BACKGROUND
process integration such as in situ product removal  has emerged as a valuable tool to increase the overall process yield and aims at minimizing costs.
ispr describes the separation of any target from the bioreaction media, e.g.
by adsorption of the target to functionalized surfaces  <cit>  in order to minimize production limitations.
these can be proteolytic degradation, inhibition of target functionality and target production  <cit> .
magnetic separation was introduced to selectively adsorb the target product to the surface of functionalized magnetic carrier particles  <cit> .
this technique allows for a high product purity in only one step minimizing overall process costs  <cit> .
potential targets can be proteins  <cit> , dna  <cit>  or microorganisms  <cit> .
in situ magnetic separation  can further increase the overall target protein yield by separating the target protein itself  <cit>  or removing unwanted molecules from the biosuspension during the bioprocess  <cit> .
ligands known from column chromatography can be employed for functionalization of the beads  <cit> .
in this work the overall impact of integrated isms on the production of his-tagged single chain fragment variable lysozyme-specific antibody fragments  d <dig>   from e. coli cultivations is investigated.
two types of particles were tested: metal-chelate and triazine-functionalized magnetic beads.
immobilized metal affinity ligands such as co2+, zn2+, ni2+ or cu2+ that chelate to covalently-bound iminodiacetic acid  are capable of specifically binding histidine residues of his-tagged target proteins.
according to the literature these ligands offer important advantages such as chemical stability, high binding capacity, protein recovery, and the possibility of matrix regeneration  <cit> .
the removal  of monoclonal antibodies from the biosuspension with magnetic metal chelate particles has been successfully evaluated by morgan et al.
<cit> .
biomimetic affinity ligands based on the triazine scaffold, as the artificial proteins a and l, could also be successfully immobilized on magnetic supports and provide a cost-efficient alternative to isolate igg antibodies  <cit> .
in the current work the triazine beads were tested for the first time to separate scfv d <dig>  fragments, corroborating evidence already obtained from theoretical studies  <cit> .
as shown by holschuh et al., antibodies were successfully captured from biosuspension with magprep® protein a functionalized magnetic beads after the cultivation process  <cit> .
lysozyme, the antigen of the d <dig> , has also been immobilized on magnetic beads to capture fv antibody fragments from clarified e. coli lysate  <cit> .
small affinity ligands such as ida charged with divalent metal ions or triazine functionalization are advantageous over biospecific ligands such as protein a due to lower manufacturing costs  <cit> , milder elution conditions, higher stability with regards to leakage and disinfection <cit> .
however, the use of divalent metal ions as ligands bears the risk to intoxicate microorganisms, especially when they are applied during cultivation  <cit> .
to our knowledge, this study is the first in order to test whether isms with metal chelate and triazine beads is compatible with the microbial production system and is suitable to increase the overall d <dig>  yield.
evaluation of isms was performed by comparison of cultivations with and without isms supported by process modeling to gain a deeper understanding of the integrated process.
methods
microorganism and media
extracellular his-tagged lysozyme-specific scfv d <dig>  antibody fragments  <cit>  were produced by the recombinant e. coli pope <dig> strain kindly provided by prof. dr. stefan dübel   <cit> .
production of d <dig>   was induced by 50 μmol/l isopropyl β-d-1-thiogalactopyranoside  while 100 mg/l na-ampicillin served as selection marker in all media.
three different media were applied.
medium  : glucose: 20–30; yeast extract: 10; casamino acids: 5; kh2po4:  <dig> ; k2hpo4:  <dig> .
the yeast extract consisted of  <dig> %  amino acids,  <dig> % and  <dig> %  cysteine and methionine, respectively.
casamino acids consisted of 100% amino acids,  <dig> %  were methionine.
medium  contained additional  <dig>  g/l mgso4∙7h2o and tracer  according to the literature  <cit>  furthermore referred to as “extra salts”: na2moo4∙4h2o:  <dig> ; cocl2∙6h2o:  <dig> ; mncl2∙4h2o:  <dig> ; cucl2∙2h2o:  <dig> ; h3bo3:  <dig> ; zn-acetate∙2h2o:  <dig>  medium  is similar to medium  with 16 g/l tryptone instead of casamino acids and was only used in shaking flask cultivations.
the reactor feed solution contained 200 g/l glucose, 20 g/l casamino acids,  <dig>  g/l na-ampicillin and 50 μmol/l iptg provided in one bottle.
chemicals were purchased from roth .
cultivation conditions in shaking flasks and bioreactor
5 ml cultures were grown as inoculum in either medium ,  or .
