Preparative Biochemistry & Biotechnology

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/lpbb20

Bioprocessing of recombinant proteins from

Escherichia coli inclusion bodies: insights from
structure-function relationship for novel
applications

Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain & Sumit K.
Singh

To cite this article: Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain & Sumit K.
Singh (2023) Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies:
insights from structure-function relationship for novel applications, Preparative Biochemistry &
Biotechnology, 53:7, 728-752, DOI: 10.1080/10826068.2022.2155835

To link to this article: https://doi.org/10.1080/10826068.2022.2155835

Published online: 19 Dec 2022.

Submit your article to this journal

Article views: 721

View related articles

View Crossmark data

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=lpbb20
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
2023, VOL. 53, NO. 7, 728–752
https://doi.org/10.1080/10826068.2022.2155835

Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies:

insights from structure-function relationship for novel applications
Kajal Kachhawahaa
, Santanu Singha
, Khyati Joshia, Priyanka Nainb, and Sumit K. Singha
a
School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, India; bDepartment of Chemical and
Bimolecular Engineering, University of Delaware, Newark, DE, USA

ABSTRACT KEYWORDS
The formation of inclusion bodies (IBs) during expression of recombinant therapeutic proteins Inclusion bodies; refolding;
using E. coli is a significant hurdle in producing high-quality, safe, and efficacious medicines. The E. coli; downstream
improved understanding of the structure-function relationship of the IBs has resulted in the devel- purification; thera-
peutic proteins
opment of novel biotechnologies that have streamlined the isolation, solubilization, refolding, and
purification of the active functional proteins from the bacterial IBs. Together, this overall effort
promises to radically improve the scope of experimental biology of therapeutic protein production
and expand new prospects in IBs usage. Notably, the IBs are increasingly used for applications in
more pristine areas such as drug delivery and material sciences. In this review, we intend to pro-
vide a comprehensive picture of the bio-processing of bacterial IBs, including assessing critical
gaps that still need to be addressed and potential solutions to overcome them. We expect this
review to be a useful resource for those working in the area of protein refolding and therapeutic
protein production.

Introduction efficacy at a large scale. Despite these developments, protein

Protein-based therapeutics have become the cornerstone of
modern-day healthcare. Both mammalian[1] and microbial
expression systems[2] are mainly utilized to express recom-
binant proteins. However, a comprehensive understanding
of the complete physiology of E. coli and the availability of a
plethora of strains that are easier to grow makes microbial
hosts the preferred platform for expressing more than 25%
of the recombinant proteins.[3]
A recombinant protein must be expressed in its native
3D form to be an effective therapeutic. However, the higher
productivity achievable with E. coli as host results in the
over-expression of recombinant proteins.[4] The overexpres-
sion of proteins invariably results in an inactive, misfolded
form of the protein, commonly referred to as inclusion
bodies (IBs).[5] This necessitates refolding the IBs under ena-
bling conditions to generate an active, functional form of
the protein.
While IBs are inherently very stable and are easy to tackle
during downstream purification, yet protein refolding under
in-vitro conditions is a non-trivial process as it involves sev-
eral structural transitions from the assembly of the amino
acid chain to the formation of the higher order structure.[6]
With the ever-growing demand for biotherapeutics for cater-
ing to several unmet medical needs, considerable attention
has been given to innovation of process development for
producing protein therapeutics of desired quality, safety, and
refolding, for a large part, is still carried out using conven-
tional time-based dilution methods.[7]
Some recent encouraging developments include the
broader acceptance among the stakeholders that the IBs are
inevitable during recombinant protein production. With this
reality, the community is striving to innovate and develop
bioprocesses that are envisioned as a universal method for
recovering active functional protein from bacterial IBs.
Further, several ways to isolate IBs have been developed that
are harsh enough to break open the bacterial cells without
disturbing the structural integrity of the proteins. The use of
mild solubilization agents has resulted in a multi-fold
improvement in the efficiency of bioprocessing of the IBs.[8]
Additionally, the advent and wide use of novel chromatog-
raphy formats such as multi-modal chromatography[9] have
opened avenues for separating molecular variants of the tar-
get product yielding highly homogenous and pure thera-
peutic proteins.
The availability of sophisticated and cutting-edge analyt-
ical tools has enabled the study of several proteins’ struc-
ture-function relationships with IB.[10] This information has
not only facilitated to design of efficient bioprocessing
schemes for IBs, but also opened newer frontiers for the
application of the IBs in areas such as drug delivery and
material sciences.[11]

CONTACT Sumit K. Singh sumit.bce@iitbhu.ac.in School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi,
221005, India.

Equal contribution
ß 2022 Taylor & Francis Group, LLC

This article discusses different types of bacterial IBs formed
during the recombinant expression of proteins and their mor-
phological, biochemical, and biophysical characteristics. We
also discuss various aspects of the bioprocessing of IBs,
including isolation, solubilization, refolding, purification, and
characterization. A great emphasis is given to innovations and
developments made in the last 5 years. Further, we analyzed
the current challenges in the bioprocessing of IBs and sug-
gested approaches to overcome these hurdles. The use of IBs
to design novel biomaterials and as a self-releasing drug deliv-
ery vehicle is also discussed. We expect that the information
consolidated in this paper will generate a deeper understand-
ing of the current developments, challenges, and opportunities
in the bioprocessing of bacterial IBs and allow them to suc-
cessfully capitalize on these aspects to develop therapeutic
proteins with consistent quality, safety, and efficacy during
commercial manufacturing.
General morphological characteristics of bacterial
inclusion bodies
Bacterial IBs are expressed in cytoplasmic or periplasmic
space inside the bacterial cells.[12,13] The diameter of IBs
spans in the range of 0.2–1.2 mm[14] encompassing round,
oval, spherical, cylindrical, and ellipsoidal shapes. IBs exist
as dense electron refractile particles of aggregated protein
during high-level expression of recombinant proteins.
Depending on the localization, the IBs exhibit amorphous or
para-crystalline nature.[15]
Inclusion bodies have a higher density (1.3 mg/mL)[16]
than many of the cellular components and thus can be easily
separated by high-speed centrifugation after cell disruption.
Despite being dense particles that are highly hydrated, IBs
have a porous architecture.[17] Another characteristic of IB
shape is the presence of either rough or smooth surfaces,
which affects the solubilization process.[18] The degree of solu-
bilization depends on the protein-protein interaction (hydro-
phobic interaction, electrostatic forces, hydrogen bonding, and
van-der Waal forces) between IB molecules. Cytoplasmic IBs
have stronger protein-protein interaction than periplasmic
IBs. Thus, periplasmic IBs are solubilized with low denaturant
concentration compared to cytoplasmic IBs.[19]
IBs lack well-defined 3D structures like functional pro-
teins. The origins of the IB formation during protein expres-
sion can be traced to the lack of post-translational
modifications such as glycosylation in E. coli.[20] The role of
glycosylation in protein folding through various cellular
compartments of a eukaryotic cell is well established.[21] The
lack of cellular organelles, such as the endoplasmic reticu-
lum and golgi apparatus in E. coli, impairs glycosylation
yielding misfolded proteins.[22] Recent studies on engineered
E. coli cells for the presence of glycosylation machinery also
indicate the formation of recombinant glycosylated pro-
teins.[23] In general, pglB N-glycosylation machinery
(obtained from C. jejuni)[24] and lsg locus machinery
(obtained from H. influenzae)[25] are commonly used
machinery for transferring glycans to the acceptor sites of
proteins in E. coli. IBs deposition in bacterial cells is
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 729
reversible[26] and achieved to its native state by downstream
processing (Figure 1).
Composition and localized cellular formation of IBs
The bacterial IBs are heterogeneous, comprising an ensem-
ble of target protein conformations and host cell protein
contaminants.[27] Various protein conformations include
amyloid-like fibrils, protofibrils, misfolded proteins, partially
folded proteins, and a fraction of the native target pro-
tein.[28] On the other hand, host cell contaminants include
membrane proteins, cytoplasmic proteins, chaperones, and
genetic material wrapped in IBs via nonspecific intermolecu-
lar interactions[29] (Figure 2). It is to be noted that conform-
ational impurities overwhelm cellular impurities as bacterial
IBs contain very low levels of host cell proteins, ribosomal
components, and DNA/RNA fragments.[30]
IBs are deposited at bacterial cells’ center and polar
end.[31] There are two hypotheses about IB formation in
bacterial cells. According to one view, the IB formation
occurs passively (without any energy expenditure) by the
bacterial nucleoid’s volumetric exclusion effect, leading to
the accumulation of IBs in the polar ends of the host bacter-
ial cell.[32] The localization of IBs within the bacterial cells
depends on nucleoid size, IBs aggregate size, and cytoplas-
mic mobility of the bacterial cell.[33] Cytoplasmic mobility
reduces the anisotropy of IB aggregates at the midpoint of
the cell (near the nucleoid border), eventually leading to
their accumulation along the polar ends of the cell.[34]
The other hypothesis postulates an energy-dependent
process[35] where IBs formation is believed to be a multi-
step process. The series of steps includes the formation of
several small aggregates that eventually agglomerates into
the large aggregate size and localization in the polar ends of
the bacterial cells. There is a substantial expenditure of
energy in the ensuing process that is modulated by chaper-
ones (DnaK and DnaJ), cytoskeleton (MreB), and proton
motive force (pmf).[36,37] Both hypotheses (energy-depend-
ent/energy-independent) are accepted and hold for various
bioprocess conditions during large-scale heterologous pro-
tein expression in E. coli.[32,35]
Classification of inclusion bodies
The bacterial IBs formed during heterologous expression[38]
of recombinant proteins, often called classical IBs, are dense
particle-like entities that completely lack functional bioactive
proteins. They also uniquely resist any protease-mediated
degradation under a dense cellular environment.[39] These
IBs usually require very high concentrations of denaturants
for solubilization and subsequent dilution of solubilized IBs
for refolding into the 3D active form of the protein.[40] The
purified form of the target protein is obtained by subjecting
the refolded output of the protein to a series of purification
steps involving orthogonal chromatography-based separation
from the product (molecular and conformational variants)
and process-related (host-cell proteins and host-cell DNA)
impurities. Overall obtaining a purified protein from
730 K. KACHHAWAHA ET AL.

Figure 1. Bioprocessing steps of recombinant proteins expressed in E. coli. (1) Induction of recombinant proteins. (2) Isolation of expressed recombinant proteins.
(3) Washing of isolated proteins which contains the IBs and cellular contaminants, washed IB pellets was removed out from cellular contaminants. (4) Solubilization
of washed IBs which will denature the IBs aggregates. (5) Refolding of solubilized IBs. (6) Purification of refolded proteins, the native proteins are separated from
the mixture of misfolded, partially folded and aggregated proteins.

Figure 2. Composition of Inclusion bodies (IBs). IBs contain cellular protein contaminants and recombinant protein conformations. (a) Cellular protein contaminants
are composed of bacterial genetic material, cytoplasmic proteins, membrane proteins, and chaperones. (b) Recombinant protein conformations are composed of
native, misfolded, amyloid like fibrils and partially folded proteins.

classical IBs is a time-consuming, cost-intensive, and tech- of IBs referred to as non-classical IBs. In contrast to the
nically complex process.[41] classical IBs, the non-classical IBs are relatively less dense
To simplify the above mentioned issues, researchers have and often contain a small fraction of bioactive proteins.[42]
attempted to express the target proteins as a particular class The non-classical IBs require relatively very mild

solubilization conditions. Active 3D correctly folded proteins
can be obtained directly from diluting solubilized proteins
without requiring additional refolding and purification
steps.[43] For example, Trinh et al. reported a feasible E.
coli-based expression platform for granulocyte colony-
stimulating factor (GCSF) production at low temperatures in
the form of non-classical IBs. With the said approach, the
GCSF was expressed, IBs were effectively isolated, solubilized
under very mild conditions, and refolded into native form
by dilution.[44]
The non-classical IBs, by their unique biophysical prop-
erties, have been utilized for a wide range of applications,
including drug delivery and the development of novel bio-
materials for tissue engineering applications.[45] For
instance, nano-pills were synthesized using the non-
classical IBs used for the sustained release of protein
drugs.[46] Similarly, pegylation of GCSF was carried out
starting from the non-classical IBs for improved pharmaco-
kinetic performance.[44]
Bioprocessing aspects of IB for therapeutic
protein production
E. coli is a primary workhorse for therapeutic protein pro-
duction. Because of the ensuing reducing environment, most
of the proteins expressed in E. coli are located in the cyto-
plasmic space. Under such reducing conditions, proteins
generally exist in the form of IBs due to non-native interac-
tions between amino acid residues leading to misfolded or
aggregated proteins.[47] According to an estimate, >80% of
recombinant proteins expressed in E. coli exist in the form
of IBs, indicating an inevitable feature of IB formation asso-
ciated with E. coli expression systems.[12]
The existing body of literature indicates that there have
been several attempts to minimize IB formation during pro-
tein expression in E. coli. However, it has been realized that
embracing the IB formation during protein expression could
offer several advantages for large-scale protein expression.
The primary benefits associated with the formation of inclu-
sion bodies are (i) expression of a very high level of protein,
more than 30% of the cellular protein,[13] (ii) easy isolation
of the inclusion bodies from cells due to differences in their
size and density as compared with cellular contaminants,[48]
(iii) lower degradation of the expressed protein, (iv) resist-
ance to proteolytic attack by cellular proteases on classical
IBs[49] and (v) homogeneity of the protein of interest in
inclusion bodies (lesser contaminants) which helps in reduc-
ing the number of purification steps to recover pure pro-
tein.[13] Being an advantageous feature during the expression
and bioprocessing of therapeutic proteins, IB formation, at
least in a theoretical sense, is no longer considered a limit-
ing bottleneck in obtaining higher titers of therapeutic pro-
teins. This review provides a comprehensive overview of the
key developments, challenges, and opportunities, including
upstream, downstream, and analytical characterization
aspects of IB bioprocessing.
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 731
Upstream processing aspects
E. coli is the most preferred host for recombinant protein
production.[50] This is attributed to its ability to express
high titers of the target proteins (50% of bacterial proteins
and 15% of non-bacterial proteins[10] in the soluble form
under relatively simple growth conditions.[51] Over 120
approved therapeutic proteins have been expressed using E.
coli as a host, which bears testimony to their prominent
roles as a potential expression system.[52]
The genome of E. coli is very simple, comprising 4400
genes.[53] This is significantly smaller than other commonly
used expression systems, such as Pichia pastoris and Chinese
Hamster Ovary (CHO) cells that are larger and more com-
plex in size.[15] The smaller genomic size of E. coli makes it
amenable to uptake diverse genes of interest without com-
promising productivity.[14] The doubling time of just
20 minutes in E. coli also facilitates faster culture campaigns
versus other expression systems, where a typical batch turn-
around time is 10–14 days.[51] With a deep understanding of
the E. coli genome, it is now possible to construct an opti-
mal expression platform for recombinant protein synthesis
using E. coli as a host cell. It can be designed in such a way
that it encompasses efficient transcription and translation
machinery.[54] In addition, it can also form proteins of inter-
est in higher amounts while maintaining the transcript’s
stability.[54]
Examples of therapeutic proteins expressed in E. coli
include insulin, growth factors, hormones, cytokines, anti-
body fragments, and nanobodies.[11,55] While E. coli systems
exhibit excellent versatility in the heterologous expression of
proteins, several issues are discussed below that render the
expression of specific proteins a non-trivial task.
Parameters affecting therapeutic protein productivity in
E. coli
Clone design. Plasmids are the most common vectors uti-
lized in recombinant DNA technology. Properties such as its
size, copy number and promotor strength significantly con-
tribute to the recombinant protein production in the host
cell. The recombinant gene dosage is defined by the copy
number of the plasmid. High gene dosage can either be
favorable or unfavorable for the expression of the recombin-
ant protein. Thus, a suitable plasmid should be selected,
keeping it mind the characteristics it possesses.[56,57] To
achieve a higher recombinant protein expression, the inter-
est gene should be cloned immediately downstream to a
strong promoter.[58] Strong transcriptional promoters con-
trol the foreign gene in various plasmids and bacteriophage
vectors.[58] These promoters do not show constitutive
expression and are governed by adding specific metabolites
or changing culture conditions.[59] These are highly regu-
lated in nature and also control the expression of the foreign
gene in such a way that they do not interfere with the nor-
mal functioning of cellular genes and are not toxic to the
host cell.[60] If these promoters are not regulated, they may
result in loss of plasmid carrying the strong promoter or
may be expressed constitutively, which shows detrimental
732 K. KACHHAWAHA ET AL.
effects on the cell.[59] The tae promotor is the most com-
monly used strong promotor.[58]
The selection of terminator sequence also plays an
important role in regulating recombinant protein synthesis.
In most organisms, TAA, TAG, and TGA are utilized as
stop codons, but in E. coli, TAA is more preferentially used
than the other two codons.[61] Efficient termination of tran-
scription may lead to lower cellular energy expenditure
reducing the host cell’s metabolic load.[41] There is one
more additional advantage of the transcription terminator as
it forms a secondary structure at the 30 end of mRNA,
which helps improve mRNA stability and thereby enhances
recombinant protein production.[62] Occasionally, the trans-
location of the protein to the periplasm is required for
proper protein folding and formation of disulfide bonds.
Signal peptides are added before target gene to direct the
movement of the protein toward the periplasm. Some plas-
mids such as p5 series of pMAL vector and pET-22 vector
contain the signal peptide at the 50 region of the MCS.
Addition of a signal peptide upstream of the target gene can
improve the recombinant protein expression.[63]
Codon optimization. Despite the excellent ability to accom-
modate a plethora of foreign genes, a major impediment
often observed during the expression of target proteins in E.
coli is codon bias. This bottleneck arises when significant
differences exist between the synonymous codon of the for-
eign genes and the host.[17] An example of such a difference
is the occurrence of rare codons such as GGA (glycine),
CUA (leucine), and AAG (lysine), which are comparatively
found in lesser frequency in E. coli than in eukaryotic
organisms.[64]
The low abundant rare codons in E. coli pose issues
regarding misincorporation/truncation of amino acids in the
newly synthesized polypeptide chain, thereby affecting over-
all yields.[17] Several strategies have been employed to solve
the issue of codon bias during heterologous protein expres-
sion in E. coli. The most common strategy used is codon
optimization in the cDNA- a method that maximizes the
codon usage of the host.[20] Codon optimization is usually
performed by site-directed mutagenesis of the rare codons
with the more abundant codons of E. coli. However, site-
directed mutagenesis is very costly and time-consuming.[65]
Therefore, a more attractive and economical approach
recently gaining attention is the co-expression of a plasmid
containing the tRNA for rare codons, thereby minimizing
the ensuing loss in yields owing to codon biases.[66] Further,
several codon bias-adjusted strains of E. coli, such as BL21
(DE3), Codon Plus, and Rosetta (DE3), are commercially
available, which effectively minimizes the issues of codon
bias during protein expression.[50]
Induction. In addition to the codon bias, low expression of
proteins could be caused by protein toxicity either before or
after induction.[15] For the former, controlling basal induc-
tion by tightly regulated promoters enhances the protein
expression multi-fold. Also, for expression vectors contain-
ing lac-based promoters, the use of defined media with
glucose as the primary carbon source is preferred.[10]
Similarly, for T7-based systems, using pLysS/pLysE bearing
E. coli BL21(DE3) cells help in reducing protein toxicity by
controlling the expression levels of T7 RNA polymerase.[13]
On the contrary, for the cases of reduced expression due to
protein toxicity after induction, using tunable promoters
and reducing induction time are some of the reported
approaches to enhance protein expression.[58] In addition,
directing the protein expression to the E. coli periplasm and
using a lower plasmid copy number during recombinant
protein expression allows for utilizing the oxidative environ-
ment of the former and reducing the metabolic burden on
the cells owing to the latter.[20] Further, increasing biomass
by optimizing and using novel media additives and provid-
ing appropriate culture conditions (aeration and anti-foam-
ing) also boosts the recombinant protein expression.[64]
Inducers such as isopropyl b- d-1-thiogalactopyranoside
(IPTG) also play an essential role in determining recombin-
ant protein yield. Inducer concentration and duration sig-
nificantly impact overall production.[67] Using a lower
concentration of inducer will lead to lower protein yield
resulting in inefficient induction, whereas using a higher
concentration of inducer will result in toxic effects on cells
and ultimately interfere with product yield.[50] It is also eco-
nomically not feasible to use higher amounts of inducer.[50]
The most suitable time for induction is the early mid-log
phase when cell density is at the maximum concentration
because cell density plays a crucial role in protein synthe-
sis.[68] Some studies have also suggested that induction can
occur in mid-log or stationary phases.[69] Reducing the
induction temperature is also a frequently used method for
obtaining soluble expressed proteins. At reduced tempera-
tures, bacteria exhibit a decreased rate of translation and
correspondingly, less likelihood of misfolding and
aggregation.[70]
Growth conditions & IB formation. Although a higher rate
of protein synthesis is desirable in therapeutic production, it
can often lead to protein aggregate formation in the form of
inclusion bodies. When foreign DNA is introduced into E.
coli, spatio-temporal regulation of its expression is lost. The
recombinant polypeptide is expressed in the E. coli micro-
environment, which may not be exactly the same as the
source in terms of pH, temperature, redox potential, cofac-
tors, and folding mechanisms. Additionally, in high level
expression, hydrophobic sections in the polypeptide are pre-
sent in large amounts and ready for interaction with related
areas during high level expression. All these parameters col-
lectively cause the protein to aggregate.[71]
Such insoluble aggregate formation can be prevented by
slowing down the protein synthesis rate by using a weak
promoter, using a low copy number plasmid, or production
at lower temperatures and extreme pH ranges.[54] Culture
conditions can be manipulated to slow down or enhance the
expression of recombinant protein in E. coli. Reducing the
growth temperature may help decrease the formation of
inclusion bodies because of the slower protein synthesis
rate.[72] Moreover, the decrease in temperature prevents
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 733

