DMD #58347 1 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 at ASPET Journals on February 15, 2018 dmd.aspetjournals.org Downloaded from
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DMD #58347
1
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
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
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
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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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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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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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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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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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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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
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).
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
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