inoculation of 195 ml of the corresponding medium followed in 500 ml baffled shaking flasks.
each culture was kept at 37°c and 150 rpm for 12 hours  while the ph decreased from ph  <dig>  to  <dig>  during cultivation.
one shaking flask culture was either used as inoculum of  <dig>  l fresh medium in the bioreactor  or further used for small scale isms experiments .
the  <dig>  l stirred tank bioreactor  was prepared for fed-batch mode equipped with an easyferm ph electrode and the oxyferm amperometric po <dig> sensor .
cultivations were performed at ph  <dig> , and the po <dig> kept above 20% oxygen saturation at 500 rpm stirrer speed.
aeration was realized by a constant flow of  <dig>  l/min into the bioreactor.
co <dig> and o <dig> off-gas analysis was performed by the exhalyzer system .
liquid offline samples could be taken manually or by using the auto sampler 851-as .
a two-stage fermentation process was established both in the reactor and shaking flasks : cells were grown in batch mode for  <dig> -13 h at 37°c until the production of d <dig>  was induced by addition of iptg and the temperature lowered to 25°c not to overload the secretory pathways of e. coli <cit> .
after induction, the reactor medium was supplemented by a continuous nutrient feed at 2–4 g/h  until the end of cultivation to avoid carbon and nitrogen limitations.
k <dig> was conducted using the same cultivation parameters and medium as in k <dig> .
shaking flask cultivations sk <dig> and sk  <dig> were performed in medium  containing tryptone instead of casamino acids, but  <dig>  g ida- <dig> beads were applied in sk <dig> while triazine beads were used in sk <dig>
particle treatment and in situ magnetic separation  procedures
two batches of polyvinyl alcohol -coated iminodiacetate -functionalized beads were used to chelate cu2+-ions and specifically bind his-tagged d <dig> : pva-ida- <dig>   <cit>  and pva-ida- <dig> .
cu2+-ions were chosen as ligands because in the case of ida-chelating ligands, the ranking of affinities of different metal ions is cu2+ > ni2+ > zn2+ ≥ co2+.
these divalent ions are preferably used for purification of histidine-tagged proteins  <cit> .
triazine-functionalized beads  were manufactured according to the literature  <cit> .
disinfection of the beads and the isms system  was done by incubation in 20-30%  ethanol for 1 hour.
ida- <dig> and ida- <dig> beads were further incubated in  <dig> - <dig>  mol/l cu2+ solution for 60 min.
elution of the pva-ida and triazine beads was performed by applying  <dig>  mol/l na2-edta/pbs buffer at ph  <dig>  or  <dig>  mol/l glycine buffer at ph  <dig>  for 1 hour, respectively.
before and after each step, at least 3- <dig> subsequent washing steps of the beads occurred in pbs buffer at ph  <dig>  .
particle treatment was performed at 22°c and 150 rpm in a 2 l bottle  and in 50 ml falcon tubes at 20 rpm .
maximum particle concentrations were 70 g/l for disinfection and washing steps as well as 19 g/l and 56 g/l for adsorption and elution steps, respectively.
adsorption was conducted in shaking flasks  for 10 min, and magnetic separation performed for another 10 min with a ndfeb block permanent magnet  according to the procedure described by käppler  <cit> .
adsorption in the closed system  was conducted in a 2 l bottle with  <dig>  l biosuspension for at least 30 min.
solid-solid–liquid separation followed for at least 15 min by means of a u-shaped permanent magnet .
in general, adsorption was conducted at 25°c and 150 rpm.
after separation, the biosuspension was further cultivated as before.
particles applied in k <dig> were regenerated and reused in k <dig>  all other beads were only used once .
magnetic particle characterization
equivalent diameters x <dig> of ida- <dig> and ida- <dig> particle agglomerates were measured in pbs buffer at ph  <dig>  using the helos analyzer .
the hydrodynamic diameter of triazine particle agglomerates was measured in double distilled h2o using the zetasizer nano zs   <cit> .
electron microscopy allowed for detailed examination of the primary particles’ structures .
magnetization properties of the beads were measured with the micromag  <dig> magnetometer .
batch adsorption studies were carried out with both particle types to determine the binding characteristics of d <dig> .
adsorption on triazine and ida beads was conducted for 20 min and 10-60 min at 22°c on a rotation shaker vv <dig> , respectively.
measurements were approximated by the langmuir adsorption model  using a nonlinear least square fitting function  to estimate the maximum adsorption load qmax*  and the dissociation constant kd .
the equilibrium concentration of d <dig>  in the medium after adsorption is cp* .