Figure 3. An illustration showing different cell compartments for protein synthesis and trafficking where cytoplasm being the most reducing environment and
both periplasmic and extracellular spaces are oxidizing in nature. The inner membrane incorporates transport proteins namely Tat and Sec complexes. Tat is capable
of transporting folded protein via TAT pathway whereas Sec is capable of transporting both unfolded/partially folded proteins via Sec-B pathway and co-transla-
tional transport via SRP pathway. N-terminal signal sequences are responsible for targeting their location and are cleaved once protein gets transported.

aggregation by reducing the hydrophobic interactions.[73]
Reduced temperature can also be responsible for the pro-
longed induction time, increasing the expressed protein’s
solubility.[58] This is due to the inactivity of most of the pro-
teases at lower temperatures, so they cannot cleave or
degrade newly synthesized proteins.[72] Several studies report
the improvement in expression of recombinant protein in E.
coli with the reduction in incubation temperature. Huang
et al., reported improvement in protein folding when the
incubation temperature was decreased from 37  C to 25  C.
This led to an increase in the yield of nonstructural antigen
protein 3 of the hepatitis C virus in E. coli from 4.15 to
11.1 mg/L.[74] In another study, higher fraction of soluble
anti-HER2 scFv was attained when the E. coli culture was
carried out at 25  C.[75] pH is also known to play an import-
ant role in the correct folding of recombinant protein. pH
manipulation influences IB formation and their physical as
well as chemical characteristics. Thus, pH should be opti-
mized in such a way that IB formation can be minimized. Li
and Cheng reported an increase in the aggregation of blue
fluorescent protein with decrease in pH.[76] In contrast, an
increase in the culture pH was shown to increase the forma-
tion of inclusion bodies during salmon growth hormone
(SGH) production using E. coli.[77] The addition of cofactors
in the growth medium may also help in the proper folding
of some proteins, and a change in pH can affect the proteo-
lytic activity and secretion of specific proteins. It has been
reported that addition of FMN in the unfolded form of fla-
vodoxin speeds up its folding process by several times.[78] In
another study, the presence of metallic cofactor Ca2þ hemin
was found to positively influence the refolding of horserad-
ish peroxidase.[79]
Strategies to minimize IB formation
Inclusion body (IB) formation is a major feature of protein
expression using E. coli that impacts productivity. IBs are
formed for various reasons, including incorrect or non-clas-
sical disulfide bond formation, inability to attain the unique
native conformation of the protein, low solubility, and lack
of glycosylation in E. coli.[51]
The current efforts to capitalize bacterial IBs without
compromising product yield principally encompasses strat-
egies to translocate the newly formed IBs from the reducing
cytoplasmic environment to relatively more oxidative peri-
plasmic or extracellular space.[80] The underlying reason is
several advantages the periplasmic and extracellular space
offers during the expression of functional therapeutic pro-
teins. For instance, periplasmic space confers resistance to
the protease-mediated degradation of the target proteins.[47]
Further, it streamlines downstream processing because very
few naturally occurring cellular proteins are present in E.
coli periplasm[81] (Figure 3).
However, since the volume of periplasm is only about
20% of the overall cell volume,[81] there is a limit to the
amount of expressed protein to be transported and accom-
modated into this space. The issue assumes greater signifi-
cance under conditions of high protein expression when the
target protein may start to leach out into the extracellular
734 K. KACHHAWAHA ET AL.
Figure 4. A simplified representation of cell-free protein expression system by using E. coli crude extract. Host cell DNA and cell debris are removed followed by
supplementing the reaction mixture with an energy source, cofactors, DNA template, and other components required for protein synthesis. The protein of interest
is expressed and further purified for bio- therapeutic development.
medium due to enhanced periplasmic permeability.[82]
Under such conditions, extracellular secretion of the
expressed proteins is the preferred alternative. The extracel-
lular secretion of the target proteins can be achieved by the
fusion of the target protein with an N-terminal signal
sequence, using chemical additives, modifying the induction
procedure, or modulating the promoter of the expres-
sion vector.[82]
Another approach to reducing IB formation is by co-
expression of molecular chaperones.[47] However, this comes
at the cost of enhanced process complexity and issues in
downstream purification of the target protein. The fusion of
solubility enhancer molecules addresses the problem of the
low solubility of the expressed protein (e.g., MBP, NusA,
SUMO, Halo, and GST).[59] Further, there have been signifi-
cant improvements in the solubilization efficiency using
detergents, urea, guanidium hydrochloride, and SDS during
in-vitro refolding in recent years.[30] The lack of post-trans-
lational modifications such as glycosylation is addressed by
co-expression of the glycosylation machinery of
Campylobacter jejuni in E. coli.[28] However, this strategy
has not been as successful as anticipated owing to complex
process machinery, the requirement of multiple vectors, and
inappropriate co-expression of proteins.[83]
A major source of the enhanced propensity of IB forma-
tion during protein expression is incorrect/scrambled disul-
fide bonding in the expressed proteins.[10] Such proteins are
usually refolded in-vitro using conditions that favor correct
disulfide bond formation (by optimizing the cell’s redox
environment), thereby facilitating the folding pathway to
attain the final native folded state.[58]
High cell density cultures
In the case of recombinant protein synthesis, cell density is
a major determinant of the overall yield of the product. Cell
density reflects the optimum cell growth by regulating cell-
to-cell signaling and communication.[84] Cell growth will be
sluggish without such interaction, leading to cell death.
Further, such cellular interactions are also responsible for
transporting small molecules required for the proliferation
of cells in a growth medium.[85] Therefore, maintaining high
cell density is a primary requisite for the growth of micro-
bial cells and the production of recombinant proteins in
high titers.[86] Cells are usually grown at high cell density by
using certain inducer molecules such as IPTG, anhydrotetra-
cycline (Atc), and appropriately optimizing the cellular
growth conditions.[87] Nevertheless, high cell density cul-
tures also lead to the accumulation of acetate and lactate,
nutrients, and metabolic load, which is detrimental to the
overall process efficiency.[88] Further, the expressed proteins
are found to be highly susceptible to proteolysis by the host
cell proteases.[30] In addition, aggregation and IB formation
also pose a major issue in successfully adopting high cell
density cultures for enhanced target protein productivity.
Cell-free protein synthesis
Cell-free synthesis of target proteins offers an attractive solu-
tion to the aforementioned issues associated with high cell
density fermentation. Under this paradigm, the cytoplasmic
constituents of the cells are used to express the target pro-
tein.[80] Unlike the cell-based protein expression, this
method does not utilize a live cell and thereby re-routing all
growth factors toward target protein synthesis (Figure 4).

The downstream purification of cell-free expressed proteins
is significantly simpler as compared to the cell-based protein
expression.[89] This is primarily attributed to the lack of the
cell membrane and relatively simpler host cell impurity pro-
files in the former. The lack of cell membrane facilitates dir-
ect extraction of target protein without the need for cell
lysis.[90] Similarly, the fact that only proteins required for
the target protein expression are included in the input cellu-
lar soup overcomes the issues of the auto-proteolysis of the
target proteins.[90] Further, the downstream recovery of the
protein post-harvest is also enhanced by minimizing product
loss at this stage of operation.[89] Several types of proteins,
such as misfolded, cytotoxic, and insoluble proteins that are
difficult to express using conventional cell-based systems,
have been successfully synthesized using cell-free systems.[89]
The ability to use small molecular additives such as denatur-
ants (sulfobetaine, substituted pyridines, and pyrroles), pro-
tein stabilizers (polyethylene glycol), and aggregation
inhibitors (L-arginine, proline) in a relatively less crowded
cellular environment in the cell-free systems facilitates the
prevention of protein aggregation and misfolding by the cor-
rect formation of disulfide bonds and 3D packing of the
proteins to its native conformation.[30] Although the cell-
free expression system provides a high-quality platform for
protein synthesis, it also offers some disadvantages as it is a
very expensive process because it requires purified cell com-
ponents for protein expression.[91] Therefore, more research
is required to lower the cost of the cell-free system, and
other strategies are to be employed for recombinant pro-
tein synthesis.
Downstream processing aspects
The recovery of biotherapeutics from IBs is a very crucial
step and is accomplished through a train of unit operations,
including isolation, solubilization, refolding, and purification
of IBs.[49] In the following sections, we discuss all aspects of
IB downstream processing, from Isolation to obtaining a
purified functional protein.
Isolation of IBs
The recombinant protein expression using bacterial systems
inadvertently leads to the formation of IBs in the E. coli
cytoplasm or periplasm.[15] In order to recover the active
protein from the IBs, the first step is to isolate the IBs and
subsequently subject them to a train of bioprocessing steps.
Isolation of IBs from the bacterial cytoplasmic soup is car-
ried out using a plethora of techniques broadly categorized
as physical, chemical, and enzymatic methods.[92,93] In sev-
eral instances of problematic IBs, a combination of any of
these methods is also employed to isolate IBs.[94] The choice
of the method is defined based on several parameters such
as scalability, efficiency, the time required for bioprocessing,
and cost-effectiveness.[95] While a detailed review of the
underlying basics of these methods is discussed elsewhere
and summarized in Table 1, we limit our discussion here to
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 735
how these techniques, when adopted for a variety of situa-
tions, impact the quality of the target product.
As a general rule of thumb, the efficient isolation of IBs
defines the overall process performance as more proteins
would be available for refolding, which would add to the
overall yield of the process. Further, any IB isolation method
used should be able to preserve the quality and structure of
the IBs, show scalability and process robustness characteris-
tics and minimize additional impurities in the target protein
product.[96] The choice of the IB isolation method signifi-
cantly impacts the quality of the target product that is being
expressed.[97] For instance, the use of lysozyme for bacterial
cell lysis inadvertently gets associated with the IBs, necessi-
tating frequent washing steps to remove additional impur-
ities during process development. Added to this, several
bacterial cells, especially gram-negative bacteria are less sus-
ceptible to lysis by lysozymes, thereby significantly affecting
the overall efficacy of cell lysis.[98] Similarly, sonication often
disturbs the active site of the protein present in the bacterial
IBs, reducing the overall reported biological activity.[99] In
contrast to these techniques, high-pressure homogenization
does not show any of the issues associated with the enzym-
atic and sonication-based methods, making it a method of
choice for IB isolation from bacterial cells.[100]
One of the major impediments during IB isolation is the
possibility of contamination of IBs with viable bacterial
cells.[101] Such issues have been solved using the combina-
torial approach of simultaneously using two orthogonal
methods for IB isolation.[102] For example, using both sonic-
ation and lysozyme treatment resulted in negligible (10–1
CFU/mL) viable cell contamination. In this approach, lyso-
zyme treatment followed by frequent washing resulted in the
removal of cellular debris, while sonication caused the
destruction of the remaining viable cell contaminants that
were originally lysozyme resistant.[103,104]
Solubilization of IBs
Subsequent to the isolation of IBs, the next step in the train
of bioprocessing is to solubilize them so that, under appro-
priate conditions, they can be refolded to their native struc-
ture. The product yield is intrinsically linked to the
solubilization efficiency. The solubilization process is aided
by adding solubilizing agents such as detergents, alkali
medium, chaotropes, organic solvents, and high pressure
that break the non-native H-bonds in the IBs.[105] The
choice of a particular method for solubilization of IBs is
generally guided by the prior knowledge of the structure-
function relationship of the proteins trapped in the IBs, if
available. However, in the lack of such information, a com-
bination of methods that maximize product recovery and
fully functional 3D folded protein is employed. The solubil-
ization process is described aptly by the homogenous layer
model.[106] According to this model, the denaturants pene-
trate the core of the IB, and the free monomeric and multi-
meric peptides diffuse back into the solution.
Typically, the turnaround time (a few to several hours)
for the solubilization process depends on the nature of
the IBs (classical or non-classical) and the choice of the
736 K. KACHHAWAHA ET AL.

Table 1. Techniques of IBs isolation from bacterial cells.

Method Technique Advantages Challenges Ref.
Physical method High pressure - No loss in biological activity of IB protein - Generates heat (approx. 1.5  C/1000 psi) [200]

(Mechanical) homogenization during cell lysis - Not suitable for heat sensitive proteins
- Applicable for both small- and large-scale
cell lysis
[93,201]
Bead mill - Efficient for microscale cell lysis - Not efficient cell lysis for large scale
- Smaller beads (0.10  0.15 mm) are optimum - Heating and power consumption increases
for bacterial cell lysis with increase in number of beads
[202]
Physical method Sonication - Effective for small scale cell lysis (below - High frequency sonication (20–40 kHz)
(Non-Mechanical) 100 mL) damages the native proteins in IBs
- Time effective (Generally, it takes 2–5 min for - Increases the temperature (approx. 50–90 K)
cell lysis) of sample which damages the proteins
[203]
Thermolysis / Heating - Efficient for isolation of heat stable proteins - Not suitable for thermolabile proteins
- Able to release cytoplasmic proteins in less - Proteins solubility changes due to
than 10 min temperature fluctuation
[204,205]
Freeze-thaw - Suitable for isolation of thermolabile proteins - Cold denaturation may occur due to
- Did not require complex instrumentation, weakening of H-bonds
thus simple and cost-effective method for - Time consuming (usually requires 3–5 cycles)
cell lysis for cell lysis
[206,207]
Chemical method Solvents (Alcohol, DMSO - Effective method for wide range of microbial - Flammable & toxic (requires special
& Toluene) cell lysis consideration)
- Suitable for classical IBs - Denatures the IB protein
- Not suitable for non-classical IBs
[207,208]
Detergents (SLS, SDS & - Effective for cytoplasmic protein release - Detergents will denature the IB protein
Triton-X-100) (more than 75% in 1 h) - Not suitable for non-classical IBs
- Variability of detergent offers the appropriate - Additional impurity of detergents added
selection of detergent for different proteins. in proteins
[209]
Alkaline lysis - Alkaline buffers (10.5–12.5 pH) efficiently lyse - High chemical cost for neutralization of alkali
the bacterial cell by hydrolyzing the
cell wall.
[206,210]
Osmotic shock - No loss in biological activity of IB protein - Product may be diluted (which can increase
during cell lysis the down streaming cost)
 1 M salt concentration is sufficient for small - Less efficient & requires pre-enzymatic
scale cell lysis (below 100 mL) treatment for cell lysis
[211]
Enzymatic method Lysozyme - No loss in biological activity of IB protein - Not very efficient for gram negative bacteria
during cell lysis - Enzymatic impurity complexes the
- Suitable for gram positive bacteria downstream process.
[212]
Lysis protein E - Upstream In-process disruption of bacterial - Quality loss (10%) in IB purity
(Bacteriophage cell
UX174-derived) - Also, bacterial ghost can be used as
desirable product which contains active
IB proteins.
[103]
Combinational Sonication & Lysozyme - No impurity of viable bacterial cell in IB - Requires frequent washing steps to remove
method protein enzymatic impurity
- Effective cell disruption technique due to
synergistic effect of combined methods
[94]
High pressure - Efficient cell lysis - Sonication with High pressure
homogenization - No viable bacteria will remain in medium homogenization is simply not possible
& sonication because cavitation is substantially inhibited
at above 100 kPa pressures.
[213]
Novel approaches High pressure jet device - High throughput and cost-effective - Complex instrumentation
(Frictional - Efficient cell lysis
shear þ cavitation
þ collision)

solubilization agent.[107] This constitutes most of the proc-
essing time required for the production of protein therapeu-
tics. Recent efforts to reduce the time required for
solubilization of IBs indicate the excellent capability of the
microwave-assisted method to carry out the process in less
than 5 minutes.[108] This method utilizes brief exposure of
the IBs treated with detergents to microwave radiation (with
a wavelength range of 300 mm–1 mm) at 25–30  C. The
exposure to microwave radiation results in the re-alignment
of the polar molecules (protein IBs) in solution, leading to
efficient heating that enhances the solubility of the proteins.
The method works for various IBs, as evidenced by the
work of Dutta et al.,[108] who showed that seven different
types of IBs were successfully solubilized in less than 2 min.
The solubilized IBs were completely unfolded and
successfully refolded into their native conformations under
appropriate conditions.[109]
One of the major challenges during the solubilization of
IBs is an aggregation of recombinant proteins. Aggregation
during the solubilization mainly occurs due to hydrophobic
interactions and non-classical disulfide bond formation.[49]
As aggregation follows higher order reaction kinetics, a high
concentration of target protein or other product-related
impurities also contributes significantly to the aggregation
during solubilization and refolding.[39]
A variety of agents have been used that assist in the solu-
bilization of the isolated IBs. These agents differ in terms of
the strength with which they mediate solubilization. For
example, while strong denaturing agents like urea and guan-
idine hydrochloride are more suitable for solubilizing
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 737

