotransformation, some

guidelines can be established based upon the literature and recent experiences, some of which

have been compiled in this review. For biotherapeutics that contain moieties that are not natural

amino acids, studies of catabolites containing these entities may require intense investigation due

to potential clinical safety issues. For biotherapeutics comprised solely of natural amino acid

sequences, biotransformation investigations may be less urgent due to the general lack of toxicity

concerns of catabolites. However, for certain modalities, circulating catabolites may have a

significant impact on drug potency and clearance. Understanding the degree of biotransformation

is crucial to the success of a drug development campaign, especially at the early stages, where

proteolytically labile sites can be stabilized through biotherapeutic re-engineering. In addition,

bioanalytical assays that are used for PK profiling must be able to detect bioactive catabolites

and exclude detection of pharmacologically-inactive catabolites in order to define the most

accurate PK exposure.

The current method of choice for analyzing biotherapeutic biotransformation is MS due to its

exquisite molecular resolution. Differential LBA can be used to screen for gross catabolic

liabilities, although pin-pointing of specific vulnerable loci is nearly impossible. In addition,

other biotransformation events, such as amino acid modifications, cannot be efficiently probed

by LBA.


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Of the biotherapeutics that are comprised only of natural amino acids, the modality that is

most prone to intermediate catabolite formation is pharmacologically-active peptides or small

proteins fused with stable half-life extenders such as mAb, fragments of mAb (e.g., Fc),

transferrin, and albumin. The stability of the half-life extender may not be conferred to the fused

peptide/protein. For replacement proteins and cytokines, biotransformation studies may be

required if enhanced circulatory stability is required that is not engendered by the native

endogeneous protein itself or if the nature of circulating catabolites must be known in order to

choose the best quantitative bioanalytical method. The modality with the least need for

examination of catabolites is mAb alone; the literature suggests that mAb do not generally form

stable, circulating catabolites.

In vivo assessment of biotransformation is presently the most informative, while in vitro

assessments, although offering some information, generally do not provide a completely accurate

correlation to that which occurs in vivo. The development of in vitro assays that are more

predictive of in vivo biotransformation is an unmet need that would help this field immensely.

Furthermore, the translation of biotransformation results across animal species and particularly to

human has not been systematically explored. This information is critical for this field as

preclinical studies must be relevant to clinical translation. Future studies should address the

extent of translation. In the worst case scenario, if the translation is less than adequate, this

would necessitate the need for further refinement of in vivo preclinical models, such as

identification and utilization of animal species with comparable proteolytic enzyme profiles to

humans, or the development of truly correlative in vitro human models.


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Acknowledgments

The author would like to thank Marc Retter for critical review of this manuscript.

Author Contributions

Wrote or contributed to the writing of the manuscript: Michael P. Hall


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Legends for Figures

Fig 1. Schematic of the biotransformation of an administered biotherapeutic. The exemplary

biotherapeutic is a bispecific molecule with pharmaco-active peptides (shown in red) fused to the

C-terminus of a monoclonal antibody (shown in blue). After dosing, biotransformation events

can occur, in this case peripheral in vivo truncation or catabolism of the fused peptide moieties

(kcatabolism), prior to systemic clearance via lysosomal degradation and/or renal excretion

(kelimination).


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DMD #58347 1 Title Page Biotransformation and In Vivo Stability of