q∗=qmax∗·cp∗cp∗+kd
offline analytical procedures
optical densities of the biosuspension were measured at 600 nm  and correlated to a bdm standard curve.
all buffers were prepared as aqueous solutions.
glucose was determined according to  <cit>  using the god-pap test kit glucose liquicolor .
measurements of total protein are based on the method by lowry modified by peterson  <cit>  , and bovine serum albumin was used as calibration standard .
cu2+ concentrations in culture supernatants were measured by inductively-coupled plasma optical emission spectrometry   <cit>  using the model jy38s, 1200 w  equipped with a u5000at + ultrasonic nebulizer .
d <dig>  was determined by means of an antigen-binding, indirect elisa-method  <cit>  using 96-well microtiter-plates .
the d <dig>  calibration standard was received from a shaking flask culture after adsorption and elution , and was stored at -20°c.
according to sds-page analysis  the elution fraction was considered to contain scfv d <dig>  only, and the d <dig>  concentration of the elution sample was then determined with the lowry method.
elisa plates were coated overnight with  <dig>  μg antigen  per well at 4°c.
soluble antibody fragments were detected with anti-c-myc 9e <dig> antibodies  and anti-mouse igg antibody conjugated with peroxidase .
after treatment with tmb  and sulfuric acid, absorbance was measured at 450 nm using a microtiter plate reader , and the blank of a sample without d <dig>   was subtracted.
elisa measurements were performed in the linear range of d <dig>  concentration.
the rate of d <dig>  degradation was examined by incubation of diluted culture supernatant at  <dig> and 37°c on a rotation shaker and taking samples hourly.
modeling the integrated bioprocess on the reactor and cellular level
mathematical models of the stirred tank reactor and the cells were established in matlab-simulink  based on differential equations , mass balances, kinetics and stoichiometries.
three substrates s,i with i = glucose , total amino acids  and cysteine  were modeled.
the model was established for two reasons.
although many assumptions were made, mainly to describe growth and product formation, it shall provide a deeper understanding of the whole bioprocess.
however, the main goal was to provide a tool that can be used to optimize the isms steps.
this is important because the amount of particles used and the number of required isms steps must be minimized because they are connected to extra costs.
a set of simulation parameters is proposed in table  <dig> and explanation of their origin is provided in the text.
calculation of the reactor volume v and the dilution rate d considered constant substrate and base flow qs,i and qbase  as well as volume reduction by the auto sampler qsampler .
the flow rate was chosen in that way that glucose was not limiting in the reactor.
dvdt=∑iqs,i+qbase−qsampler
d=∑iqs,i+qbasev
a) estimated by minimizing least squares for eqs.
a1) 3- <dig>  a2) 2- <dig>  a3) values valid for 37°c; for 25°c calculation according to equation 3- <dig>
b) estimated from literature.
c) estimated manually.
d) according to measurements.
e) set manually.
calculations of the bdm concentration cx  took the specific growth rate μ of the microorganisms into consideration which was obtained from equation 3- <dig>
dcxdt=μ·cx−d·cx
substrate concentrations cs,i were derived from equation 3- <dig> where rs,i are the specific substrate uptake rates which were calculated from equation 3- <dig>  the composition of the substrate feed  can be taken from microorganism and media section.
when i = cysteine is considered as substrate, the assumption was made that cysteine could be synthesized by the cells at a fixed rate rcys when additional sulfur 2so4) was present in the medium  and cysteine provided in the medium was exhausted after approx.
40 h of cultivation.
otherwise no cysteine was synthesized .
dcs,idt=−rs,i·cx−d·cs,i+d·cs,i,f+rcys·cx
carbon dioxide production  depends on the specific carbon dioxide production rate rco <dig> of the microorganisms.
hereby, the co <dig> flux into the reactor was neglected as it is insignificant in comparison to the co <dig> produced.
dmco2dt=qco2=rco2·cx·v
concentrations of d <dig>  and other proteins cp,i and cp,j were calculated from equation 3- <dig>  while rp,i,j describe protein formation rates  that were estimated manually by approximation of the measurements, rp,i,deg represents d <dig>  degradation which was determined experimentally.
adsorption and removal of proteins from the bioreactor is considered by the term Γ˙gijm−2h− <dig> that takes particle properties such as the bet-surface aspec  into account  <cit> .
the total protein content was calculated as cp,tot = cp,i + cp,j
dcp,i,jdt=rp,i,j·cx−d·cp,i,j−rp,i,deg·cp,i−Γ˙·mmp·aspecv
adsorption kinetics of proteins to a solid matrix is determined by the transport rate ktr of the protein to the surface and the adsorption rate ka <cit> .