Table 2. List of fusion tags to improve solubility and expression of recombinant proteins in E. coli.
Amino acid
Fusion tags Protein Organism Type Isoelectric point chain length Size (kDa) Ref.
[214]
MBP Maltose-binding protein E. coli chaperone based tag 4.97 396 40
[215]
GST Glutathione-S-transferase S. japonicum chaperone based tag 6.51 211 26
[216]
Trx Thioredoxin E. coli chaperone based tag 4.46 109 12
[217]
Spy Spheroplast protein Y E. coli chaperone based tag 9.45 161 18
SUMO Small ubiquitin modified Homo sapiens chaperone based tag 4.96 100 11 [218]

Fh8 F. hepatica antigen (8 kDa) F. hepatica Peptide tag – 69 8 [219]

[220]
Halo (DhA) Mutated dehalogenase Rhodococcus Peptide/affinity tag 4.83 293 33
[221]
NusA N-utilization substance A E. coli Fusion protein 4.44 495 54
[222]
Ecotin E. coli trypsin inhibitor E. coli Fusion protein 6.53 162 18
[223]
GB1 IgG domain B1 of protein G streptococcus Fusion protein 4.68 593 63
[224]
Skp Seventeen kilodalton protein E. coli Fusion protein 8.86 161 17

classical IBs,[12] their use is limited owing to the damaging
effect they exert on the secondary and high-order structures
of the IBs. Further, strong denaturants also contribute to the
formation of soluble and insoluble aggregates. The non-
denaturants like DMSO and n-propanol, on the other hand,
solubilize the non-classical IBs. As these agents have mild
effects, the IBs retain their bioactivity and generally do not
require a downstream refolding step.[110] This, in turn, saves
both time and cost involved in the bioprocessing of the IBs.
Despite their advantages, non-denaturing agents often fail to
solubilize tough/classical IBs. The disadvantages associated
with the denaturing and non-denaturing agents necessitated
using mild solubilization agents that could solubilize both
classical and non-classical IBs and preserve their residual
3 D structure, if any. Some mild solubilization methods
include subjecting the IBs to heat, alkaline pH, acid, organic
solvents, freeze-thaw, and high hydrostatic pressure.[18]
Nevertheless, these methods introduce artifacts by modifying
amino acid side chains, acid-mediated cleavage, and the
introduction of impurities emanating from the use of
organic solvents and detergents. Further, it is difficult to
make an appropriate decision as to which mild solubilization
method would be useful in solubilizing a given IB.[111]
Efforts to develop correlations between various attributes of
solubilizing agents and bacterial IBs are currently in pro-
gress to overcome the uncertainty in choosing appropriate
mild solubilization agents during the bioprocessing of bac-
terial IBs.[112] In addition, a combination of mild solubiliza-
tion methods, such as simultaneously subjecting IBs to
extreme pH and pressure/temperature conditions, can also
generate conditions wherein the IBs get solubilized without
altering the existing native-3D structure of the proteins.[113]
While the use of mild solubilization methods is yet to
become a norm in the industry, their successful adoption
would eventually pave the way for a reduction in the time
and cost involved in the production of protein therapeutics.
However, efforts have been made for effective solubiliza-
tion of the IBs, i.e., getting more soluble IBs with less or no
formation of aggregates. Recently, the nucleocapsid fragment
IB of SARS-CoV-2 expressed in E. coli was efficiently solubi-
lized under mild denaturing conditions, i.e., high pressure at
2.4 kbar and alkaline pH 9.[114] High-pressure solubilizes
protein aggregates by weakening hydrophobic and ionic
bonds, and alkaline pH promotes solubilization by
electrostatic repulsion. Similarly, the IBs of hEGF protein
were expressed as CBD-Ssp DnaB-hEGF protein in E. coli. It
was also solubilized via a mild denaturation method by the
addition of urea followed by freeze-thaw. Urea breaks the
hydrophobic interactions of IBs peptides. Freeze-thaw allows
the IBs peptides to separate due to the formation of ice crys-
tals.[115] Another effort to get solubilized IBs is adding
fusion tags to the expression vectors.[116] These Fusion tags
(Table 2) will increase the solubility and inhibits the aggre-
gation of recombinant proteins by electrostatic repulsion
between fusion tags, the interaction of hydrophobic portion
of fusion tags with recombinant proteins, and attraction of
chaperones (trigger factor, GroEL, DnaK and ClpB) by
fusion tags to mediate chaperones-assisted solubilization and
refolding.[117] Recently a new retro-protein XXA has been
developed as a fusion tag for the expression of soluble IBs.
Retro-protein XXA is derived from antifreeze protein AXX
and contains 192 amino acids. This retro-protein XXA
fusion tag is more efficient than other fusion tags because it
can express Chrono, Notch2NL, and nCLu soluble proteins,
which are difficult for other fusion tags.[118] Fusion tags are
removed by enzymatic cleavage (enterokinase, aminopepti-
dase) or chemical cleavage (formic acid).[119]
Refolding of IBs
The thermodynamic hypothesis of protein folding states all
that a protein needs to fold into its native 3 D structure is
its primary amino acid sequence.[6] However, this is true
only under dilute conditions. Within the cells (in-vivo), the
protein translation occurs under very crowded conditions
and is generally assisted by molecular chaperones. However,
the in-vivo protein folding is non-trivial, and hence methods
of refolding proteins outside the cell (in-vitro) are
preferred.[49]
In-vitro protein folding is done by decreasing the
denaturant concentration of the solubilized IB, thereby driv-
ing the equilibrium toward the native folded state of the
protein. However, the transition from the solubilized,
unfolded, to the natively folded form of the protein is asso-
ciated with instances of protein misfolding and aggrega-
tion.[120] As such, aggregation and protein misfolding
constitute the biggest challenge during protein refolding as
it impacts the final product’s overall process economics and
quality. Protein refolding is a strong function of the initial
738 K. KACHHAWAHA ET AL.
concentration of the unfolded protein and follows first-order
kinetics. On the contrary, protein aggregation is a higher-
order process and is accelerated at the higher initial concen-
tration of the proteins.[121] Thus, protein refolding is
strongly favored under dilute conditions (approx.
0.01–0.1 mg/mL), wherein the molecular collisions between
the unfolded and folding intermediates are minimized. The
stability of the protein folding intermediates is enhanced by
the addition of several molecular excipients (folding
enhancers and aggregation suppressors) such as sucrose, L-
arginine, ethyl ammonium nitrate (EAN), trifluoroethanol,
cyclodextrins, and alginates.[122]
Several physicochemical factors, such as pressure, tem-
perature, pH, and ionic strength of the refolding buffer, also
determine the efficiency of protein refolding.[123] For
example, successful refolding of b-lactamase from IBs was
done at 2  103 bar for 48 hours.[124] Under this condition,
the protein aggregate dissociates and assumes the native
folded conformation of the protein. Similarly, existing data
from a refolding database[125,126] indicates the existence of
two temperature conditions for refolding under low temper-
atures (0–5  C) and under ambient conditions (25–30  C).
Likewise, the optimum pH for a variety of IB refolding lies
in the broad range of 5–9, with the most common pH of
8–8.5. It is important to note that refolding should not be
carried out under pI conditions to avoid precipitation.
Table 3 lists the commonly used methods of protein
refolding and their associated advantages/disadvantages. As
evident from the table, each refolding condition must be
optimized on a case-to-case basis depending on the propen-
sity of the IB to aggregate and the scale of the operation.
For example, while dilution of denaturants or chromatog-
raphy-based processes is an attractive option for protein
refolding at a laboratory scale, the large volume of buffer
used necessitates an additional concentration step, which
adds to the cost and time of bioprocessing. Likewise, refold-
ing proteins at an industrial scale should also yield a high
concentration of refolded protein output. A dialysis-based
method is suitable for obtaining a high concentration of the
refolded protein output.[127] However, the rate-limiting step
of removing denaturants from the membrane makes dialy-
sis-based refolding exceedingly time-consuming.[7] This step
could be hastened by increasing the membrane surface area
(i.e., membrane area per volume of denatured protein solu-
tion). Kato et al. achieved this objective by employing a
chemical engineering approach wherein they incorporated a
dialysis membrane into a microchannel. They successfully
refolded a model protein (known to be difficult to refold)
with an eight-fold concentration instead of the dilution-
based method in under 20 min using this custom-designed
dialysis setup.[128,129] However, the size of such refolding
apparatus limits the volume of solubilized IB that could be
handled. Also, rapid mixing of the solubilized IB and refold-
ing buffer in the microchannel may result in protein aggre-
gation. The use of aggregation suppressors such as arginine,
allantoin, and hydantoin could prevent aggregate formation
in such instances.[130]
Further, our understanding of the protein folding mech-
anism, conditions of refolding, and the characterization
methods involving biophysical and biochemical approaches
are still incomplete. Consequently, there is still a need for
the development of a universal refolding buffer that could
assist in refolding IBs of any characteristics back into their
native folded state. However, practices have been made to
develop the correlation between the refolding parameters
(such as temperature and pH) and methods of refolding.
These databases can assist in the better prediction of protein
refolding conditions.[125]
Purification of refolded proteins
The refolded output generally would be a cocktail of cor-
rectly folded, misfolded, truncated, and aggregated forms of
target protein.[28] It is of utmost necessity to separate the
correctly folded form of the protein from the other con-
formational impurities for the development of a safe and
efficacious biotherapeutic. While the truncated and aggre-
gated form of the protein could easily be removed using size
exclusion chromatography (SEC),[131] it is not an attractive
option at the industrial scale. The SEC is characterized by
small loading (usually <5% of the column volume) and
operation under slow flow rates (50 cm/h), which exorbi-
tantly adds to the overall cost and time of bioprocessing.[132]
The biggest challenge, however, is the separation of the mis-
folded forms from the correctly folded proteins due to
nearly the same Physio-chemical properties. The currently
used approaches to separate protein isoforms include multi-
modal chromatography, which uses two orthogonal dimen-
sions of separation based on the intrinsic properties of the
proteins.[133] The excellent separation efficiency offered by
multi-modal chromatography is often marred by difficulties
in optimizing the separation conditions and issues with the
method’s reproducibility.[134]
Recently, affinity-based purification using Ni-NTA chem-
istry has been used to separate protein isomers during
refolding.[135] The advantage of this method is that the same
buffer used for purification of IBs can be used for refolding
after diluting into refolding buffer, which allows the protein
to specifically bind to the resin in a denatured state. Upon
elution under refolding conditions, active function proteins
in milligram amounts could be received per liter of the cell
culture. Other novel chromatographic modes include the use
of monolithic[136] and membrane-based chromatography[137]
which also provides avenues for high-throughput separation
of protein isoforms. However, these methods have low-bind-
ing capacity during separation and therefore are less sought
after at industrial scales.[138] The amalgamation of nano-
technology with separation science has resulted in the devel-
opment of nano-fiber-based chromatography systems that
have much higher binding capacities than monoliths/mem-
brane-based systems.[139,140] However, such systems still
need to be validated for robustness, efficiency, and ease of
operation before they can be rolled out for large-scale usage
in the purification of correctly folded proteins.
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
Table 3. Techniques of refolding of solubilized IBs.
Methods of refolding Technique Sub-type Advantages
Conventional Dilution Single step dilution - Simple procedure and ease of
Methods operation
- Applicable for small scale refolding
- Time effective
Batch dilution - Less formation of protein
aggregates
- Applicable for large scale refolding
Dialysis One-step Dialysis - Simple method
Multi-step Dialysis - High refolding efficiency as
compared to one-step dialysis
Chemical additives Stabilizers, Aggregation - Stabilizers prevents the reverse
inhibitors, transition of native proteins
Chaotropes - Decreases the rate of aggregation
- Chaotropes promotes the
denaturation and solubilization
Non-conventional Microfluidic Chip - Efficient protein refolding due
Methods controlled and regulated diffusion
in microchannels
- Rapid process
On-column Size exclusion -Simple and automated process
chromatography -Combined refolding and purification
steps are performed
Periodic Counter-current - Economical process
Chromatographic - Continuous process utilizing
system multiple subsequent columns, so
chromatography resins is
efficiently used
- Specific buffer consumption is less
as compared to other similar
techniques
- Higher protein refolding efficiency
(11mg/hour)
Hydrostatic pressure - Able to refold Hydrophobic proteins
- High hydrostatic pressure
(100–300 MPa) efficiently
solubilizes the protein by
disrupting inter and intra
hydrophobic interaction between
protein molecules
Differential scanning Fluorescence - Cost effective method
fluorimetry based method - Used to track both protein folding
state and thermal stability.
- Can provide reliable data even in
unpurified chemical reactions.
Microchannel dialysis - High concentration of protein can
be refolded
- Low residence time for dialysis
- Greater membrane surface area as
compared to conventional dialysis
Analytical characterization of solubilized IBs/ details of the structure and homogeneity of IBs/refolded
refolded proteins proteins are briefly discussed:
IBs are heterogeneous entities exhibiting significant diversity
Structural characterization
in their shape, architecture, organization, and function. In
view of these properties, a panel of analytical tools is often Circular dichroism
employed to derive the detailed fingerprint of the IBs. In the CD spectroscopy is a nondestructive technique that is usu-
following section, various analytical tools that can provide ally implemented to monitor IB refolding to detect
739
Disadvantages Ref.
[7,225]
- Suitable for low concentration of
protein (approx. 0.1 mg/mL)
- Large volume of buffers and
reactors increases the refolding
cost
- Non-uniform mixing leads to
aggregate formation
- Time-consuming in contrast to
single step dilution
- Additional step of protein
concentration is required
[7,226]
- Rapid decrease in denaturant
concentration increases the
aggregate formation
- Interaction of denatured protein
with dialysis membrane
- Time consuming
- Small membrane surface area slows
down the denaturant removal
from dialysis bag
[227]
- Chemical additives increases the
overall production cost
- Imparts the chemical impurity
- Additional purification step needs
to be performed
[228]
- Not suitable for the large-scale
protein refolding
- Sometimes protein aggregates will
disrupt the laminar flow in
microchannels
[132]
-Large volume of buffer required
-Large dilution factor reduces the
protein concentration
-High time consumption due to
column retention time
[229]
- Continuous columns efficiency
requires frequent usage
- Sophisticated columns, chances of
mechanical breakdown and
microbial contamination
[230]
- Requires preliminary treatment of
proteins with detergents and
denaturants
[231]
- Chances of false positive and
negative results are higher
- Thermal shift may interfere with
the native state of protein sample
[128]
- Incorporation of dialysis membrane
into microchannel is challenging
- Design and fabrication of
microchannels is complex process
740 K. KACHHAWAHA ET AL.
conformational changes in the secondary (in the far-UV
region between 190 and 250 nm) and tertiary structure (in
the near-UV region between 260 and 300 nm) of the pro-
tein.[141,142] The absorption spectrum in the far-UV region
is attributed to the amide bonds, while in the near-UV
region, it is dominated by the absorption by the side chains
of the aromatic amino acids (buried within the core of the
protein).[143] While far- UV CD measures spectra of both a
helices and b-sheets, it is mostly applied for distinguishing
the refolded proteins rich in a helix from denatured protein.
The major issue in successful implementation of CD spec-
troscopy during monitoring of protein refolding is the need
of high sample concentration, i.e., 0.1 mg for far-UV and
1 mg for near-UV.[143] Recent studies have also reported the
use of CD for real-time, in-situ analysis of the relationship
between protein aggregation and the ensuing conformational
changes, if any.[144] However, the use of CD should be done
with caution as the resultant CD signal is a result of the sig-
nals emanating from different parts of the molecules, and
since these signals possess directionality, the overall signal
may not reflect the actual conformational changes in the
molecule.[145] Further, far-UV CD is also limited by intrinsic
insolubility of proteins, which causes high level of light scat-
tering disturbances and low signal-to noise ratio.[146]
Fluorescence spectroscopy
It is a nondestructive technique used for the real-time evalu-
ation of aggregation mechanisms, from monomers to the
formation of large aggregates. It also detects protein self-
assembly.[147] It is a highly sensitive technique where the
fluorophores used can be either intrinsic or extrinsic. In
intrinsic fluorophores, the fluorescent properties of aromatic
amino acids are used for detection. In extrinsic fluorophore,
fluorescence is observed from the externally used fluorescent
probes that can covalently bind with the hydrophobic
patches or chemically attach to the molecule.[148] Some of
the commonly used extrinsic fluorescence dyes include
Thioflavin T, Congo Red, Nile Red.[149] These dyes give less
fluorescence when present in an aqueous environment, and
their intensity is increased once they are bound with the
hydrophobic patch, which may be present in protein
aggregate.[147]
The high sensitivity of the extrinsic fluorescence makes it
ideal choice for high-throughput screening during protein
refolding. The variation in surface hydrophobicity is
reflected as increased fluorescence intensity during protein
refolding.[145] However, the main limitation of using extrin-
sic dyes is that it could alter the protein structure and affect
the overall process of detection. On the other hand, quan-
tum yield of detection using intrinsic fluorescence method is
significantly lower than the extrinsic fluores-
cence methods.[148]
Nuclear magnetic resonance (NMR) spectroscopy
NMR is used to determine the time needed to establish the
native state of the protein during refolding. The advantage
with NMR spectroscopy is that it can be used in real-time
to monitor higher order structure (tertiary and quaternary)
of a protein.[145] However, NMR can be used to characterize
the structural dynamics of only relatively smaller proteins
(<40kDa) and are not suitable of proteins with larger sizes
such as mAbs. Additionally, for NMR analysis, the protein
samples should be highly pure and concentrated (0.5 mM
or higher), thereby limiting its application in protein refold-
ing studies.[150,151] For instance, NMR analysis of IBs exhibit
additional resonance due to the presence of phospholipids
from the E. coli membrane, other proteins, or RNA necessi-
tating purification prior to NMR analysis.[152]
Another approach to decrease the NMR spectra complex-
ity is isotope labeling. Utilization of 13C- and 15N-labeled
amino acids corresponding to the residue pairs allows for
the structural and thermodynamic characterization of inter-
mediately folded protein conformers since it allows us to
examine a broad range of a protein’s conformational space
with a relatively neutral disturbance.[152] For instance,
Kamatari et al., investigated the atomic resolution structures
and thermodynamic properties of several partially folded
conformers under varying pressure using NMR spectros-
copy.[153] Water proton transverse relaxation rate R2(1H2O)
measurement by NMR is a strong noninvasive method for
the detection of inclusion bodies. The water proton NMR
(wNMR) technique is an indirect technique that draws its
conclusions from water–protein interactions and exhibits
excellent sensitivity toward protein aggregate formation.[154]
Dynamic light scattering
Dynamic light scattering (DLS) is a technique used for the
measurement of the surface charge of the proteins.[145]
Owing to this property, DLS can effectively measure the
magnitude of the electrostatic repulsion/attraction between
two molecules. Thus, it is a good technique to measure pro-
tein aggregates in size range of 1 nm  5 mm.[155] In this
technique, monomers are not separated from the aggregate,
and their average size is used for the estimation of protein
aggregate. This is more effective in the analysis if the sample
contains some visible particles.[155] For better detection, it is
very important to keep the working conditions constant
such as temperature, viscosity, and interaction between
aggregated particles.[156] This technique has been used for
the detection of protein aggregate formation by heat and the
suppression of aggregate formation using heat shock pro-
teins like a-crystallin. It has also been used for analysis of
the nonspecific protein aggregation formation by the action
of detergents.[157]
There are several advantages of using dynamic light scat-
tering for aggregate characterization because it is highly sen-
sitive for the detection of even a small quantity of
aggregates, and it is a high throughput technique. In this
technique, both reversible and irreversible aggregates can be
easily characterized. The software used for the measurement
of aggregate size is Zetasizer which is very user-friendly. It
helps in the extraction of maximum possible information
from the aggregate sample and a more accurate representa-
tion of data. The procedures for measurement are highly
automated and thus do not require high degree of training

