An advanced liquid chromatography=mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance
(DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were
observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification
processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to
patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP
composition of two upstream samples was also analyzed and used to demonstrate that HCPs
that carry through purification processes to be detectable in DS are not always among those
that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating
process development as well as more rationally assessing potential safety risks posed by
C 2013 American Institute of Chemical Engineers Biotechnol.
individual, identified HCPs. V
Prog., 29:951957, 2013
Keywords: mass spectrometry, host cell proteins, biotherapeutics
Introduction
Modern recombinant biotherapeutics are typically produced in non-human cell lines. Despite rigorous purification,
low levels of host cell protein (HCP) impurities can remain
in the final drug product (DP). Residual HCPs represent
potential safety risks for patients, including immunogenicity,1 adjuvant activity,2,3 decreased product stability due to
enzymatic activity,4 or, more theoretically, direct biological
activity.5 To reduce such concerns, clearance of HCPs to
levels deemed safe is required by regulatory agencies.6 This
could be considered even more important for todays highdose therapeutics, such as antibody products, which are often
dosed at 100 mg, in contrast to the lower doses used for
first-generation biotherapeutics such as insulin and growth
hormone (10 mg=dose). With high-dose products, a patient
might receive impurity HCPs at levels comparable to the
active protein in first-generation biotherapeutics. Truly meaningful a priori evaluation of potential risks associated with
residual HCPs in DPs requires both identification and quantification of the individual HCPs present, as individual proteins can vary widely with respect to attributes that might
generate safety concerns (e.g., immunogenicity). In addition,
Current Address of Matthew R. Schenauer: 1 DNA Way, South San
Francisco, CA 94080.
Additional Supporting Information may be found in the online version
of this article.
Correspondence concerning this article should be addressed to A.M.
Goetze at goetzeam@amgen.com.
C 2013 American Institute of Chemical Engineers
V
952
Refold Conc:
1:
2:
3:
4:
13
Precipitation
A
B
C
13
A
C
13
A
C
E
D
53
A
C
E
D
953
Figure 1. Venn diagram showing distribution of identified DS HCPs among different Pb products and processes. Superscripts on the
Pb process numbers refer to the number of lots of DS analyzed for each process followed by the total number of MSE
acquisitions of those lots. For brevity, only the top 10 (previously identified) HCPs in Pb 2 are shown.10
20
96
<LOD
<LOD
26
391
195
166
8
73
5
2
3
5
1
2
Average quantification results for DnaK and total HCP by MSE and
Total HCP by MSE is estimated as the sum of all identified individual
HCPs. All numbers are in ppm (w=w). LOD 5 limit of detection.
954
composition and total HCP level had changed. Such an impurity profile difference could represent an increased safety
risk for patients, although no effort was made to assess this.
In this case, MSE made this information available, whereas
the use of HCP ELISA did not.
Four Process 2 DS lots were analyzed quantitatively by
MSE; they were chosen to represent the extremes in DnaK
levels as measured by ELISA. Nine of the eleven HCPs identified for Pb1, Process 2 in Figure 1 were identified in all
four of these lots, while two were identified in three lots. Figure 2 plots the MSE-determined concentrations of the nine
most abundant Process 2 HCPs against the DnaK ELISAdetermined DnaK concentration, which itself shows very
good quantitative correlation with MSE.10 The other eight
HCPs appear to fall into two classes: those whose concentrations scale in proportion to that of DnaK (MiaB, AsnA, and
NarP) and those whose concentrations are approximately constant, independent of the DnaK levels (HsIU, YhbS, YdhR,
and NfuA). These two groups of proteins did not exhibit
obvious differences in pI or amino acid composition. It is
conceivable that the proteins that scale similarly to DnaK
might be removed from the product with similar efficiency at
the same key chromatographic step(s) as DnaK, whereas the
remainder might be cleared at step(s) that have less impact
on DnaK concentration.