equation 3- <dig> is obtained for the assumption ka ≫ ktr with the surface-specific load of protein on the particle Γ. hereby, ktr was estimated by minimizing least squares for equation 3- <dig>  kd and q*max were estimated from equation 2- <dig>
dΓdt=Γ˙=1000·ktr·cp,i−kd·Γqmax∗aspec−Γ
the substrate uptake was considered as the first step in the metabolic network and was modeled by michaelis-menten kinetics .
km was taken from the literature with the assumption km,aa,cys ≈ km,nh4+ <cit> .
rs,i=rs,i,max·cs,ics,i+km
maximum substrate uptake rates were calculated by equation 3- <dig> where yx,s,i is the integral yield coefficient for bdm on each substrate i that was obtained from measurements  or estimated manually .
rs,i,max=μmaxyx,s,i
μmax was experimentally determined for t = 37°c and estimated by equation 3- <dig> for t = 25°c  <cit> .
hereby, aarrh.
is a pre-exponential factor that was estimated manually, ea the activation energy  <cit> , and r =  <dig>  j mol-1 k- <dig> the universal gas constant  <cit> .
μmax=aarrh.·exp−ear·t+ <dig> k
d <dig>  production was modeled by a constant specific production rate rp,i,0 >  <dig> after induction in medium   and after an additional lag phase  in medium  , 3 mg g-1 h- <dig>  and 6 mg g-1 h- <dig> ).
production of proteins beside d <dig>  was constantly modeled as rp,j,0 >  <dig> .
rp,i,j=rp,i,j,o
the total mass balance for glucose in the anabolism  and catabolism  is modeled as equation 3- <dig>   rglu=rana+rcata
component balances of carbon , amino acids and cysteine in the anabolism are shown in equations 3- <dig> - 3- <dig>  hereby, e is the molar or mass fraction of a chemical compound within another compound as indicated.
the following values were taken from the literature: ec,x <cit> , ec,p,i,j <cit> , eaa,x <cit> , ecys,yeast , ecys,x <cit> , ecys,p,i ≈ 6∙ <dig>  g/mol/26000 g/mol  <cit> .
to obtain eaa,p,i,j the assumption was made that proteins only consist of amino acids while ecys,p,j was estimated manually.
ec,glu·rana=ec,x·μ+ec,p,i·rp,i+ec,p,j·rp,j
raa=eaa,x·μ+eaa,p,i·rp,i+eaa,p,j·rp,j
rcys=ecys,x·μ+ecys,p,i·rp,i+ecyc,p,j·rp,j
the carbon balance for the respiration is presented in equation 3- <dig>
ec,co2·rco2=ec,glu·rcata
the energy balance couples anabolism and catabolism of the cell, but intracellular atp levels were not quantified.
hence, the carbon dioxide production rate rco <dig> was modeled as a function of specific growth as well as protein production rates .
yield coefficients on atp as well as the maintenance rate rm were taken from the literature  <cit>  and normalized .
the maintenance rate mf · rm  refers to intracellular energy requirements.
estimates for mf were received from  <cit> .
rco2=yco <dig> atpyx,atp·μ+yco <dig> atpyp,i,atp·rp,i+yco <dig> atpyp,j,atp·rp,j+mf·rm
yco <dig> x=yco <dig> atpyx,atp;yco <dig> p,i=yco <dig> atpyp,i,atp;yco <dig> p,j=yco <dig> atpyp,j,atp
mathematic modeling of d <dig>  production requires solutions for nine rates r by means of  <dig> available equations .
thus, the system would be over-determined if all equations were used  <cit> , and rates might possibly contradict each other.
however, there can only be one specific growth rate that relies upon the limiting substrate and its uptake rate.
thus, the three theoretical substrate uptake rates rglu, raa, rcys were determined separately before the system matrix was calculated.
if glucose is assumed to be limiting, the remaining eight rates were calculated by finding a solution for the equation system a∙s = b with the stoichiometric matrix a and the vectors s =  and b. finally, the set of rates corresponding to the minimum solution of μ was chosen for further calculation .
two different parameter sets for k5-k <dig>  and k9-k <dig>  were applied .
the main difference is found in the parameters rp,i, <dig> that were obtained due to different d <dig>  production rates and rcys due to different medium compositions.