to use the instrument.[156] Unlike extrinsic or intrinsic fluor-
escence methods, DLS being non-intrusive has minimal
chances of assay induced aggregation, thereby significantly
reducing the chances of measurement artifacts.
DLS also has some disadvantages, such as the data
obtained for analysis being semi-quantitative. As a result of
its sensitivity toward detecting even small quantities of
aggregate samples, it sometimes also detects dust particles
and other contaminants present in the sample and gives
false positive results.[158] This also has the significant draw-
back of being unable to distinguish between scattering
amounts from various aggregate molecules.[156]
Cryo-electron microscopy
It is a variant of transmission electron microscopy which
nowadays is widely used for the structure prediction of mac-
romolecules. This technique uses flash-freezing solutions of
proteins and samples are cooled up to their freezing temper-
atures.[159] Protein samples are kept in an environment of
vitreous water (ice with the properties of water) and are
bombarded with electrons.[157] The image is reconstructed
with the help of multiple individual microscopic images of
molecules and is used for the elucidation of the 3D structure
and shape of the protein aggregate sample.[160]
It offers several advantages, such as it does not require
crystallization and yields near-atomic level resolution of the
molecular structures being examined. As there is practically
no upper limit of size for EM, it is an ideal tool for protein
aggregate analysis.[161] For example, cryo-EM has been used
to elucidate the overall fibril structure showing the number
of protofilaments involved in aggregate formation and the
degree of twist present in amyloid formation. It also gives
information about the atomic structure of fibrils. It is a
next-generation method in which the electrons are bom-
barded in such a way that they form a thin, sensitive layer
(usually a carbon film), thereby preventing signals from get-
ting scattered in the surroundings, which may result in poor
image formation.[162]
While cryo-EM offers better resolution than other con-
temporary techniques like x-ray crystallography, however, it
has lower contrast. Several strategies, including ‘negative
staining’ (the sample is immersed in a layer of heavy metal),
are used to improve the contrast during cryo-EM measure-
ment of the samples.[161] Further, the lower contrast results
in a low signal-to-noise ratio, necessitating advanced detec-
tion systems and image processing systems for accurate
detection of sample images.[161]
While recent advancements and automation techniques
have solved many issues related to image resolution and
sample preparation, major issues still exist. In particular,
cryo-EM instrumentation is very costly and reaches many
millions of dollars, exceeding the grasp of individual investi-
gators and universities.[162] Further, cryo-EM maintenance is
very expensive and demanding.[162] One potential solution
for these issues is the establishment of national and inter-
national facilities that operates on a cost-sharing basis.
These are starting to emerge in the US, UK, Germany,
Sweden, and India.
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 741
Homogeneity of IBs
Reversed-phase liquid chromatography
The separation in reversed phase chromatography is based
on the differences in the hydrophobicity of various species
in a sample. Hence, the method is capable of separating the
product related impurities such as oxidized and reduced spe-
cies formed during protein refolding.[163] However, since
RP-HPLC is carried out under denaturing conditions, no
information is obtained about the secondary and tertiary
structure of the protein. Therefore, the information obtained
is merely chemical in nature. Further, the high column tem-
perature used during RP-HPLC might induce protein aggre-
gation. Nevertheless, RP-HPLC being very fast, robust, and
high-resolution method is widely used to assess the product
homogeneity by measuring the levels of various product-
related impurities.[151]
Size exclusion chromatography
Size Exclusion Chromatography (SEC) is another method to
assess the levels of aggregation and fragmentation during
protein refolding. Hence, the method is highly suitable to
distinguish size- related variants such as dimers, oligomers,
and fragments. The aggregation/misfolding is detected using
UV-based detectors/fluorescence/multi-angle light scattering
(MALS).[164] Recently, SEC coupled with mass spectrometry
under native conditions has become possible to enhance the
coverage and depth of information obtained.[165] The ubi-
quitous use of the SEC in the characterization of protein
aggregates is evident from the fact that it has been estab-
lished as a quality control method for protein aggre-
gate analysis.
Despite their widespread usage in the characterization of
protein aggregates, it suffers from certain limitations. The
biggest issue in the adoption of SEC as a method of choice
in industrial setup is attributed to low volumes of sample
loading (2–5% of a column volume) and large amounts of
buffer requirement.[132] Further, secondary interactions on
the column also limit the resolution and performance
obtained while using SEC. The UV-based detectors also
show issues of sensitivity while discriminating between big-
ger vs. smaller aggregates.[132] These factors necessitate
orthogonal analytical tools to be used for the analysis of IBs.
Capillary electrophoresis
Capillary electrophoresis, also called high-efficiency capillary
electrophoresis, is a class of liquid phase micro-separation
analysis method. It is used for the separation of ions on the
basis of their electrophoretic mobility under an applied volt-
age. The mobility of these ions depends upon the molecule
charge, size and viscosity. The mobility of the molecules is
directly proportional to the applied electric field. Capillary
electrophoresis is utilized very commonly due to faster
results and high-resolution separation.[166] Garza et al.
developed an IB solubilization method and determined the
purity of recombinant proteins in inclusion bodies using
capillary gel electrophoresis. It is based on the utilization of
742 K. KACHHAWAHA ET AL.

a single-component solution which is completely compatible Novel applications of IBs

with capillary gel electrophoresis analysis. In this method,
IBs sample were prepared and separated from recombinant
proteins and other impurities. This allows capillary gel elec-
trophoresis to be regarded as a suitable analytical tool to
obtain the quantitative process information during IB
refolding.[167]
Peptide mapping
Peptide mapping is a useful method for the identification of
product-related impurities formed during protein refolding.
The biggest advantage of peptide mapping is when used
with mass spectrometry to confidently identify site-specific
modifications on the proteins.[168] In a recent application of
peptide mapping for studying protein refolding, the dynam-
ics of covalent and non-covalent interactions during the
refolding of the light chain, heavy chain, and antibody frag-
ment were studied. It was found that covalent interaction
(disulfide bond formation) was the major driving force for
protein refolding.[169] Further, multi-attribute monitoring
(MAM) workflows are currently widely used for the discov-
ery of unknown intermediates formed during the protein
refolding. This allows for better optimization of the protein
refolding process to enhance yields.[170]
Table 4 lists other characterization techniques that are
used for the analysis of bacterial IBs and protein refolding.
The choice of the technique would depend on the detection
limits, sensitivity, resolution, and reproducibility of the tool.
With the adoption of the QbD–approach in bio-manufactur-
ing, the tools that could yield real-time monitoring and ana-
lysis would continue to be in demand and the focus of
further research.
IBs as a vehicle for drug delivery
Therapeutic proteins are susceptible to degradation via mul-
tiple mechanisms during purification, formulation, and stor-
age. The degraded protein product has attenuated efficacy
and is even immunogenic, posing a serious threat to the
lives of the patients.[171] Protein aggregation is the most
common route causing protein degradation. Being immuno-
genic, protein aggregates elicit an anti-drug antibody (ADA)
response upon the administration that may interfere with
the absorption, distribution, metabolism, and excretion
(ADME) profiles of the drug.[155]
The recombinant protein expression using E. coli inad-
vertently entails the formation of target proteins in the form
of inclusion bodies (IBs). Existing evidence about the archi-
tecture and morphology of the IBs suggests the presence of
functional native-like structures within the overall IB net-
work.[172] The presence of these structures within the bacter-
ial IBs has inspired the packaging of active protein-based
drugs in the form of IBs and is being used as a novel self-
releasing drug delivery system.[173] However, the large
batch-to-batch variability in the morphological features of
IBs during protein biomanufacturing using microbial expres-
sion systems eludes their wider applicability as the potential
drug delivery platform. The presence of bacterial contamin-
ation in such IBs poses regulatory challenges in terms of
accruing approvals for market authorization of such
novel platforms.
Despite these insurmountable challenges, innovations
have been made by engineering artificial IBs for clinical use.
As opposed to the natural IBs formed from direct bacterial
cell cultures, artificial IBs exhibit batch-to-batch consistency

Table 4. List of techniques.

Technique
Fluorescence microscopy
Circular Dichroism
Cryo electron microscopy
Atomic Force microscopy
ATR-FTIR
Confocal spectroscopy
Native Mass spectrometry
1 [238]
H- NMR
All Atom Molecular Dynamic Simulation
(AA-MD)
SEC-ESI-MS
Dynamic light scattering
Size exclusion chromatography
Peculiar feature Ref.
[232]
- Do not need prior sample filtration and can detect turbid (in vaccines) and concentrated
(subcutaneous injection) protein solution. Detect highly concentrated protein solutions without
dilution.
- Helps in stability testing of formulation by detecting small aggregates (seeds) formation even
before their fibrillar structure formation.
- Allows characterization in-situ even in presence of excipients.
[144]
- Quantitative analysis of secondary and tertiary structure of optically active protein aggregates.
- Study relationship between molecular aggregation and conformational changes.
[233]
- Atomic level structure prediction of macromolecules.
[234]
- It is capable of characterizing even a single molecule of protein aggregate.
- It supports the examination of untreated specimens even directly from tissue sample.
[235]
- Capable of detecting very low quantity of samples and also applicable for insoluble and
impure sample.
[236]
- Monitor protein misfolding and aggregates in living cells.
- Quantitatively measures protein oligomerization.
[237]
- Able to detect native quaternary structure of protein aggregate.
- Monitors presence of coexisting classes of protein aggregates.
- Detects the self-assembly of both hydrophobic and hydrophilic peptides.
[238]
- Provides information about the amino acid residues associated with peptide-peptide interaction in
aggregate formation.
- Predicts the different aspects of protein aggregation behavior.
- Detects protein- excipients interaction.
[239]
- Differentiates between the oligomers present in protein samples and those formed during the
detection process.
[155]
- Highly sensitive technique for small quantity of aggregate sample.
[164]
- Qualitative and quantitative evaluation of protein aggregates.
- Monitoring of modified protein such as protein-drug conjugates and PEGylated proteins.
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 743

Figure 5. Artificial Inclusion Bodies (ArtIBs) production and brief characteristics of ArtIBs. (a) Showing the general procedure followed for Artificial Inclusion Bodies
(ArtIBs) production. Soluble proteins are linked with the divalent cations (ZnCl2, CaCl2, and MnCl2), followed by incubation for sufficient fabrication of proteins. The
fabricated protein contains both soluble and insoluble faction which are separated by centrifugation. The soluble fraction of protein was discarded and insoluble
fraction was used as ArtIBs. The ArtIBs will release the functional proteins as mimicking the action of secretory granules. (b) Characteristic properties of ArtIBs which
makes effective and suitable agents for therapeutics development.

and are devoid of any bacterial contaminations.[41] Further,
their synthesis routes are amenable to optimization of pro-
tein-protein interactions that may assist in easily releasing
active proteins under physiological conditions
(Figure 5).[174] Over time, we envision the use of bacterial
IBs as an attractive platform to package the glycoprotein
(for instance, IgGs) expressed using mammalian systems
and being used on various delivery routes, including much
restricted oral routes of protein drug administration.
IBs as immobilized catalysts
The ability of IBs to withstand harsh physicochemical condi-
tions (pH, temperature, and solvents) renders them a useful
candidate as immobilized catalysts with wide applications in
the biotechnology, pharmaceutical, and cosmetic indus-
try.[42] The wider applicability of immobilized catalyzed ver-
sus the traditional soluble catalyst includes superior
mechanical, thermal, and functional stability. In addition to
this, the former exhibits superior multi-enzyme functional-
ity, enantioselectivity, protease resistance, re-usability, and
easy downstream purification characteristics than
the latter.[175]
The IBs immobilization is carried out by primarily three
methods: carrier immobilization, entrapment of enzymes to
polymer network, and cross-linking of enzyme aggregates
with linker agents (carrier-free immobilization).
Carrier immobilization involves the attachment of par-
tially soluble enzymes to an insoluble solid support or
carriers (resins, microparticles, silica gels, graphene oxide,
carbon nanotubes, and nano-porous materials) via physical
(adsorption), ionic, or covalent bonding.[176] However, there
are some challenges associated with carrier immobilization.
Firstly, the leakage of partially soluble IBs from carriers due
to weak or improper binding. Secondly, nonspecific inter-
action between the active site of partially soluble enzymes
and the carrier decreases the catalytic activity of immobi-
lized IBs. Furthermore, carrier immobilization requires a
high amount of non-catalytic mass (carrier/support mater-
ial), which affects the yield and quality.[177]
The above-mentioned drawbacks of carrier-based immo-
bilization are overcome by entrapment or encapsulation of
the IBs in an insoluble polymer network made up of gelatin
beads, silica sol-gel, and hollow fiber or microcapsules. Since
the active site of the IB does not participate in any inter-
action with the encapsulating network, the structure and
function of the IBs remain in toto.[176,178] However, due to
mass transfer limitations owing to a bulky polymer network,
the catalytic efficiency of the immobilized IBs is reduced.
Carrier-free immobilization is based on cross-linking of
IBs with the help of linkers such as glutaraldehyde.[179] In
contrast to the entrapment and carrier-based immobilization
methods, this technique does not need any bulky polymer
matrix, thereby overcoming the mass-transfer-induced
reduction in the catalytic efficiency of the immobilized IBs.
In addition, the carrier-free method is a low-cost technique
for the immobilization of IBs yielding high purity catalyst
systems (due to the absence of the carrier support that
744 K. KACHHAWAHA ET AL.
potentially ends up as a process impurity), enhancing its
prospects of industrial applicability multi-fold.[180] The
cross-linking of IBs enhances their mechanical and thermal
stability and allows them to withstand extremely harsh pro-
cess conditions without affecting their catalytic activity.[176]
For instance, the carrier-free immobilization of the hyaluro-
nidase active IBs enhanced the cell permeability for drug
absorption, enhancing its pharmacokinetic/pharmacody-
namic (PK/PD) profile.[181] In addition, a plethora of
enzymes, including kinases, lyases, aldoses, oxidases, reduc-
tases, and phosphorylases, have been used as carrier-free or
self-immobilized formats for enhanced catalytic
activity.[182,183]
Another version of the carrier-free immobilized IBs that
are gaining prominence is the catalytically active IBs
(CatIBs). The CatIBs are solid and amorphous in nature and
contain a specific amount of catalytic activity.[184] This class
of IBs is heterogeneously produced inside the bacterial cell
(E. coli) and formed by the fusion of aggregation-prone
domain (fusion tags) to the N/C-terminus of the target pro-
tein.[185] The fusion tags do not interfere with the native
structure of the target protein (immobilized IBs). The
CatIBs have been widely used for the biosynthesis of cadav-
erine from the CatIBs of lysine decarboxylase (LDC).[186] A
recent application also suggests the co-immobilization of dif-
ferent enzymes within the same CatIBs to catalyze multistep
reactions.[187] Similarly, co-immobilization of CatIBs was
carried out in E. coli via co-expression of two fluorescent
proteins, which catalyzes a two-step reaction for the produc-
tion of (1 R,2R)-1-phenylpropane-1,2-diol (PPD).[188]
IBs as biocompatible and mechanically stable materials
IBs extoll the virtues of high mechanical stability due to
their ability to withstand cell lysis conditions during isola-
tion, proteolytic stability, and bio-adhesiveness property. IBs,
unlike active proteins, are not susceptible to changes in their
size, geometry, and biological activity during long-term stor-
age or incubation.[189] In addition, the porous nature of the
IBs renders them an excellent candidate for the development
of novel biomaterials as they hardly offer any mass-transfer
resistance to the ensuing flow regimes.[11] These characteris-
tics make them an excellent choice for developing novel
materials possessing superior elasticity, adhesives, and self-
assembling peptides spanning a wide range of
applications.[190]
One of the well-recognized uses of IBs as biomaterials is
their ability to be utilized as a drug delivery agent for sus-
tained drug release.[55] The ability emanates from its prop-
erty to mimic the endocrine release action of hormones[191]
and the ability to cross the membrane with minimal tox-
icity.[174] An example of such a system is proteinase-K-
resistant amyloid IBs.[192] These are self-releasing chemically
homogenous IBs, and themselves act as both the scaffold
material and the target drug substance. Another application
of IBs as biomaterials is for eliciting stimuli for cell prolifer-
ation and differentiation by providing adequate support for
cell adhesion during cell culture.[193] Ovalbumin-derived IBs
were used as an adjuvant for an immunogenic response dur-
ing vaccine development as IBs have potential dendritic cell
maturation capacity.[194] However, optimal cell colonization
is achieved by multi-factorial process optimization of param-
eters such as surface topography, size, wettability, and cellu-
lar migration.[195]
Current status and future prospects
The current breakthrough in the enhancement of the overall
efficacy of recovery of the active protein from bacterial IBs
could, to a large part, be attributed to the use of computa-
tional methods to analyze protein structure and identify the
potential product-related variants/impurities that could be
formed during refolding. Further, the availability of struc-
ture-function relationship data has enabled the identification
of critical quality attributes (CQAs) for the target prod-
uct.[196] The emphasis on the development of analytical
methods with improved precision, accuracy, robustness, and
sensitivity has opened avenues for a deeper understanding
of bacterial IBs and their bioprocessing.[197] For instance,
researchers have employed a combination of statistical
design of experiments (DOE) and high throughput process
development (HTPD) to explore a wide characterization
space for various process parameters and raw material
attributes under consideration.[198] In addition, process
understanding gleaned through these tools issued to create
appropriate real-time monitoring and control tools that
potentially serve as process analytical technology (PAT)
tool.[199] Further, use of these advanced methods of moni-
toring and analysis of bacterial IBs, the current endeavors
are to integrate the bioprocessing along with other estab-
lished unit operations during bio-manufacturing for con-
tinuous bio-processing to yield a highly productive
manufacturing process.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the Indian Institute of Technology
(Banaras Hindu University) Varanasi’s Seed Grant Ref. No IIT(BHU)/
Budget/19-(14)/2021-22/0975 dated 31-November-2021 to SKS.
References
[1] Moremen, K. W.; Ramiah, A.; Stuart, M.; Steel, J.; Meng, L.;
Forouhar, F.; Moniz, H. A.; Gahlay, G.; Gao, Z.; Chapla, D.;
et al. Expression System for Structural and Functional Studies
of Human Glycosylation Enzymes. Nat. Chem. Biol. 2018, 14,
156–162. DOI: 10.1038/nchembio.2539.
[2] Gileadi, O. Recombinant Protein Expression in E. Coli : A
Historical Perspective. Methods Mol. Biol. 2017, 1586, 3–10.
[3] Walsh, G. Biopharmaceutical Benchmarks 2018. Nat.
Biotechnol. 2018, 36, 1136–1145. DOI: 10.1038/nbt.4305.
[4] Buscajoni, L.; Martinetz, M. C.; Berkemeyer, M.; Brocard, C.
Refolding in the Modern Biopharmaceutical Industry.