Process 3 was the result of adding a new purification step
to Process 2 (Table 1), with the specific goal of reducing residual DnaK. Following identification of DnaK, differences
in physiochemical properties between DnaK and Pb1 were
readily exploited for DnaK removal with the addition of a
tailored chromatography step. As a result, DnaK levels were
reduced from 73 ppm to 5 ppm by DnaK ELISA and from
96 ppm to below detection by MSE. In contrast, the HCP
ELISA was unable to detect a significant HCP level difference in Process 3 compared to Processes 1 or 2 (Table 2). In
addition to greatly decreasing residual DnaK in Process 3,
the spectrum of identified, non-DnaK HCPs was reduced to
a smaller subset of those identified in Process 2 DP (Figure
1). The latter observation is reasonable since Process 3 differed from Process 2 only by the addition of a single unit
operation. With such a change, the quantity and spectrum of
HCPs might be expected to decrease, but no new HCP(s)
955
Table 3. Compilation of all HCPs Identified in Pb1, Pb2 or Pb3 DS Along with Status of Each Proteins Identification in Upstream Samples
All Null Lysate
250 Most Abundant
Unit Op 1
Entry
Identified E coli Protein
MW
ECPs (1539 Total)
Null Lysate ECPs
Pool of Pb1, Pr2
ASNA
CH60
CLPB
DNAK
ERPA
FLMA
FNR
GRCA
HINT
HNS
HSLU
IDH
MIAB
MPRA
NARP
NFUA
NIFU
PFLB
PHOP
PTKB
RIMM
RL3
SUCC
YBEL
YDHR
YHBS
36651
57329
95585
68984
12101
6108
27967
14284
13241
15540
49594
45757
53663
20564
23575
20998
13849
85357
25535
10222
20605
22244
41393
18797
11288
18534
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y23
Y51
Y8
N
N
N
Y240
Y234
Y60
N
Y5
N
N
N
Y171
Y227
Y49
Y224
Y145
Y45
Y95
Y24
N
Y226
N
Y
Y
N
Y
Y
N
N
N
N
N
Y
N
Y
N
Y
N
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Superscript in column 250 Most Abundant Null gives rank among top 250 identified ECPs.
956
Conclusions
This study represents, to our knowledge, only the second
detailed MS characterization of the HCP content of a biotherapeutic DS=DP (following10), and the first that correlates
residual HCP content with changes in the purification process steps used. It demonstrates not only that MS has sufficient sensitivity to identify and quantify multiple residual
HCPs in highly purified Pb biotherapeutics from E. coli, but
also that the composition of residual HCPs in a single therapeutics DS may sensitively reflect purification process
changes. Significant differences in both composition and
quantity of individual HCPs as a result of process changes
were monitored by MSE. The technique sheds light on overall changes in HCP impurity profiles, while quantification of
a specific HCP, DnaK, using this technology was corroborated by a quantitative DnaK-specific ELISA. In contrast, a
multiproduct E. coli-specific HCP ELISA failed to detect
significant changes in DnaK levels as well as major changes
in overall HCP composition and quantities. These observations likely reflect the fact that a single analyte ELISA is capable of good accuracy, whereas quantification with a
multianalyte ELISA, like that for HCP in the present study,
can be problematic and even fail to detect key components
depending on the initial immunoreactivity of HCPs in the
immunogen and complement of HCPs that eventually persist
into DS. If residual HCPs present safety risks, our study
indicates that patient safety may not always be adequately
assured with sole reliance on a traditional HCP ELISA. One
potential lesson is that whereas the biotechnology industry
typically uses multiple high-resolution bioanalytical methods
to analyze the DP itself, this rigor is conventionally not
matched by the assay methodologies used to monitor residual HCP. In our study, a validated HCP ELISA was clearly
unable to detect DnaK, a HCP that presented possible safety
concerns, as well as other HCPs. As demonstrated in the
present study, high-resolution LC=MS methodologies are
now capable of providing more comprehensive, and accurate,
DS HCP characterization, thereby facilitating rational assessment of potential safety risks posed by individual, identified
HCPs. This information can also be used to accelerate and
improve process development by intelligently removing
newly identified HCPs of concern by exploiting each components hitherto unknown physiochemical properties. In conclusion, the deeper understanding of product quality with
regard to HCPs provided by a method such as MSE, not only
addresses the expectations of the Quality by Design initiative,19,20 but also provides a viable path forward in addressing HCP comparability for biosimilars as well as in other
manufacturing changes.
Acknowledgments
The authors would like to acknowledge Ken Chen, Hai
Pan, and Gang Huang for the original identification of DnaK
as an impurity in Pbs, Susan Callahan for DnaK ELISA
development and support, and Amy Hu, Brian Williamson,
and Oliver Kaltenbrunner for process development.
Notation
E
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