RESULTS
magnetic particle characterization
equivalent diameters of particle agglomerates x50 =  <dig>   <cit>  and  <dig>  μm were found for ida- <dig> and ida- <dig> beads in pbs buffer at ph  <dig> .
the hydrodynamic diameter of triazine bead agglomerates was  <dig>  ±  <dig>  μm  <cit> .
results obtained from electron microscopy also revealed much smaller primary particles .
thus, these particles have a very high surface-to-volume ratio which is desirable to obtain a high protein binding capacity  <cit> .
it is known from the literature that magnetic particles with a diameter ≥  <dig> -1 μm can be easily separated by simple magnetic separators , while separation of smaller particles is much more efficient when high gradient magnetic separators are used   <cit> .
this applies because the magnetic force which is experienced by the particles through the external magnetic field is proportional to the particle volume and the gradient of the magnetic field strength inside the separation chamber  <cit> .
especially at higher viscosities of the cultivation medium  magnetic particle agglomerates are favorable due to shorter separation times  <cit> .
since particle agglomeration can occur spontaneously in the medium, especially at high particle concentrations  <cit> , it is important for in situ protein separation that no biomass, i.e.
microorganisms, is included or adsorbed in/to those agglomerates.
losses of biomass, i.e.
escherichia coli cells, after the isms step are discussed in comparison of the total d <dig>  yield in processes with and without isms section.
saturation magnetization ms =  <dig> a m2 kg- <dig> <cit>  and  <dig>  ±  <dig>  a m2 kg- <dig> were measured for ida- <dig> and ida- <dig> beads, and ms =  <dig>  ±  <dig>  a m2 kg- <dig> for triazine beads.
the remanent magnetization was low at mr =  <dig> - <dig>  a m2 kg- <dig>  thus particles were considered superparamagnetic.
it is important to keep in mind that magnetic particles or agglomerates thereof have superparamagnetic properties only when the size of each individual magnetite core is between 15–50 nm  <cit> .
adsorption of the target protein scfv d <dig>  to the beads was quantified by measuring isotherms  that could be approximated by the langmuir adsorption model .
in the experiments maximum loads of qmax* =  <dig>  g/g and  <dig>  g/g were found for ida- <dig> and ida- <dig> beads, respectively, when medium  containing extra salts was used .
in medium  without extra salts, qmax* was significantly decreased to ~  <dig> - <dig>  g/g .
different maximum loads might be based on different particle production processes and are not further discussed.
highest loads of ida beads were achieved for 60 min incubation in cu2+-solution and 60 min adsorption.
for triazine beads we found qmax* =  <dig>  g/g in medium  with extra salts .
according to the low kd-values obtained from the isotherms , all beads can be categorized as affinity adsorbents  <cit> .
the maximum obtained load for ida- <dig> beads qmax* =  <dig>  g/g is very high compared to qmax* =  <dig>  g/g for green fluorescent protein  <cit> .
possibly, d <dig>  concentrations were overestimated by the elisa procedure in general  due to incomplete elution or loss of d <dig>  activity in the elution buffer .
however, the proposed elisa method is sufficient to compare the results provided in this study among each other.
degradation of the scfv d <dig>  in cultivation media
the stability of the d <dig>  was tested in cell-free cultivation medium up to 50 h of incubation, but no degradation or any loss of d <dig>  activity could be detected .
although the d <dig>  fragments were found to be stable in cell-free medium, isms was applied to study its impact on the whole bioprocess in presence of the microorganisms with the particle systems characterized in magnetic particle characterization section.
production of d <dig>  and isms in bioreactor cultivations
process performance in complex medium with extra salts
two reference cultivations k <dig> and k <dig> were conducted without and two cultivations k <dig> and k <dig> carried out with isms to investigate the performance of  isms .
results of k <dig> were similar to those in k <dig> and are not shown.
before induction, cells were grown in medium  up to a bio dry mass concentration of 5– <dig>  g/l at a maximum specific growth rate of μmax =  <dig> - <dig>  h- <dig> while glucose was rapidly consumed but not limiting .
after induction, bdm concentrations increased up to  <dig> - <dig>  g/l at the end of k <dig>  k <dig> and k <dig>  offline bdm and glucose concentrations were similar in cultivations with and without isms .
d <dig>  was only produced after induction: in k <dig>  k <dig> and k <dig>  d <dig>  production was delayed by 30 hours .
further investigations are required to clarify whether the delay was due to inhibition of d <dig>  production by the additional salts provided in the medium.
although cultivations k <dig>  k <dig> and k <dig> were performed in the same medium under the same conditions, maximum obtained d <dig>  concentrations differed significantly before the isms step .
according to harrison, different leakage rates of the d <dig>  fragments from the periplasmic space into the medium might be responsible for those differences  <cit> .