Biotechnol. Adv. 2022, 61, 108050–108050. DOI: 10.1016/j.bio-
techadv.2022.108050.
[5] Hwang, P. M.; Pan, J. S.; Sykes, B. D. Targeted Expression,
Purification, and Cleavage of Fusion Proteins from Inclusion
Bodies in Escherichia Coli. FEBS Lett. 2014, 588, 247–252.
DOI: 10.1016/j.febslet.2013.09.028.
[6] Hirata, F.; Sugita, M.; Yoshida, M.; Akasaka, K. Perspective:
Structural Fluctuation of Protein and Anfinsen’s
Thermodynamic Hypothesis. J. Chem. Phys. 2018, 148,
020901. DOI: 10.1063/1.5013104.
[7] Mirhosseini, S. A.; Latifi, A. M.; Hosseini, H. M.; Seidmoradi,
R.; Aghamollaei, H.; Farnoosh, G. The Efficient Solubilization
and Refolding of Recombinant Organophosphorus Hydrolyses
Inclusion Bodies Produced in Escherichia Coli. J. Apple
Biotechnol. Rep. 2019, 6, 20–25. DOI: 10.29252/JABR.06.01.04.
[8] Sarker, A.; Rathore, A. S.; Gupta, R. D. Evaluation of ScFv
Protein Recovery from E. coli by in Vitro Refolding and Mild
Solubilization Process. Microb. Cell Fact. 2019, 18, 1–12. DOI:
10.1186/s12934-019-1053-9.
[9] Halan, V.; Maity, S.; Bhambure, R.; Rathore, A. S. Multimodal
Chromatography for Purification of Biotherapeutics – a
Review. Curr. Protein Pept. Sci. 2019, 20, 4–13. DOI: 10.2174/
1389203718666171020103559.
[10] Rosano, G. L.; Ceccarelli, E. A. Recombinant Protein
Expression in Escherichia Coli: Advances and Challenges.
Front Microbiol. 2014, 1–17.
[11] Corchero, J. L.; V
azquez, E.; Garc
ıa-Fruit
os, E.; Ferrer-
Miralles, N.; Villaverde, A. Recombinant Protein Materials for
Bioengineering and Nanomedicine. Nanomedicine (Lond)
2014, 9, 2817–2828. DOI: 10.2217/nnm.14.153.
[12] Singhvi, P.; Saneja, A.; Srichandan, S.; Panda, A. K. Bacterial
Inclusion Bodies: A Treasure Trove of Bioactive Proteins.
Trends Biotechnol. 2020, 38, 474–486. DOI: 10.1016/j.tibtech.
2019.12.011.
[13] Huang, C. J.; Lin, H.; Yang, X. Industrial Production of
Recombinant Therapeutics in Escherichia Coli and Its Recent
Advancements. J. Ind. Microbiol. Biotechnol. 2012, 39,
383–399. DOI: 10.1007/s10295-011-1082-9.
[14] Margreiter, G.; Messner, P.; Caldwell, K. D.; Bayer, K. Size
Characterization of Inclusion Bodies by Sedimentation Field-
Flow Fractionation. J. Biotechnol. 2008, 138, 67–73. DOI: 10.
1016/j.jbiotec.2008.07.1995.
[15] Bhatwa, A.; Wang, W.; Hassan, Y. I.; Abraham, N.; Li, X. Z.;
Zhou, T. Challenges Associated with the Formation of
Recombinant Protein Inclusion Bodies in Escherichia Coli and
Strategies to Address Them for Industrial Applications. Front
Bioeng. Biotechnol. 2021, 1–18.
[16] Taylor, G.; Hoare, M.; Gray, D. R.; Marston, F. A. O. Size and
Density of Protein Inclusion Bodies. Bio/Technology 1986, 4,
553–557.
[17] Humer, D.; Spadiut, O. Wanted: More Monitoring and
Control During Inclusion Body Processing. World J. Microbiol.
Biotechnol. 2018, 34, 1–9. DOI: 10.1007/s11274-018-2541-5.
[18] Singh, A.; Upadhyay, V.; Upadhyay, A. K.; Singh, S. M.;
Panda, A. K. Protein Recovery from Inclusion Bodies of
Escherichia Coli Using Mild Solubilization Process. Microb.
Cell Fact. 2015, 14, 10. DOI: 10.1186/s12934-015-0222-8.
[19] Khodabakhsh, F.; Zia, M. F.; Moazen, F.; Rabbani, M.;
Sadeghi, H. M. M. Comparison of the Cytoplasmic and
Periplasmic Production of Reteplase in Escherichia coli. Prep.
Biochem. Biotechnol. 2013, 43, 613–623. DOI: 10.1080/
10826068.2013.764896.
[20] Jaroentomeechai, T.; Stark, J. C.; Natarajan, A.; Glasscock,
C. J.; Yates, L. E.; Hsu, K. J.; Mrksich, M.; Jewett, M. C.;
Delisa, M. P. Single-Pot Glycoprotein Biosynthesis Using a
Cell-Free Transcription-Translation System Enriched With
Glycosylation Machinery. Nat. Commun. 2018, 9, 1–11.
[21] Schjoldager, K. T.; Narimatsu, Y.; Joshi, H. J.; Clausen, H.
Global View of Human Protein Glycosylation Pathways and
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 745
Functions. Nat. Rev. Mol. Cell Biol. 2020, 21, 729–749. DOI:
10.1038/s41580-020-00294-x.
[22] Clausen, H.; Wandall, H. H.; DeLisa, M. P.; Stanley, P.;
Schnaar, R. L. Chapter 56: Glycosylation Engineering. In
Essentials of Glycobiology: Varki, A., Cummings, R. D., Esko, J.
D., Stanley, P., Hart, G. W., Aebi, M., Mohnen, D., Kinoshita,
T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., Seeberger,
P. H., Eds.; Cold Spring Harbor Laboratory Press: New York,
2022; pp 1–16.
[23] Strutton, B.; Jaff
e, S. R. P.; Pandhal, J.; Wright, P. C.
Producing a Glycosylating Escherichia Coli Cell Factory: The
Placement of the Bacterial Oligosaccharyl Transferase pglB
onto the Genome. Biochem. Biophys. Res. Commun. 2018, 495,
686–692. DOI: 10.1016/j.bbrc.2017.11.023.
[24] Yates, L. E.; Natarajan, A.; Li, M.; Hale, M. E.; Mills, D. C.;
DeLisa, M. P. Glyco-Recoded Escherichia Coli:
Recombineering-Based Genome Editing of Native
Polysaccharide Biosynthesis Gene Clusters. Metab. Eng. 2019,
53, 59–68. DOI: 10.1016/j.ymben.2019.02.002.
[25] Benz, I.; Schmidt, M. A. Never Say Never Again: Protein
Glycosylation in Pathogenic Bacteria. Mol. Microbiol. 2002, 45,
267–276. DOI: 10.1046/j.1365-2958.2002.03030.x.
[26] Carri
o, M. M.; Villaverde, A. Protein Aggregation as Bacterial
Inclusion Bodies is Reversible. FEBS Lett. 2001, 489, 29–33.
DOI: 10.1016/s0014-5793(01)02073-7.
[27] S
anchez, J. M.; Carratal
a, J. V.; Gifre-Renom, L.; Ar
ıs, A.;
Garcia-Fruit
os, E.; Ferrer-Miralles, N. Quality Control of
Proteins Solubilized from Inclusion Bodies. Methods Mol. Biol.
2022, 2406, 469–477. DOI: 10.1007/978-1-0716-1859-2_28.
[28] Wang, L. Towards Revealing the Structure of Bacterial
Inclusion Bodies. Prion 2009, 3, 139–145. DOI: 10.4161/pri.3.
3.9922.
[29] J€
urgen, B.; Breitenstein, A.; Urlacher, V.; B€ uttner, K.; Lin, H.;
Hecker, M.; Schweder, T.; Neubauer, P. Quality Control of
Inclusion Bodies in Escherichia Coli. Microb. Cell Fact. 2010,
9, 13. DOI: 10.1186/1475-2859-9-41.
[30] Siew, Y. Y.; Zhang, W. Downstream Processing of
Recombinant Human Insulin and Its Analogues Production
from E. coli Inclusion Bodies. Bioresour. Bioprocess 2021, 8,
1–27.
[31] Rinas, U.; Garcia-Fruit
os, E.; Corchero, J. L.; V
azquez, E.;
Seras-Franzoso, J.; Villaverde, A. Bacterial Inclusion Bodies:
Discovering Their Better Half. Trends Biochem. Sci. 2017, 42,
726–737. DOI: 10.1016/j.tibs.2017.01.005.
[32] Coquel, A. S.; Jacob, J. P.; Primet, M.; Demarez, A.; Dimiccoli,
M.; Julou, T.; Moisan, L.; Lindner, A. B.; Berry, H.
Localization of Protein Aggregation in Escherichia Coli is
Governed by Diffusion and Nucleoid Macromolecular
Crowding Effect. PLoS Comput. Biol. 2013, 9, e1003038. DOI:
10.1371/journal.pcbi.1003038.
[33] Oliveira, S. M. D.; Neeli-Venkata, R.; Goncalves, N. S. M.;
Santinha, J. A.; Martins, L.; Tran, H.; M€akel€a, J.; Gupta, A.;
Barandas, M.; H€akkinen, A.; et al. Increased Cytoplasm
Viscosity Hampers Aggregate Polar Segregation in Escherichia
Coli. Mol. Microbiol. 2016, 99, 686–699. DOI: 10.1111/mmi.
13257.
[34] Laloux, G.; Jacobs-Wagner, C. How Do Bacteria Localize
Proteins to the Cell Pole? J. Cell Sci. 2014, 127, 11–19.
[35] Pu, Y.; Li, Y.; Jin, X.; Tian, T.; Ma, Q.; Zhao, Z.; Lin, S.;
Yuan , Chen, Z.; Li, B.; Yao, G.; et al. ATP-Dependent
Dynamic Protein Aggregation Regulates Bacterial Dormancy
Depth Critical for Antibiotic Tolerance. Mol. Cell 2019, 73,
143–156.e4. DOI: 10.1016/j.molcel.2018.10.022.
[36] Rokney, A.; Shagan, M.; Kessel, M.; Smith, Y.; Rosenshine, I.;
Oppenheim, AB. E. coli Transports Aggregated Proteins to the
Poles by a Specific and Energy-Dependent Process. J. Mol.
Biol. 2009, 392, 589–601. DOI: 10.1016/j.jmb.2009.07.009.
[37] Govers, S. K.; Dutr
e, P.; Aertsen, A. In Vivo Disassembly and
Reassembly of Protein Aggregates in Escherichia Coli. J.
Bacteriol. 2014, 196, 2325–2332. DOI: 10.1128/JB.01549-14.
746 K. KACHHAWAHA ET AL.
[38] Sun, X.; Shen, W.; Gao, Y.; Cai, M.; Zhou, M.; Zhang, Y.
Heterologous Expression and Purification of a Marine
Alginate Lyase in Escherichia Coli. Protein Expr. Purif. 2019,
153, 97–104. DOI: 10.1016/j.pep.2018.09.002.
[39] Upadhyay, A. K.; Murmu, A.; Singh, A.; Panda, A. K. Kinetics
of Inclusion Body Formation and Its Correlation With the
Characteristics of Protein Aggregates in Escherichia Coli. PLoS
One 2012, 7, e33951. DOI: 10.1371/journal.pone.0033951.
[40] Hashemzadeh, M. S.; Mohammadi, M.; Ghaleh, H. E. G.;
Sharti, M.; Choopani, A.; Panda, A. K. Expression,
Solubilization, Refolding and Final Purification of
Recombinant Proteins as Expressed in the Form of “Classical
Inclusion Bodies” in E. Coli. Protein Pept. Lett. 2021, 28,
122–130. DOI: 10.2174/0929866527999200729182831.
[41] Slouka, C.; Kopp, J.; Spadiut, O.; Herwig, C. Perspectives of
Inclusion Bodies for Bio-Based Products: Curse or Blessing?
Appl. Microbiol. Biotechnol. 2019, 103, 1143–1153. DOI: 10.
1007/s00253-018-9569-1.
[42] Belkov
a, M.; K€oszagov
a, R.; Nah
alka, J.; Belkov
a, M. M. Active
Inclusion Bodies: The Unexpected Journey. J. Microb. Biotech.
Food Sci. 2022, 12, e5951. DOI: 10.55251/jmbfs.5951.
[43] Peternel, S.; Bele, M.; Gaberc-Porekar, V.; Menart, V.
Nonclassical Inclusion Bodies in Escherichia Coli. Microb. Cell
Fact. 2006, 5, P23–2. DOI: 10.1186/1475-2859-5-S1-P23.
[44] Trinh, N. T. M.; Thuoc, T. L.; Thao, D. T. P. Production of
PEGylated GCSF from Non-Classical Inclusion Bodies
Expressed in Escherichia Coli. Avicenna J. Med. Biotechnol.
2021, 13, 192–200.
[45] Peternel, S. Designing Non-Classical Inclusion Bodies. In
Ribosomal Proteins and Protein Engineering; Ortendhal V.,
Salchow H., Eds; Nova Science Publishers: New York, 2010;
pp. 1–20.
[46] Villaverde, A.; Garc
ıa-Fruit
os, E.; Rinas, U.; Seras-Franzoso, J.;
Kosoy, A.; Corchero, J. L.; Vazquez, E. Packaging Protein
Drugs as Bacterial Inclusion Bodies for Therapeutic
Applications. Microb. Cell Fact. 2012, 11, 76. DOI: 10.1186/
1475-2859-11-76.
[47] Hannig, G.; Makrides, S. C. Strategies for Optimizing
Heterologous Protein Expression in Escherichia Coli. Trends
Biotechnol. 1998, 16, 54–60. DOI: 10.1016/s0167-
7799(97)01155-4.
[48] Ventura, S.; Villaverde, A. Protein Quality in Bacterial
Inclusion Bodies. Trends Biotechnol. 2006, 24, 179–185. DOI:
10.1016/j.tibtech.2006.02.007.
[49] Singhvi, P.; Panda, A. K. Solubilization and Refolding of
Inclusion Body Proteins. Methods Mol. Biol. 2022, 2406,
371–387. DOI: 10.1007/978-1-0716-1859-2_22.
[50] Rosano, G. L.; Morales, E. S.; Ceccarelli, E. A. New Tools for
Recombinant Protein Production in Escherichia Coli: A 5-Year
Update. Protein Sci. 2019, 28, 1412–1422. DOI: 10.1002/pro.
3668.
[51] Phoeurk, C.; Mushtaq, A. U.; Rogne, P.; Wolf-Watz, M.
Milligram Scale Expression, Refolding, and Purification of
Bombyx mori Cocoonase Using a Recombinant E. coli System.
Protein Expr Purif 2021, 186, 105919. DOI: 10.1016/j.pep.2021.
105919.
[52] Sanchez-Garcia, L.; Mart
ın, L.; Mangues, R.; Ferrer-Miralles,
N.; V
azquez, E.; Villaverde, A. Recombinant Pharmaceuticals
from Microbial Cells: A 2015 Update. Microb. Cell Fact 2016,
15, 1–7. DOI: 10.1186/s12934-016-0437-3.
[53] de Marco, A.; Ferrer-Miralles, N.; Garcia-Fruit
os, E.; Mitraki,
A.; Peternel, S.; Rinas, U.; Trujillo-Rold
an, M. A.; Valdez-
Cruz, N. A.; V
azquez, E.; Villaverde, A. Bacterial Inclusion
Bodies Are Industrially Exploitable Amyloids. FEMS Microbiol.
Rev. 2019, 43, 53–72. DOI: 10.1093/femsre/fuy038.
[54] Schumann, W.; Ferreira, L. C. S. Production of Recombinant
Proteins in Escherichia Coli. Genet. Mol. Biol 2004, 27,
442–453. DOI: 10.1590/S1415-47572004000300022.
[55] Seras-Franzoso, J.; Peebo, K.; Luis Corchero, J.; Tsimbouri,
P. M.; Unzueta, U.; Rinas, U.; Dalby, M. J.; Vazquez, E.;
Garc
ıa-Fruit
os, E.; Villaverde, A. A Nanostructured Bacterial
Bioscaffold for the Sustained Bottom-Up Delivery of Protein
Drugs. Nanomedicine (Lond). 2013, 8, 1587–1599. DOI: 10.
2217/nnm.12.188.
[56] Berlec, A.; Strukelj, B. Current State and Recent Advances in
Biopharmaceutical Production in Escherichia Coli, Yeasts and
Mammalian Cells. J. Ind. Microbiol. Biotechnol. 2013, 40,
257–274. DOI: 10.1007/s10295-013-1235-0.
[57] Carneiro, S.; Ferreira, E. C.; Rocha, I. Metabolic Responses to
Recombinant Bioprocesses in Escherichia Coli. J. Biotechnol.
2013, 164, 396–408. DOI: 10.1016/j.jbiotec.2012.08.026.
[58] Fakruddin, M.; Mazumdar, R. M.; Mannan, K. S. b.;
Chowdhury, A.; Hossain, M. N. Critical Factors Affecting the
Success of Cloning, Expression, and Mass Production of
Enzymes by Recombinant E. coli. ISRN Biotechnol. 2013,
590587, 1–7.
[59] Huleani, S.; Roberts, M. R.; Beales, L.; Papaioannou, E. H.
Escherichia Coli as an Antibody Expression Host for the
Production of Diagnostic Proteins: Significance and
Expression. Crit. Rev. Biotechnol. 2022, 42, 756–773. DOI: 10.
1080/07388551.2021.1967871.
[60] Joseph, B. C.; Pichaimuthu, S.; Srimeenakshi, S. An Overview
of the Parameters for Recombinant Protein Expression in
Escherichia Coli. J. Cell. Sci. Ther 2015, 6, 1–7. DOI: 10.4172/
2157-7013.1000221.
[61] Korkmaz, G.; Holm, M.; Wiens, T.; Sanyal, S. Comprehensive
Analysis of Stop Codon Usage in Bacteria and Its Correlation
with Release Factor Abundance. J. Biol. Chem. 2014, 289,
30334–30342. DOI: 10.1074/jbc.M114.606632.
[62] Liu, L.; Yang, H.; Shin, H.; Chen, R. R.; Li, J.; Du, G.; Chen, J.
How to Achieve High-Level Expression of Microbial Enzymes.
Bioengineered 2013, 4, 212–223. DOI: 10.4161/bioe.24761.
[64] Gopal, G. J.; Kumar, A. Strategies for the Production of
Recombinant Protein in Escherichia Coli. Protein. J. 2013, 32,
419–425. DOI: 10.1007/s10930-013-9502-5.
[64] Baeshen, M. N.; Al-Hejin, A. M.; Bora, R. S.; Ahmed,
M. M. M.; Ramadan, H. A. I.; Saini, K. S.; Baeshen, N. A.;
Redwan, E. M. Production of Biopharmaceuticals in E. Coli:
Current Scenario and Future Perspectives. J. Microbiol.
Biotechnol. 2015, 25, 953–962. DOI: 10.4014/jmb.1412.12079.
[65] Sørensen, H. P.; Mortensen, K. K. Advanced Genetic Strategies
for Recombinant Protein Expression in Escherichia Coli. J.
Biotechnol. 2005, 115, 113–128. DOI: 10.1016/j.jbiotec.2004.08.
004.
[66] Mauro, V. P.; Chappell, S. A. A Critical Analysis of Codon
Optimization in Human Therapeutics Optimizing Codon
Usage for Increased Protein Expression. Trends. Mol. Med.
2014, 20, 604–613. DOI: 10.1016/j.molmed.2014.09.003.
[67] M€uhlmann, M.; Forsten, E.; Noack, S.; B€ uchs, J. Optimizing
Recombinant Protein Expression via Automated Induction
Profiling in Microtiter Plates at Different Temperatures.
Microb. Cell. Fact 2017, 16, 1–12. DOI: 10.1186/s12934-017-
0832-4.
[68] Hayat, S. M. G.; Farahani, N.; Golichenari, B.; Sahebkar, A.
Recombinant Protein Expression in Escherichia Coli (E.Coli):
What We Need to Know. Curr. Pharm. Des. 2018, 24,
718–725. DOI: 10.2174/1381612824666180131121940.
[69] Papaneophytou, C. P.; Kontopidis, G. Statistical Approaches to
Maximize Recombinant Protein Expression in Escherichia Coli:
A General Review. Protein. Expr. Purif. 2014, 94, 22–32. DOI:
10.1016/j.pep.2013.10.016.
[70] Tsai, W. C.; Wu, T. C.; Chiang, B. L.; Wen, H. W. Cloning,
Expression, and Purification of Recombinant Major Mango
Allergen Man i 1 in Escherichia Coli. Protein. Expr. Purif.
2017, 130, 35–43. DOI: 10.1016/j.pep.2016.06.009.
[71] Rosano, G. L.; Ceccarelli, E. A. Recombinant Protein
Expression in Escherichia Coli: Advances and Challenges.
Front. Microbiol. 2014, 5, 172.
[72] Khow, O.; Suntrarachun, S. Strategies for Production of Active
Eukaryotic Proteins in Bacterial Expression System. Asian.