furthermore, the obtained extracellular d <dig>  titers are much higher compared to those reported in literature   <cit> .
although d <dig>  concentrations might have been overestimated within the elisa procedure, the impact of isms on the process could be studied well.
for better comparison of processes with and without isms, d <dig>  concentrations were normalized to the final d <dig>  concentration before the isms step.
in k <dig> and k <dig> 92% and 98% of the overall d <dig>  were separated from the medium by isms, respectively .
however, no significant production was observed after separation in k <dig> and k <dig>  instead, d <dig>  activities decreased towards zero until the end of cultivations.
this effect cannot be attributed to significant degradation of the d <dig>  fragments in the medium since no d <dig>  degradation could be quantified.
thus, d <dig>  production was supposed to be inhibited as a consequence of the isms step.
further explanation is provided in the section response of the microorganisms to the isms steps.
during all cultivations, protein other than d <dig>  was continuously produced and secreted into the medium up to 2 g/l.
process performance in complex medium without extra salts
two reference cultivations k <dig> and k <dig> were conducted without isms .
bdm and glucose concentrations  as well as total protein concentrations are comparable to those obtained from cultivations in medium containing extra salts .
however, after induction, bdm concentrations remained constant in k <dig> or slightly decreased in k10-k <dig>  d <dig>  was immediately produced after isms in k <dig> , and maximum d <dig>  concentrations before the isms step were lower compared to the results obtained from k5-k <dig> , and they did not increase above  <dig> - <dig>  g/l at the end of cultivations .
investigations of the culture medium suggest that the lower overall bdm and d <dig>  concentrations are based on potential sulfur limitation in medium  after induction .
only methionine was supplied by the feed being theoretically sufficient to deliver the sulfur required for d <dig>  production.
sulfur is a compound of cysteine that is required for biomass and protein synthesis.
calculations were based on  <dig> %  sulfur and  <dig> %  cysteine content of the bdm  <cit>  and d <dig>  fragments  <cit> , respectively.
in k <dig> 78% of the overall d <dig>  were separated from the medium by isms .
in contrast to k <dig> and k <dig>  d <dig>  was produced in k <dig> after isms reaching 50% of the maximum level before separation.
validation of the process simulation by offline data
simulation of bdm and glucose concentrations over cultivation time could be validated by offline measurements for all cultivations .
as it was observed in all cultivations, biomass growth already declined before induction.
the experimentally unknown limitation was modeled as cysteine, i.e.
sulfur limitation , and exceptionally as glucose limitation in k <dig> after 98 hours.
cells were considered capable of producing cysteine from sulfate that was abundant in the medium of k <dig>  k <dig> and k <dig> to fulfill the requirements of d <dig>  production and biomass growth , but that was not abundant in k <dig>  k <dig> and k <dig> .
this can be an explanation for decreased bdm and d <dig>  titers in cultivations k9-k <dig>  degradation of d <dig>  in the medium was set to zero .
significant differences between the d <dig>  offline data and the simulation occurred after separation in k <dig> and k <dig>  while d <dig>  was reproduced in the simulation, no d <dig>  reproduction occurred in the offline data .
simulated d <dig>  and total protein courses could be validated well with experimental data in k9-k <dig> including the isms step.
d <dig>  production rates were comparable before and after isms  but were not elevated compared to the reference cultivations k <dig> and k <dig> .
similar results were obtained for cultivations with and without isms in shaking flask cultivation ).
in the best case similar d <dig>  production rates before and after isms were expected because no degradation of the d <dig>  was observed.
in the response of the microorganisms to the isms steps an explanation is given why the production of d <dig>  might have been limited in k <dig> and k <dig> after the isms steps.
response of the microorganisms to the isms steps
measurements of co <dig> in the off-gas and cu2+ concentrations in the medium were conducted to study the culture viability before and after isms.
decreased or no d <dig>  production after isms correlates well with a decreased respiratory capacity of the cells: in k <dig> and k <dig>  the co <dig> fluxes in the off-gas stream dropped from  <dig>  to  <dig>  g/h and from  <dig>  to  <dig>  g/h after the initial isms step indicating a decreased metabolic activity.
in k <dig>  qco <dig> recovered up to  <dig>  g/h after 87 h of cultivation .
interestingly, after 87 h of cultivation, bdm concentrations almost doubled although no significant d <dig>  production was observed any more.
this implies that cells were either inhibited to produce d <dig>  after isms or the process suffered from microbial contamination.
the second isms step in k <dig> did not evoke a drop of the co <dig> signal at 116 h. in k <dig>  no significant drop of the co <dig> signal was observed indicating that the metabolic activity was not decreased by isms .
overestimation of the co <dig> flux by the simulation  might be based on the fact that no overflow metabolism  was taken into account in the model  <cit> .
measurements of cu2+ concentrations in the medium revealed significant copper contamination of up to 180 mg/l and 300 mg/l after the first and second isms step in k <dig>  respectively .
copper accumulation in the medium due to insufficient washing steps after copper-loading of ida particles seems unlikely because four subsequent washing steps were carried out.
partial leakage of copper from the beads most likely occurred during their application in the medium.
this suggestion is supported by high availability of up to  <dig>  mmol free ida groups on the particle surface per gram pva-ida- <dig> beads that theoretically chelate equimolar amounts of cu2+ ions .
these findings imply that cell metabolism and thus production of d <dig>  by recombinant e. coli can be strongly inhibited by free cu2+ ions.
cu2+ ions might be reduced in the fermentation medium to cu+ ions that are able to structurally damage proteins and decrease their activity due to highly reactive radical intermediates  <cit> .
copper concentrations below 120 mg/l as in k <dig> seem to be less inhibiting, and d <dig>  production was less diminished in k <dig> compared to k <dig> and k <dig> where copper concentrations exceeded 140 mg/l.
in sk <dig>  the copper concentration was not measured before and after isms.
however, due to similar concentrations of cu2+-loaded ida- <dig> particles in the biosuspension during the isms step , it can be assumed that the copper concentration in the biosuspension after isms in sk <dig> did not exceed the value obtained in k <dig> .
in literature, the minimal inhibitory concentration of cu2+, ni2+, co2+ and zn2+ ions on e. coli cells is  <dig>  mmol/l   <cit> .
further research can clarify if a potential d <dig>  production inhibition can be circumvented by using other divalent metal ions: ni2+ and co2+ ions are reported to be less carcinogenic than cu2+ ions  <cit>  but are still potentially harmful if intended for therapeutic purposes  <cit> .
since zn2+ is redox-inactive and less toxic than cu2+, it might be tested as metal ligand to bind his-tagged d <dig>   <cit> .
another approach would be to use tetradendate chelating agents such as nitrilotriacetic acid  instead of tridendate ligands such as ida to obtain more stable complexes with the metal ion  <cit>  and potentially avoid excessive leakage of me2+ into the medium.
cultivation and isms in shaking flasks by means of triazine beads
additional isms experiments were conducted in shaking flasks using triazine-functionalized beads.
in both complex media  and  , up to 57-79% of d <dig>  was separated from the medium, and d <dig>  was produced after isms.
for both media, similar linearized d <dig>  production rates rp,i,0 =  <dig> - <dig>  g g-1 h- <dig> were found.
these results are comparable to those obtained for ida- <dig> beads used in sk <dig> .
however, no copper or other heavy metal ions were required for adsorption using the triazine beads.
we have thus shown experimentally that scfv d <dig>  antibody fragments can be selectively separated from biosuspension by gum arabic coated magnetic carrier particles functionalized with the triazine ligand 22/ <dig>  a biomimetic of protein a. until now, binding of those beads has only been reported to full igg antibodies  <cit>  although it has been shown recently that ligand 22/ <dig> can also bind to the fab portion of igg by molecular modeling studies  <cit> .
comparison of the total d <dig>  yield in processes with and without isms
as can be seen from figure  <dig>  isms did not yield extra benefit for total d <dig>  production since the total accumulated d <dig>  concentrations with and without isms were equal in the best case.
this result has been confirmed by simulation including theoretical multi step isms.
as shown for extracellular protease that was degraded in biosuspension  <cit>  such a model can be useful to optimize the number of isms steps before the experiment is actually performed.
neither an inhibition of d <dig>  in the medium on d <dig>  production itself nor losses of d <dig>  degradation were observed in the extracellular medium.
while the total d <dig>  yield without isms was set yno isms =  <dig>  the yield with isms was yisms =  <dig> - <dig>  and  <dig> - <dig>  for ida and triazine beads, respectively.
elution efficiencies between  <dig> and 100% were received for both pva-ida- <dig> and - <dig> particle charges.
it remains unclear whether low elution efficiencies are to be attributed to incomplete elution or loss of activity in the elution buffer although edta is reported to be a strong eluent which extracts the metal ions from ida and disrupts interactions between proteins and chelating ligands  <cit> .
adsorption and elution efficiency of his-tagged proteins from ida-functionalized magnetic beads can depend on the chelated metal ion: in literature, multi-subunit adsorption of a his-tagged protein onto chelated cu2+ was reported which significantly limited elution efficiency  <cit> .