Pac. J. Trop. Biomed. 2012, 2, 159–162. DOI: 10.1016/S2221-
1691(11)60213-X.
[73] Coskun, K. A.; Yurekli, N.; Abay, E. C.; Tutar, M.; Al, M.;
Tutar, Y.; Coskun, K. A.; Yurekli, N.; Abay, E. C.; Tutar, M.,
Chapter 1: Structure- and Design-Based Difficulties in
Recombinant Protein Purification in Bacterial Expression. In
Protein Detection; Tutar, Y., Tutar, L., Eds.; IntechOpen
Limited: London, 2022; pp 1–19.
[74] Huang, C. J.; Peng, H. L.; Patel, A. K.; Singhania, R. R.; Dong,
C. d.; Cheng, C. Y. Effects of Lower Temperature on
Expression and Biochemical Characteristics of HCV NS3
Antigen Recombinant Protein. Catalysts 2021, 11, 1297. DOI:
10.3390/catal11111297.
[75] Farshdari, F.; Ahmadzadeh, M.; Nematollahi, L.; Mohit, E. The
Improvement of Anti-HER2 ScFv Soluble Expression in
Escherichia Coli. Braz. J. Pharm. Sci. 2020, 56, 17861. DOI: 10.
1590/s2175-97902019000317861.
[76] Li, R. Y.; Cheng, C. Y. Investigation of Inclusion Body
Formation in Recombinant Escherichia Coli with a Bioimaging
System. J. Biosci. Bioeng. 2009, 107, 512–515. DOI: 10.1016/j.
jbiosc.2009.01.025.
[77] Sugimoto, S.; Yokoo, Y.; Hatakeyama, N.; Yotsuji, A.; Teshiba,
S.; Hagino, H. Higher Culture PH is Preferable for Inclusion
Body Formation of Recombinant Salmon Growth Hormone in
Esherichia Coli. Biotechnol. Lett. 1991, 13, 385–388. DOI: 10.
1007/BF01030987.
[78] Wittung-Stafshede, P. Role of Cofactors in Protein Folding.
Acc. Chem. Res. 2002, 35, 201–208. DOI: 10.1021/ar010106e.
[79] Bae, S. W.; Eom, D.; Mai, N. L.; Koo, Y. M. Refolding of
Horseradish Peroxidase is Enhanced in Presence of Metal
Cofactors and Ionic Liquids. Biotechnol. J. 2016, 11, 464–472.
DOI: 10.1002/biot.201500142.
[80] Graumann, K.; Premstaller, A. Manufacturing of Recombinant
Therapeutic Proteins in Microbial Systems. Biotechnol. J. 2006,
1, 164–186. DOI: 10.1002/biot.200500051.
[81] Sandomenico, A.; Sivaccumar, J. P.; Ruvo, M. Evolution of
Escherichia Coli Expression System in Producing Antibody
Recombinant Fragments. Int. J. Mol. Sci. 2020, 21, 1–39.
[82] Kastenhofer, J.; Rajamanickam, V.; Libiseller-Egger, J.; Spadiut,
O. Monitoring and Control of E. coli Cell Integrity. J.
Biotechnol. 2021, 329, 1–12. DOI: 10.1016/j.jbiotec.2021.01.
009.
[83] Tripathi, N. K.; Shrivastava, A. Recent Developments in
Bioprocessing of Recombinant Proteins: Expression Hosts and
Process Development. Front. Bioeng. Biotechnol. 2019, 7, 1–35.
DOI: 10.3389/fbioe.2019.00420.
[84] Bellani, C. F.; Ajeian, J.; Duffy, L.; Miotto, M.; Groenewegen,
L.; Connon, C. J. Scale-Up Technologies for the Manufacture
of Adherent Cells. Front. Nutr. 2020, 7, 1–14.
[85] Neurohr, G. E.; Amon, A. Relevance and Regulation of Cell
Density. Trends. Cell. Biol. 2020, 30, 213–225. DOI: 10.1016/j.
tcb.2019.12.006.
[86] Subramaniam, R.; Thirumal, V.; Chistoserdov, A.; Bajpai, R.;
Bader, J.; Popovic, M. High-Density Cultivation in the
Production of Microbial Products. Chem. Biochem. Eng. Q
2019, 32, 451–464. DOI: 10.15255/CABEQ.2018.1394.
[87] Gupta, S. K.; Shukla, P. Advanced Technologies for Improved
Expression of Recombinant Proteins in Bacteria: Perspectives
and Applications. Crit. Rev. Biotechnol. 2016, 36, 1089–1098.
DOI: 10.3109/07388551.2015.1084264.
[88] Panda, A. K. Bioprocessing of Therapeutic Proteins from the
Inclusion Bodies of Escherichia Coli. Adv. Biochem. Eng.
Biotechnol. 2003, 85, 43–93. DOI: 10.1007/3-540-36466-8_3.
[89] Chiba, C. H.; Knirsch, M. C.; Azzoni, A. R.; Moreira, A. R.;
Stephano, M. A. Cell-Free Protein Synthesis: Advances on
Production Process for Biopharmaceuticals and
Immunobiological Products. Biotechniques 2021, 70, 126–133.
DOI: 10.2144/btn-2020-0155.
[90] Khambhati, K.; Bhattacharjee, G.; Gohil, N.; Braddick, D.;
Kulkarni, V.; Singh, V. Exploring the Potential of Cell-Free
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 747
Protein Synthesis for Extending the Abilities of Biological
Systems. Front. Bioeng. Biotechnol. 2019, 7, 1–16.
[91] Guzman-Chavez, F.; Arce, A.; Adhikari, A.; Vadhin, S.;
Pedroza-Garcia, J. A.; Gandini, C.; Ajioka, J. W.; Molloy, J.;
Sanchez-Nieto, S.; Varner, J. D.; et al. Constructing Cell-Free
Expression Systems for Low-Cost Access. ACS. Synth. Biol.
2022, 11, 1114–1128. DOI: 10.1021/acssynbio.1c00342.
[92] Islam, M. S.; Aryasomayajula, A.; Selvaganapathy, P. R. A
Review on Macroscale and Microscale Cell Lysis Methods.
Micromachines 2017, 8, 83. DOI: 10.3390/mi8030083.
[93] Gomes, T. A.; Zanette, C. M.; Spier, M. R. An Overview of
Cell Disruption Methods for Intracellular Biomolecules
Recovery. Prep. Biochem. Biotechnol. 2020, 50, 635–654. DOI:
10.1080/10826068.2020.1728696.
[94] Pyatkovskyy, T. I.; Shynkaryk, M. v.; Mohamed, H. M.;
Yousef, A. E.; Sastry, S. K. Effects of Combined High Pressure
(HPP), Pulsed Electric Field (PEF) and Sonication Treatments
on Inactivation of Listeria Innocua. J. Food. Eng. 2018, 233,
49–56. DOI: 10.1016/j.jfoodeng.2018.04.002.
[95] Harrison, S. T. L. Bacterial Cell Disruption: A Key Unit
Operation in the Recovery of Intracellular Products.
Biotechnol. Adv 1991, 9, 217–240. DOI: 10.1016/0734-
9750(91)90005-G.
[96] Asenjo, J. A. Product Release and Recovery. Sep. Processes
Biotechnol. Part II 2020, 1, 142.
[97] Haddad, L.; Babaeipour, V.; Mofid, M. R. The Effect of Cell
Disruption Techniques and Chaotropic Agents on the
Downstream Purification Process of Mecasermin Produced as
Inclusion Body in E. coli. Res. Pharm. Sci. 2015, 10, 553–561.
[98] Primo, E. D.; Otero, L. H.; Ruiz, F.; Klinke, S.; Giordano, W.
The Disruptive Effect of Lysozyme on the Bacterial Cell Wall
Explored by an In-Silico Structural Outlook. Biochem. Mol.
Biol. Educ. 2018, 46, 83–90. DOI: 10.1002/bmb.21092.
[99] Martins, M.; Ooi, C. W.; Neves, M. C.; Pereira, J. F. B.;
Coutinho, J. A. P.; Ventura, S. P. M. Extraction of
Recombinant Proteins from Escherichia Coli by Cell
Disruption With Aqueous Solutions of Surface-Active
Compounds. J. Chem. Technol. Biotechnol. 2018, 93,
1864–1870. DOI: 10.1002/jctb.5596.
[100] Pekarsky, A.; Spadiut, O.; Rajamanickam, V.; Wurm, D. J. A
Fast and Simple Approach to Optimize the Unit Operation
High Pressure Homogenization - A Case Study for a Soluble
Therapeutic Protein in E. Coli. Prep. Biochem. Biotechnol.
2019, 49, 74–81. DOI: 10.1080/10826068.2018.1536988.
[101] Sharma, M.; Tyagi, J. L.; Poluri, K. M. Quantifying Bacterial
Cell Lysis Using GFP Based Fluorimetric Assay. Int. J. Biol.
Macromol. 2019, 138, 881–889. DOI: 10.1016/j.ijbiomac.2019.
07.172.
[102] Baumann, P.; Hubbuch, J. Downstream Process Development
Strategies for Effective Bioprocesses: Trends, Progress, and
Combinatorial Approaches. Eng. Life. Sci. 2017, 17,
1142–1158. DOI: 10.1002/elsc.201600033.
[103] Rodr
ıguez-Carmona, E.; Cano-Garrido, O.; Seras-Franzoso, J.;
Villaverde, A.; Garc
ıa-Fruit
os, E. Isolation of Cell-Free
Bacterial Inclusion Bodies. Microb. Cell. Fact 2010, 9, 1–9.
DOI: 10.1186/1475-2859-9-71.
[104] Rueda, F.; Cano-Garrido, O.; Mamat, U.; Wilke, K.; Seras-
Franzoso, J.; Garc
ıa-Fruit

os, E.; Villaverde, A. Production of
Functional Inclusion Bodies in Endotoxin-Free Escherichia
Coli. Appl. Microbiol. Biotechnol. 2014, 98, 9229–9238. DOI:
10.1007/s00253-014-6008-9.
[105] Singh, A.; Upadhyay, V.; Panda, A. K. Solubilization and
Refolding of Inclusion Body Proteins. Methods. Mol. Biol.
2015, 1258, 283–291. DOI: 10.1007/978-1-4939-2205-5_15.
[106] Walther, C.; Mayer, S.; Sekot, G.; Antos, D.; Hahn, R.;
Jungbauer, A.; D€ urauer, A. Mechanism and Model for
Solubilization of Inclusion Bodies. Chem. Eng. Sci. 2013, 101,
631–641. DOI: 10.1016/j.ces.2013.07.026.
[107] Fischer, B.; Sumner, I.; Goodenough, P. Isolation,
Renaturation, and Formation of Disulfide Bonds of Eukaryotic
748 K. KACHHAWAHA ET AL.
Proteins Expressed in Escherichia Coli as Inclusion Bodies.
Biotechnol. Bioeng. 1993, 41, 3–13. DOI: 10.1002/bit.
260410103.
[108] Datta, I.; Gautam, S.; Gupta, M. N. Microwave Assisted
Solubilization of Inclusion Bodies. Sustain. Chem. Processes.
2013, 1, 1–7.
[109] Mukherjee, J.; Nath Gupta, M. Paradigm Shifts in Our Views
on Inclusion Bodies. CBE 2015, 3, 47–55. DOI: 10.2174/
2212711902666150302212051.
[110] Tsumoto, K.; Abe, R.; Ejima, D.; Arakawa, T. Non-Denaturing
Solubilization of Inclusion Bodies. Curr. Pharm. Biotechnol.
2010, 11, 309–312. DOI: 10.2174/138920110791111924.
[111] Mohammadian, A.; Kaghazian, H.; Kavianpour, A.; Jalalirad,
R. Solubilization of Inclusion Body Proteins Using Low and
Very Low Concentrations of Chemicals: Implications of Novel
Combined Chemical Treatment Designs in Enhancement of
Post-Solubilization Target Protein Purity and Biological
Activity. J. Chem. Technol. Biotechnol. 2018, 93, 1579–1587.
DOI: 10.1002/jctb.5525.
[112] Padhiar, A. A.; Chanda, W.; Joseph, T. P.; Guo, X.; Liu, M.;
Sha, L.; Batool, S.; Gao, Y.; Zhang, W.; Huang, M.; et al.
Comparative Study to Develop a Single Method for Retrieving
Wide Class of Recombinant Proteins from Classical Inclusion
Bodies. Appl. Microbiol. Biotechnol. 2018, 102, 2363–2377.
DOI: 10.1007/s00253-018-8754-6.
[113] Rosa Da Silva, C. M.; Chura-Chambi, R. M.; Ramos Pereira,
L.; Cordeiro, Y.; de Souza Ferreira, L. C.; Morganti, L.
Association of High Pressure and Alkaline Condition for
Solubilization of Inclusion Bodies and Refolding of the NS1
Protein from Zika Virus. BMC. Biotechnol. 2018, 18, 1–10.
DOI: 10.1186/s12896-018-0486-2.
[114] Chura-Chambi, R. M.; de Brand~ao Prieto-Da-Silva, A. R.; di
Lela, M. M.; Oliveira, J. E.; Abreu, P. E. A.; Meireles, L. R.; de
Andrade Junior, H. F.; Morganti, L. High Level SARS-CoV-2
Nucleocapsid Refolding Using Mild Condition for Inclusion
Bodies Solubilization: Application of High Pressure at PH 9.0.
PLoS. One 2022, 17, e0262591. DOI: 10.1371/journal.pone.
0262591.
[115] Maksum, I. P.; Yosua, Y.; Nabiel, A.; Pratiwi, R. D.; Sriwidodo,
S.; Soedjanaatmadja, U. M. S. Refolding of Bioactive Human
Epidermal Growth Factor from E. coli BL21(DE3) Inclusion
Bodies & Evaluations on Its In Vitro & In Vivo Bioactivity.
Heliyon 2022, 8, e09306. DOI: 10.1016/j.heliyon.2022.e09306.
[116] Costa, S.; Almeida, A.; Castro, A.; Domingues, L. Fusion Tags
for Protein Solubility, Purification, and Immunogenicity in
Escherichia Coli: The Novel Fh8 System. Front. Microbiol.
2014, 5, 63.
[117] Fatima, K.; Naqvi, F.; Younas, H. A Review: Molecular
Chaperone-Mediated Folding, Unfolding and Disaggregation
of Expressed Recombinant Proteins. Cell. Biochem. Biophys.
2021, 79, 153–174. DOI: 10.1007/s12013-021-00970-5.
[118] Xie, X.; Wu, P.; Huang, X.; Bai, W. F.; Li, B.; Shi, N. Retro-
Protein XXA is a Remarkable Solubilizing Fusion Tag for
Inclusion Bodies. Microb. Cell. Fact 2022, 21, 15. DOI: 10.
1186/s12934-022-01776-7.
[119] Ki, M. R.; Pack, S. P. Fusion Tags to Enhance Heterologous
Protein Expression. Appl. Microbiol. Biotechnol. 2020, 104,
2411–2425. DOI: 10.1007/s00253-020-10402-8.
[120] Schramm, F. D.; Schroeder, K.; Jonas, K. Protein Aggregation
in Bacteria. FEMS. Microbiol. Rev. 2020, 44, 54–72. DOI: 10.
1093/femsre/fuz026.
[121] Hirota, N.; Edskes, H.; Hall, D. Unified Theoretical
Description of the Kinetics of Protein Aggregation. Biophys.
Rev. 2019, 11, 191–208. DOI: 10.1007/s12551-019-00506-5.
[122] Akiba, H.; Tsumoto, K. Expression in Bacteria and Refolding.
In Advance Methods in Structural Biology; Senda, T., Maenaka,
K., Eds.; Springer Protocols Handbooks: Tokyo, 2016; pp.
3–23.
[123] Al-Ayoubi, S. R.; Schummel, P. H.; Golub, M.; Peters, J.;
Winter, R. Influence of Cosolvents, Self-Crowding,
Temperature and Pressure on the Sub-Nanosecond Dynamics
and Folding Stability of Lysozyme. Phys. Chem. Chem. Phys.
2017, 19, 14230–14237. DOI: 10.1039/C7CP00705A.
[124] Phelps, D. J.; Hesterberg, L. K. Protein Disaggregation and
Refolding Using High Hydrostatic Pressure. J. Chem. Technol.
Biotechnol. 2007, 82, 610–613. DOI: 10.1002/jctb.1707.
[125] Mizutani, H.; Sugawara, H.; Buckle, A. M.; Sangawa, T.;
Miyazono, K. I.; Ohtsuka, J.; Nagata, K.; Shojima, T.; Nosaki,
S.; Xu, Y.; et al. REFOLDdb: A New and Sustainable Gateway
to Experimental Protocols for Protein Refolding. BMC. Struct.
Biol. 2018, 17, 8. DOI: 10.1186/s12900-017-0074-z.
[126] Chow, M. K. M.; Amin, A. A.; Fulton, K. F.; Fernando, T.;
Kamau, L.; Batty, C.; Louca, M.; Ho, S.; Whisstock, J. C.;
Bottomley, S. P.; et al. The REFOLD Database: A Tool for the
Optimization of Protein Expression and Refolding. Nucleic.
Acids Res. 2006, 34, D207–D212. DOI: 10.1093/nar/gkj080.
[127] Yamaguchi, H.; Miyazaki, M. Refolding Techniques for
Recovering Biologically Active Recombinant Proteins from
Inclusion Bodies. Biomolecules 2014, 4, 235–251. DOI: 10.
3390/biom4010235.
[128] Kato, A.; Ohashi, H. Quick Refolding of High-Concentration
Proteins Via Microchannel Dialysis. Ind. Eng. Chem. Res.
2021, 60, 10076–10082. DOI: 10.1021/acs.iecr.1c00410.
[129] Kashanian, F.; Masoudi, M. M.; Shamloo, A.; Habibi-Rezaei,
M.; Moosavi-Movahedi, A. A. Modeling, Simulation, and
Employing Dilution–Dialysis Microfluidic Chip (DDMC) for
Heightening Proteins Refolding Efficiency. Bioprocess. Biosyst.
Eng. 2018, 41, 707–714. DOI: 10.1007/s00449-018-1904-5.
[130] Nishinami, S.; Yoshizawa, S.; Arakawa, T.; Shiraki, K.
Allantoin and Hydantoin as New Protein Aggregation
Suppressors. Int. J. Biol. Macromol. 2018, 114, 497–503. DOI:
10.1016/j.ijbiomac.2018.03.011.
[131] Brusotti, G.; Calleri, E.; Colombo, R.; Massolini, G.; Rinaldi,
F.; Temporini, C. Advances on Size Exclusion
Chromatography and Applications on the Analysis of Protein
Biopharmaceuticals and Protein Aggregates: A Mini Review.
Chromatographia 2018, 81, 3–23. DOI: 10.1007/s10337-017-
3380-5.
[132] Burgess, R. R. A Brief Practical Review of Size Exclusion
Chromatography: Rules of Thumb, Limitations, and
Troubleshooting. Protein. Expr. Purif. 2018, 150, 81–85. DOI:
10.1016/j.pep.2018.05.007.
[133] Zhang, K.; Liu, X. Reprint of “Mixed-Mode Chromatography
in Pharmaceutical and Biopharmaceutical Applications”. J.
Pharm. Biomed. Anal. 2016, 130, 19–34. DOI: 10.1016/j.jpba.
2016.09.013.
[134] Eriksson, K. O., Chapter 19: Hydrophobic Interaction
Chromatography. In Biopharmaceutical Processing:
Development, Design, and Implementation of Manufacturing
Processes; Jagschies, G., Lindskog, E., Ła˛cki, K., Galliher, P.,
Eds.; Elsevier: Amsterdam, 2017; pp. 401–408.
[135] Kaur, J.; Singh, A.; Panda, A. K.; Lal, R. Protocol for In-Vitro
Purification and Refolding of Hexachlorocyclohexane
Degrading Enzyme Haloalkane Dehalogenase LinB from
Inclusion Bodies. Enzyme. Microb. Technol. 2021, 146, 109760.
DOI: 10.1016/j.enzmictec.2021.109760.
[136] Ma, S.; Li, Y.; Ma, C.; Wang, Y.; Ou, J.; Ye, M. Challenges and
Advances in the Fabrication of Monolithic Bioseparation
Materials and Their Applications in Proteomics Research. Adv.
Mater. 2019, 31, 1902023–1902027. DOI: 10.1002/adma.
201902023.
[137] Hoffmann, D.; Leber, J.; Loewe, D.; Lothert, K.; Oppermann,
T.; Zitzmann, J.; Weidner, T.; Salzig, D.; Wolff, M.; Czermak,
P., Chapter 5: Purification of New Biologicals Using
Membrane-Based Processes. In Current Trends and Future
Developments on (Bio-) Membranes: Membrane Processes in the
Pharmaceutical and Biotechnological Field; Basile, A.,
Charcosset, C., Eds.; Elsevier: Amsterdam, 2019; pp. 123–150.
[138] Poddar, S.; Sharmeen, S.; Hage, D. S. Affinity Monolith
Chromatography: A Review of General Principles and Recent

Developments. Electrophoresis 2021, 42, 2577–2598. DOI: 10.
1002/elps.202100163.
[139] Dods, S. R.; Hardick, O.; Stevens, B.; Bracewell, D. G.
Fabricating Electrospun Cellulose Nanofibre Adsorbents for
Ion-Exchange Chromatography. J. Chromatogr. A 2015, 1376,
74–83. DOI: 10.1016/j.chroma.2014.12.010.
[140] Fu, Q.; Duan, C.; Yan, Z.; Si, Y.; Liu, L.; Yu, J.; Ding, B.
Electrospun Nanofibrous Composite Materials: A Versatile
Platform for High Efficiency Protein Adsorption and
Separation. Compos. Commun. 2018, 8, 92–100. DOI: 10.1016/
j.coco.2017.11.007.
[141] Miles, A. J.; Janes, R. W.; Wallace, B. A. Tools and Methods
for Circular Dichroism Spectroscopy of Proteins: A Tutorial
Review. Chem. Soc. Rev. 2021, 50, 8400–8413. DOI: 10.1039/
d0cs00558d.
[142] Pathak, M.; Dixit, S.; Muthukumar, S.; Rathore, A. S.
Analytical Characterization of In Vitro Refolding in the
Quality by Design Paradigm: Refolding of Recombinant
Human Granulocyte Colony Stimulating Factor. J. Pharm.
Biomed. Anal. 2016, 126, 124–131. DOI: 10.1016/j.jpba.2016.
05.001.
[143] Ling, C.; Zhang, J.; Lin, D.; Tao, A. Approaches for the
Generation of Active Papain-like Cysteine Proteases
from Inclusion Bodies of Escherichia Coli. World. J.
Microbiol. Biotechnol. 2015, 31, 681–690. DOI: 10.1007/
s11274-015-1804-7.
[144] Zhang, H.; Zheng, X.; Kwok, R. T. K.; Wang, J.; Leung,
N. L. C.; Shi, L.; Sun, J. Z.; Tang, Z.; Lam, J. W. Y.; Qin, A.;
et al. In Situ Monitoring of Molecular Aggregation Using
Circular Dichroism. Nat. Commun. 2018, 9, 1–9. DOI: 10.
1038/s41467-018-07299-3.
[145] Kelly, S. M.; Price, N. C. The Use of Circular Dichroism in
the Investigation of Protein Structure and Function. Curr.
Protein. Pept. Sci. 2000, 1, 349–384. DOI: 10.2174/
1389203003381315.
[146] Gatti-Lafranconi, P.; Natalello, A.; Ami, D.; Doglia, S. M.;
Lotti, M. Concepts and Tools to Exploit the Potential of
Bacterial Inclusion Bodies in Protein Science and
Biotechnology. FEBS. J. 2011, 278, 2408–2418. DOI: 10.1111/j.
1742-4658.2011.08163.x.
[147] Pignataro, M. F.; Herrera, M. G.; Dodero, V. I. Evaluation of
Peptide/Protein Self-Assembly and Aggregation by
Spectroscopic Methods. Molecules 2020, 25, 4854–4140. DOI:
10.3390/molecules25204854.
[148] Dodero, V. I.; Messina, P. V., Chapter 4: Analyzing the
Solution State of Protein Structure, Interactions, and Ligands
by Spectroscopic Methods. In Proteins in Solution and at
Interfaces: Methods and Applications in Biotechnology and
Materials Science; Ruso, J. M., Pi~ neiro, A., Eds.; Wiley: New
Jersey, 2013; pp. 73–98.
[149] Hawe, A.; Sutter, M.; Jiskoot, W. Extrinsic Fluorescent Dyes as
Tools for Protein Characterization. Pharm. Res. 2008, 25,
1487–1499. DOI: 10.1007/s11095-007-9516-9.
[150] Tokunaga, Y.; Takeuchi, K. Role of NMR in High Ordered
Structure Characterization of Monoclonal Antibodies. Int. J.
Mol. Sci. 2021, 22, 1–12.
[151] Humer, D.; Spadiut, O. Wanted: More Monitoring and
Control During Inclusion Body Processing. World. J.
Microbiol. Biotechnol. 2018, 34, 1–9. DOI: 10.1007/s11274-018-
2541-5.
[152] Schaefer, A.; Naser, D.; Siebeneichler, B.; Tarasca, M. V.;
Meiering, E. M. Methodological Advances and Strategies for
High Resolution Structure Determination of Cellular Protein
Aggregates. J. Biol. Chem. 2022, 298, 102197. DOI: 10.1016/j.
jbc.2022.102197.
[153] Kamatari, Y. O.; Kitahara, R.; Yamada, H.; Yokoyama, S.;
Akasaka, K. High-Pressure NMR Spectroscopy for
Characterizing Folding Intermediates and Denatured States of
Proteins. Methods 2004, 34, 133–143. DOI: 10.1016/j.ymeth.
2004.03.010.
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 749
[154] Taraban, M. B.; Depaz, R. A.; Lobo, B.; Yu, Y. B. Use of
Water Proton NMR to Characterize Protein Aggregates:
Gauging the Response and Sensitivity. Anal. Chem. 2019, 91,
4107–4115. DOI: 10.1021/acs.analchem.8b05733.
[155] Bansal, R.; Gupta, S.; Rathore, A. S. Analytical Platform for
Monitoring Aggregation of Monoclonal Antibody
Therapeutics. Pharm. Res. 2019, 36, 1–11. DOI: 10.1007/
s11095-019-2690-8.
[156] Stetefeld, J.; McKenna, S. A.; Patel, T. R. Dynamic Light
Scattering: A Practical Guide and Applications in Biomedical
Sciences. Biophys. Rev. 2016, 8, 409–427. DOI: 10.1007/
s12551-016-0218-6.
[157] Aggregates, P. Protein Aggregates. Biophys. J. 2008, 94,
933–946.
[158] Philo, J. S. Is Any Measurement Method Optimal for All
Aggregate Sizes and Types? AAPS J. 2006, 8, E564–E571. DOI:
10.1208/aapsj080365.
[159] Tatkiewicz, W.; Elizondo, E.; Moreno, E.; D
ıez-Gil, C.;
Ventosa, N.; Veciana, J.; Ratera, I. Methods for
Characterization of Protein Aggregates. Methods. Mol. Biol.
2015, 1258, 387–401. DOI: 10.1007/978-1-4939-2205-5_22.
[160] Kumar, M.; Pant, A.; Bansal, R.; Pandey, A.; Gomes, J.; Khare,
K.; Singh Rathore, A.; Banerjee, M. Electron Microscopy-Based
Semi-Automated Characterization of Aggregation in
Monoclonal Antibody Products. Comput. Struct. Biotechnol. J.
2020, 18, 1458–1465. DOI: 10.1016/j.csbj.2020.06.009.
[161] Hurtley, S. M. Continuing the Resolution Revolution. Science
2018, 360, 280.11–282. DOI: 10.1126/science.360.6386.280-k.
[162] Almeida, Z. L.; Brito, R. M. M. Structure and Aggregation
Mechanisms in Amyloids. Molecules 2020, 25, 1195–1130.
DOI: 10.3390/molecules25051195.
[163] Schluter, H. Reversed-Phase Chromatography. J. Chromatogr.
A 2000, 61, 147–234.
[164] Hong, P.; Koza, S.; Bouvier, E. S. P. A Review Size-Exclusion
Chromatography for the Analysis of Protein Biotherapeutics
and Their Aggregates. J. Liq. Chromatogr. Relat. Technol.
2012, 35, 2923–2950. DOI: 10.1080/10826076.2012.743724.
[165] Haberger, M.; Leiss, M.; Heidenreich, A.-K.; Pester, O.;
Hafenmair, G.; Hook, M.; Bonnington, L.; Wegele, H.; Haindl,
M.; Reusch, D.; Bulau, P. Rapid Characterization of
Biotherapeutic Proteins by Size-Exclusion Chromatography
Coupled to Native Mass Spectrometry. MABS 2016, 8,
331–339. DOI: 10.1080/19420862.2015.1122150.
[166] Castagnola, M. Capillary Electrophoresis: Principles, Practice
and Applications. J. Chromatogr. A 1992, 52, 118.
[167] Espinosa-de la Garza, C. E.; Perdomo-Ab
undez, F. C.;
Campos-Garc
ıa, V. R.; P
erez, N. O.; Flores-Ortiz, L. F.;
Medina-Rivero, E. Capillary Gel Electrophoresis for the
Quantification and Purity Determination of Recombinant
Proteins in Inclusion Bodies. Electrophoresis 2013, 34,
2754–2759. DOI: 10.1002/elps.201300232.
[168] Formolo, T.; Ly, M.; Levy, M.; Kilpatrick, L.; Lute, S.; Phinney,
K.; Marzilli, L.; Brorson, K.; Boyne, M.; Davis, D.
Determination of the NISTmAb Primary Structure. ACS Symp.
Ser. 2015, 1201, 1–62.
[169] Gani, K.; Chirmade, T.; Ughade, S.; Thulasiram, H.;
Bhambure, R. Understanding Unfolding and Refolding of the
Antibody Fragment (Fab) III: Mapping Covalent and Non-
Covalent Interactions During In-Vitro Refolding of Light
Chain, Heavy Chain, and Fab. Biochem. Eng. J. 2022, 187,
108644. DOI: 10.1016/j.bej.2022.108644.
[170] Puranik, A.; Rasam, P.; Dandekar, P.; Jain, R. Development
and Optimization of a LC-MS Based Multi-Attribute Method
(MAM) Workflow for Characterization of Therapeutic Fc-
Fusion Protein. Anal. Biochem. 2023, 660, 114969. DOI: 10.
1016/j.ab.2022.114969.
[171] Rosenberg, A. S. Effects of Protein Aggregates: An
Immunologic Perspective. AAPS. J 2006, 8, E501–E507. DOI:
10.1208/aapsj080359.
750 K. KACHHAWAHA ET AL.
[172] Garc
ıa-Fruit
os, E.; V
azquez, E.; D
ıez-Gil, C.; Corchero, J. L.;
Seras-Franzoso, J.; Ratera, I.; Veciana, J.; Villaverde, A.
Bacterial Inclusion Bodies: Making Gold from Waste. Trends
Biotechnol 2012, 30, 65–70. DOI: 10.1016/j.tibtech.2011.09.003.
[173] Serna, N.; Cano-Garrido, O.; S
anchez, J. M.; S
anchez-Chardi,
A.; S
anchez-Garc
ıa, L.; L
opez-Laguna, H.; Fern
andez, E.;
V
azquez, E.; Villaverde, A. Release of Functional Fibroblast
Growth Factor-2 from Artificial Inclusion Bodies. J. Control
Release 2020, 327, 61–69. DOI: 10.1016/j.jconrel.2020.08.007.
[174] S
anchez, J. M.; L


opez-Laguna, H.; Alamo, P.; Serna, N.;
S
anchez-Chardi, A.; Nolan, V.; Cano-Garrido, O.; Casanova,
I.; Unzueta, U.; Vazquez, E.; et al. Artificial Inclusion Bodies
for Clinical Development. Adv. Sci. (Weinh) 2020, 7, 1902420.
DOI: 10.1002/advs.201902420.
[175] Liese, A.; Hilterhaus, L. Evaluation of Immobilized Enzymes
for Industrial Applications. Chem. Soc. Rev. 2013, 42,
6236–6249. DOI: 10.1039/c3cs35511j.
[176] Rehm, F. B. H.; Chen, S.; Rehm, B. H. A. Enzyme Engineering
for In Situ Immobilization. Molecules 2016, 21, 1370. DOI: 10.
3390/molecules21101370.
[177] Santos, J. C. S. D.; Barbosa, O.; Ortiz, C.; Berenguer-Murcia,
A.; Rodrigues, R. C.; Fernandez-Lafuente, R. Importance of
the Support Properties for Immobilization or Purification of
Enzymes. ChemCatChem 2015, 7, 2413–2432. DOI: 10.1002/
cctc.201500310.
[178] Mestrom, L.; Marsden, S. R.; McMillan, D. G. G.; Schoevaart,
R.; Hagedoorn, P. L.; Hanefeld, U. Comparison of Enzymes
Immobilised on Immobeads and Inclusion Bodies: A Case
Study of a Trehalose Transferase. ChemCatChem 2020, 12,
3249–3256. DOI: 10.1002/cctc.202000241.
[179] Migneault, I.; Dartiguenave, C.; Bertrand, M. J.; Waldron,
K. C. Glutaraldehyde: Behavior in Aqueous Solution, Reaction
with Proteins, and Application to Enzyme Crosslinking.
Biotechniques 2004, 37, 790–802. DOI: 10.2144/04375RV01.
[180] Hrab
arov
a, E.; Achbergerov
a, L.; Nah
alka, J. Insoluble Protein
Applications: The Use of Bacterial Inclusion Bodies as
Biocatalysts. Methods. Mol. Biol. 2015, 1258, 411–422. DOI:
10.1007/978-1-4939-2205-5_24.
[181] Guo, X.; Liu, F.; Zhu, X.; Su, Y.; Ling, P. Expression of a
Novel Hyaluronidase from Streptococcus Zooepidemicus in
Escherichia Coli and Its Application for the Preparation of HA
Oligosaccharides. Carbohydr. Polym. 2009, 77, 254–260. DOI:
10.1016/j.carbpol.2008.12.036.
[182] Kloss, R.; Karmainski, T.; J€ager, V. D.; Hahn, D.; Gr€ unberger,
A.; Baumgart, M.; Krauss, U.; Jaeger, K. E.; Wiechert, W.;
Pohl, M. Tailor-Made Catalytically Active Inclusion Bodies for
Different Applications in Biocatalysis. Catal. Sci. Technol.
2018, 8, 5816–5826. DOI: 10.1039/C8CY01891J.
[183] Han, H.; Zeng, W.; Zhang, G.; Zhou, J. Active Tyrosine
Phenol-Lyase Aggregates Induced by Terminally Attached
Functional Peptides in Escherichia Coli. J. Ind. Microbiol.
Biotechnol. 2020, 47, 563–571. DOI: 10.1007/s10295-020-
02294-4.
[184] J€ager, V. D.; Lamm, R.; K€ € uc€
usters, K.; Olç€ u, G.; Oldiges, M.;
Jaeger, K. E.; B€ uchs, J.; Krauss, U. Catalytically-Active
Inclusion Bodies for Biotechnology—General Concepts,
Optimization, and Application. Appl. Microbiol. Biotechnol.
2020, 104, 7313–7329. DOI: 10.1007/s00253-020-10760-3.
[185] € uc€
Olç€ u, G.; Baumer, B.; K€ usters, K.; M€ollenhoff, K.; Oldiges,
M.; Pietruszka, J.; Jaeger, K. E.; Krauss, U. Catalytically Active
Inclusion Bodies-Benchmarking and Application in Flow
Chemistry. ACS. Synth. Biol. 2022, 11, 1881–1896. DOI: 10.
1021/acssynbio.2c00035.
[186] Ma, W.; Chen, K.; Li, Y.; Hao, N.; Wang, X.; Ouyang, P.
Advances in Cadaverine Bacterial Production and Its
Applications. Engineering 2017, 3, 308–317. DOI: 10.1016/J.
ENG.2017.03.012.
[187] Han, G. H.; Seong, W.; Fu, Y.; Yoon, P. K.; Kim, S. K.; Yeom,
S. J.; Lee, D. H.; Lee, S. G. Leucine Zipper-Mediated Targeting
of Multi-Enzyme Cascade Reactions to Inclusion Bodies in
Escherichia Coli for Enhanced Production of 1-Butanol. Metab.
Eng. 2017, 40, 41–49. DOI: 10.1016/j.ymben.2016.12.012.
[188] J€ager, V. D.; Lamm, R.; Kloß, R.; Kaganovitch, E.; Gr€ unberger,
A.; Pohl, M.; B€ uchs, J.; Jaeger, K. E.; Krauss, U. A Synthetic
Reaction Cascade Implemented by Colocalization of Two
Proteins Within Catalytically Active Inclusion Bodies. ACS.
Synth. Biol. 2018, 7, 2282–2295. DOI: 10.1021/acssynbio.
8b00274.
[189] Mart
ınez-Miguel, M.; Tatkiewicz, W.; K€ ober, M.; Ventosa, N.;
Veciana, J.; Guasch, J.; Ratera, I. Methods for Processing
Protein Aggregates Into Surfaces. Methods. Mol. Biol. 2022,
2406, 517–530. DOI: 10.1007/978-1-0716-1859-2_31.
[190] Gifre-Renom, L.; Seras-Franzoso, J.; Rafael, D.; Andrade, F.;
Cano-Garrido, O.; Martinez-Trucharte, F.; Ugarte-Berzal, E.;
Martens, E.; Boon, L.; Villaverde, A.; et al. The Biological
Potential Hidden in Inclusion Bodies. Pharmaceutics 2020, 12,
157. DOI: 10.3390/pharmaceutics12020157.
[191] C
espedes, M. V.; Fern
andez, Y.; Unzueta, U.; Mendoza, R.;


Seras-Franzoso, J.; S
anchez-Chardi, A.; Alamo, P.; Toledo-
Rubio, V.; Ferrer-Miralles, N.; V
azquez, E.; et al. Bacterial
Mimetics of Endocrine Secretory Granules as Immobilized
In Vivo Depots for Functional Protein Drugs. Sci. Rep. 2016,
6, 1–10.
[192] Cano-Garrido, O.; Rodr
ıguez-Carmona, E.; D
ıez-Gil, C.;
V
azquez, E.; Elizondo, E.; Cubarsi, R.; Seras-Franzoso, J.;
Corchero, J. L.; Rinas, U.; Ratera, I.; et al. Supramolecular
Organization of Protein-Releasing Functional Amyloids Solved
in Bacterial Inclusion Bodies. Acta. Biomater. 2013, 9,
6134–6142. DOI: 10.1016/j.actbio.2012.11.033.
[193] Seras-Franzoso, J.; D
ıez-Gil, C.; Vazquez, E.; Garc
ıa-Fruit
os,
E.; Cubarsi, R.; Ratera, I.; Veciana, J.; Villaverde, A.
Bioadhesiveness and Efficient Mechanotransduction Stimuli
Synergistically Provided by Bacterial Inclusion Bodies as
Scaffolds for Tissue Engineering. Nanomedicine (Lond) 2012,
7, 79–93. DOI: 10.2217/nnm.11.83.
[194] Schetters, S. T. T.; Jong, W. S. P.; Kruijssen, L. J. W.; van den
Berg van Saparoea, H. B.; Engels, S.; Unger, W. W. J.;
Houben, D.; den Haan, J. M. M.; Luirink, J.; Kooyk, Y. V.
Bacterial Inclusion Bodies Function as Vehicles for Dendritic
Cell-Mediated T Cell Responses. jCell. Mol. Immunol 2020, 17,
415–417. DOI: 10.1038/s41423-019-0298-x.
[195] Mart
ınez-Miguel, M.; Kyvik, A. R.; M Ernst, L.; Mart
ınez-
Moreno, A.; Cano-Garrido, O.; Garcia-Fruit
os, E.; Vazquez, E.;
Ventosa, N.; Guasch, J.; Veciana, J.; et al. Stable Anchoring of
Bacteria-Based Protein Nanoparticles for Surface Enhanced
Cell Guidance. J. Mater. Chem. B 2020, 8, 5080–5088. DOI:
10.1039/d0tb00702a.
[196] Kontoravdi, C.; Jimenez del Val, I. Computational Tools for
Predicting and Controlling the Glycosylation of
Biopharmaceuticals. Curr. Opin. Chem. Eng 2018, 22, 89–97.
DOI: 10.1016/j.coche.2018.08.007.
[197] Smiatek, J.; Jung, A.; Bluhmki, E. Towards a Digital
Bioprocess Replica: Computational Approaches in
Biopharmaceutical Development and Manufacturing. Trends.
Biotechnol 2020, 38, 1141–1153. DOI: 10.1016/j.tibtech.2020.
05.008.
[198] Keulen, D.; Geldhof, G.; Bussy, O. l.; Pabst, M.; Ottens, M.
Recent Advances to Accelerate Purification Process
Development: A Review with a Focus on Vaccines. J.
Chromatogr. A 2022, 1676, 463195. DOI: 10.1016/j.chroma.
2022.463195.
[199] von Stosch, M.; Hamelink, J.-M.; Oliveira, R. Hybrid Modeling
as a QbD/PAT Tool in Process Development: An Industrial E.
coli Case Study. Bioprocess. Biosyst. Eng 2016, 39, 773–784.
DOI: 10.1007/s00449-016-1557-1.
[200] Geciova, J.; Bury, D.; Jelen, P. Methods for Disruption of
Microbial Cells for Potential Use in the Dairy Industry—a
Review. Int. Dairy J 2002, 12, 541–553. DOI: 10.1016/S0958-
6946(02)00038-9.

[201] Walther, C.; Kellner, M.; Berkemeyer, M.; Brocard, C.;
D€urauer, A. A Microscale Bacterial Cell Disruption Technique
as First Step for Automated and Miniaturized Process
Development. Process. Biochem. 2017, 59, 207–215. DOI: 10.
1016/j.procbio.2017.05.013.
[202] Starke, R.; Jehmlich, N.; Alfaro, T.; Dohnalkova, A.; Capek, P.;
Bell, S. L.; Hofmockel, K. S. Incomplete Cell Disruption of
Resistant Microbes. Sci. Rep 2019, 9, 1–5. DOI: 10.1038/
s41598-019-42188-9.
[203] Haryati, T.; Widhiastuty, M. P.; Warganegara, F. M.;
Akhmaloka . Response Surface Methodology for Optimization
Membrane Disruption Using Thermolysis in Lipase Lk2 and
Lk3. J. Pure Appl. Microbiol 2022, 16, 1274–1283. DOI: 10.
22207/JPAM.16.2.56.
[204] Berrill, A.; Biddlecombe, J.; Bracewell, D., Chapter 13: Product
Quality during Manufacture and Supply. In Peptide and
Protein Delivery; Walle, C. V. D., Ed.; Elsevier: Amsterdam,
2011; pp. 313–339.
[205] Grabski, A. C. Advances in Preparation of Biological Extracts
for Protein Purification. Methods Enzymol 2009, 463, 285–303.
DOI: 10.1016/S0076-6879(09)63018-4.
[206] Stanbury, P. F.; Whitaker, A.; Hall, S. J. Chapter 10: The
Recovery and Purification of Fermentation Products. In
Principles of Fermentation Technology; Butterworth-
Heimeman; Elsevier Science: Burlington MA, 2017; pp.
619–686.
[207] Peternel, S. Bacterial Cell Disruption: A Crucial Step in
Protein Production. N Biotechnol 2013, 30, 250–254. DOI: 10.
1016/j.nbt.2011.09.005.
[208] Stanbury, P. F.; Whitaker, A.; Hall, S. J., Chapter 3: The
Isolation and Improvement of Industrially Important
Microorganisms. In Principles of Fermentation
Technology;Third Edition; Butterworth-Heimeman; Elsevier
Science: Burlington MA, 2016; pp. 75–211.
[209] Hoffmann, D.; Ebrahimi, M.; Gerlach, D.; Salzig, D.; Czermak,
P. Reassessment of Inclusion Body-Based Production as a
Versatile Opportunity for Difficult-to-Express Recombinant
Proteins. Crit. Rev. Biotechnol 2018, 38, 729–744. DOI: 10.
1080/07388551.2017.1398134.
[210] Dodd, C. E. R. Characteristics of Foodborne Hazard and
Diseases: Microbial Stress Response. Encyclopedia Food Saf.
2014, 1, 183–187.
[211] Salazar, O.; Asenjo, J. A. Enzymatic Lysis of Microbial Cells.
Biotechnol. Lett. 2007, 29, 985–994. DOI: 10.1007/s10529-007-
9345-2.
[212] Ehgartner, D.; Sagmeister, P.; Langemann, T.; Meitz, A.;
Lubitz, W.; Herwig, C. A Novel Method to Recover Inclusion
Body Protein from Recombinant E. coli Fed-Batch Processes
Based on Phage UX174-Derived Lysis Protein E. Appl.
Microbiol. Biotechnol. 2017, 101, 5603–5614. DOI: 10.1007/
s00253-017-8281-x.
[213] Xie, L.; Terada, A.; Hosomi, M. Disentangling the Multiple
Effects of a Novel High Pressure Jet Device Upon Bacterial
Cell Disruption. Chem. Eng. J. 2017, 323, 105–113. DOI: 10.
1016/j.cej.2017.04.067.
[214] Cheong, D. E.; Choi, H. J.; Yoo, S. K.; Lee, H. D.; Kim, G. J. A
Designed Fusion Tag for Soluble Expression and Selective
Separation of Extracellular Domains of Fibroblast Growth
Factor Receptors. Sci. Rep. 2021, 11, 10. DOI: 10.1038/s41598-
021-01029-4.
[215] Zhou, L.; Liu, Z.; Xu, G.; Li, L.; Xuan, K.; Xu, Y.; Zhang, R.
Expression of Melittin in Fusion with GST in Escherichia Coli
and Its Purification as a Pure Peptide with Good Bacteriostatic
Efficacy. ACS Omega 2020, 5, 9251–9258. DOI: 10.1021/acso-
mega.0c00085.
[216] Rasooli, F.; Hashemi, A. Efficient Expression of EpEX in the
Cytoplasm of Escherichia Coli Using Thioredoxin Fusion
Protein. Res. Pharm. Sci. 2019, 14, 554–565. DOI: 10.4103/
1735-5362.272564.
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 751
[217] Yamamoto, K.; Ogasawara, H.; Ishihama, A. Involvement of
Multiple Transcription Factors for Metal-Induced Spy Gene
Expression in Escherichia Coli. J. Biotechnol. 2008, 133,
196–200. DOI: 10.1016/j.jbiotec.2007.08.002.
[218] Chen, Q. Chi; Liu, L.; Yu, T. Y.; Tang, L.; Yin, M. Li; Zhu,
W. H.; Jiang, X. Yun.; Wang, H. Y. High-Level Expression and
Purification of Melittin in Escherichia Coli Using SUMO
Fusion Partner. Int. J. Pept. Res. Ther. 2021, 27, 9–15. DOI:
10.1007/s10989-020-10060-4.
[219] Kim, Y. S.; Lee, H. J.; Han, M. Ho; Yoon, N. Kyung; Kim,
Y. Chun; Ahn, J. Effective Production of Human Growth
Factors in Escherichia Coli by Fusing With Small Protein
6HFh8. Microb. Cell Fact. 2021, 20, 16. DOI: 10.1186/s12934-
020-01502-1.
[220] Liu, M.; Chen, Z.; Huang, S.; Chu, S.; Issaro, N.; Tian, H.; Hu,
H.; Jiang, C. Effective Protection on Acute Liver Injury by
Halo Tag-Flanked Recombinant Fibroblast Growth Factor 7.
Biotechnol. J. 2018, 13, 1700411. DOI: 10.1002/biot.201700411.
[221] Hemmati, S.; Ranjbari, J. Soluble Form Production of
Recombinant Human Insulin-Like Growth Factor-1 by NusA
Fusion Partner in E. coli. Trends. Peptide Protein Sci. 2019, 4,
1–5.
[222] Paal, M.; Heel, T.; Schneider, R.; Auer, B. A Novel Ecotin-
Ubiquitin-Tag (ECUT) for Efficient, Soluble Peptide
Production in the Periplasm of Escherichia Coli. Microb. Cell
Fact. 2009, 8, 1–9. DOI: 10.1186/1475-2859-8-7.
[223] Zheng, X.; Wu, X.; Fu, X.; Dai, D.; Wang, F. Expression and
Purification of Human Epidermal Growth Factor (HEGF)
Fused with GB1. Biotechnol. Biotechnol. Equip. 2016, 30,
813–818. DOI: 10.1080/13102818.2016.1166984.
[224] Chamachi, N.; Hartmann, A.; Ma, M. Q.; Krainer, G.; Schlierf,
M. Chaperones Skp and SurA Dynamically Expand Unfolded
Outer Membrane Protein X and Synergistically Disassemble
Oligomeric Aggregates. Proc. Natl. Acad. Sci. U S A 2021, 119,
1–11.
[225] Ban, B.; Sharma, M.; Shetty, J. Optimization of Methods for
the Production and Refolding of Biologically Active Disulfide
Bond-Rich Antibody Fragments in Microbial Hosts. Antibodies
2020, 9, 39. DOI: 10.3390/antib9030039.
[226] Sørensen, H. P.; Sperling-Petersen, H. U.; Mortensen, K. K.
Dialysis Strategies for Protein Refolding: Preparative
Streptavidin Production. Protein Expr. Purif. 2003, 31,
149–154. DOI: 10.1016/s1046-5928(03)00133-5.
[227] Yamaguchi, S.; Yamamoto, E.; Mannen, T.; Nagamune, T.;
Nagamune, T. Protein Refolding Using Chemical Refolding
Additives. Biotechnol. J. 2013, 8, 17–31. DOI: 10.1002/biot.
201200025.
[228] Yamaguchi, H.; Miyazaki, M. Microfluidic Chips with Multi-
Junctions: An Advanced Tool in Recovering Proteins from
Inclusion Bodies. Bioeng. Bugs 2015, 6, 1–4. DOI: 10.4161/
21655979.2014.987022.
[229] Godawat, R.; Brower, K.; Jain, S.; Konstantinov, K.; Riske, F.;
Warikoo, V. Periodic Counter-Current Chromatography –
Design and Operational Considerations for Integrated and
Continuous Purification of Proteins. Biotechnol. J. 2012, 7,
1496–1508. DOI: 10.1002/biot.201200068.
[230] Wang, Q.; Zhang, C.; Li, Z.; Guo, F.; Zhang, J.; Liu, Y.; Su, Z.
High Hydrostatic Pressure Refolding of Highly Hydrophobic
Protein: A Case Study of Recombinant Human Interferon
b-1b from Inclusion Bodies. Biochem. Eng. J. 2021, 172,
108055. DOI: 10.1016/j.bej.2021.108055.
[231] Gao, K.; Oerlemans, R.; Groves, M. R. Theory and
Applications of Differential Scanning Fluorimetry in Early-
Stage Drug Discovery. Biophys. Rev. 2020, 12, 85–104. DOI:
10.1007/s12551-020-00619-2.
[232] Demeule, B.; Gurny, R.; Arvinte, T. Detection and
Characterization of Protein Aggregates by Fluorescence
Microscopy. Int. J. Pharm. 2007, 329, 37–45. DOI: 10.1016/j.
ijpharm.2006.08.024.
752 K. KACHHAWAHA ET AL.
[233] Fitzpatrick, A. W.; Saibil, H. R. Cryo-EM of Amyloid Fibrils
and Cellular Aggregates. Curr. Opin. Struct. Biol. 2019, 58,
34–42. DOI: 10.1016/j.sbi.2019.05.003.
[234] Ruggeri, F. S.; Sneideris, T.; Vendruscolo, M.; Knowles, T. P. J.
Atomic Force Microscopy for Single Molecule Characterisation
of Protein Aggregation. Arch. Biochem. Biophys. 2019, 664,
134–148. DOI: 10.1016/j.abb.2019.02.001.
[235] Tiernan, H.; Byrne, B.; Kazarian, S. G. ATR-FTIR
Spectroscopy and Spectroscopic Imaging for the Analysis of
Biopharmaceuticals. Spectrochim. Acta. A Mol. Biomol.
Spectrosc 2020, 241, 118636. DOI: 10.1016/j.saa.2020.118636.
[236] Gambin, Y.; Polinkovsky, M.; Francois, B.; Giles, N.; Bhumkar,
A.; Sierecki, E. Confocal Spectroscopy to Study Dimerization,
Oligomerization and Aggregation of Proteins: A Practical
Guide. Int. J. Mol. Sci. 2016, 17, 1–20.
[237] Tong, W.; Wang, G. How Can Native Mass Spectrometry
Contribute to Characterization of Biomacromolecular Higher-
Order Structure and Interactions? Methods 2018, 144, 3–13.
DOI: 10.1016/j.ymeth.2018.04.025.
[238] Hjalte, J.; Hossain, S.; Hugerth, A.; Sj€
ogren, H.; Wahlgren, M.;
Larsson, P.; Lundberg, D. Aggregation Behavior of Structurally
Similar Therapeutic Peptides Investigated by 1H NMR and
All-Atom Molecular Dynamics Simulations. Mol. Pharm. 2022,
19, 904–917. DOI: 10.1021/acs.molpharmaceut.1c00883.
[239] Desligniere, E.; Ley, M.; Bourguet, M.; Ehkirch, A.;
Botzanowski, T.; Erb, S.; Hernandez-Alba, O.; Cianf
erani, S.
Pushing the Limits of Native MS: Online SEC-Native MS for
Structural Biology Applications. Int. J. Mass Spectrom. 2021,
461, 116502. DOI: 10.1016/j.ijms.2020.116502.