furthermore, the isolated target protein can potentially be structurally damaged in presence of reduced cu+ ions which leads to reduced activity  <cit> .
this implies to test other divalent metal ions such as ni2+, zn2+ or co2+ which might lead to higher elution efficiencies  <cit> .
elution efficiencies of d <dig>  from triazine-functionalized beads only reached 6%.
in comparison, elution efficiencies of 35% were obtained for igg  <cit> , and the elution protocol needs further improvement.
sds-page should be applied to control the purity of the elution samples.
losses of biomass due to the adsorption procedure were 9 ± 2% and 21 ± 4% for ida and triazine beads, respectively.
this indicates that biomass is more attracted to adsorb to the triazine beads.
coating the beads with an extra layer of biopolymer such as alginate might minimize the risk of bio mass adsorption  <cit> .
the application of a high gradient magnetic separator can further help to minimize biomass losses because formation of one single compact filter cake with inclusion of biomass is avoided.
CONCLUSIONS
results have shown that pva-ida particles are well suited to separate and purify his-tagged proteins from complex media .
however, their application for isms is limited due to the release of chelated copper ions into the medium which have shown to inhibit d <dig>  production above ccu > 120 mg/l.
other less toxic divalent metal ions should be tested and validated as ligands for the ida-beads.
but it must be shown in semi-continuous isms experiments whether this particle type proofs to be compatible with the bioprocess or is better suited for separation of his-tagged proteins after cultivation as it is well known from the literature.
in the case of separation of antibodies and their fragments, triazine-functionalized magnetic particles were found to be an excellent low-cost alternative to protein a-functionalized beads.
although no negative impact of these beads was observed on the bioprocess , further semi-continuous isms experiments are necessary.
in the best case, similar d <dig>  production rates were observed before and after in situ separation when ida- or triazine-functionalized particle types were applied.
no degradation of d <dig>  was measured in biosuspension and, thus, isms did not increase the total yield of the d <dig> -production process as was shown by experiments and simulation.
under consideration of the obtained results and proposed improvements on pva-ida particle performance, both pva-ida and triazine-functionalized magnetic beads can serve as isms platform technology to obtain highly purified product in only one separation step.
in situ magnetic separation can bring most benefit to those production processes where antibodies or their fragments lack stability in the medium or are subject to degradation.
these processes need to be identified.
this study provides a route how to proceed with experiments and optimize the process by simulation: small scale experiments should be conducted first and followed by scale up.
when the kinetics of degradation or lack of stability of the target are quantified and implemented into the model, the latter can be applied to optimize isms in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required isms steps.
abbreviations
greek symbols
μ: specific growth rate, g g-1 h-1; Γ: surface-specific particle load, g m-2;
latin symbols
aarrh: arrhenius pre-factor; aspec: bet-surface, m2 g-1; b: magnetic flux density, t; c: concentration, g l-1; d: dilution rate, h-1; e: mass or molar fraction; ea: activation energy, j mol-1; ka: adsorption rate, m h-1; kd: dissociation constant, g l-1; km: michaelis-menten constant, g l-1; ktr: transport rate, m h-1; m: saturation magnetization, a m2 kg-1; m: mass, g; mf: maintenance factor; q: adsorption load, g g-1; q: flow to/from reactor, l/h, g/h; r: specific uptake/production/maintenance rate, g g-1 h-1; t: cultivation time, h; t: temperature,°c; v: reactor volume, l; x50: equivalent diameter, m; yx,s,i: integral yield coefficient, g g-1; y: overall product yield; yx,s,i: differential yield coefficient, g g- <dig> ;
indices
*: equilibrium; 0: initial condition; aa: amino acids; ana: anabolism; atp: adenosine triphosphate; c: carbon; cata: catabolism; co2: carbon dioxide; cu2+: divalent copper ions; cys: cysteine; deg: degradation; f: feed; glu: glucose; isms: in situ magnetic separation; m: maintenance; max: maximum; mp: magnetic particles; p,i: scfv d <dig> ; p,j: other proteins besides d <dig> ; r: remanent; s: saturation; s,i: substrate; x: biomass.
;
natural constant
r: universal gas constant,  <dig>  g mol-1 k.;
other abbreviations
bdm: bio dry mass; bet: brunauer-emmett-teller; isms: in situ magnetic separation; iptg: isopropyl β-d-1-thiogalactopyranoside; scfv: single chain fragment variable; pbs: phosphate buffered saline; mpbst: modified phosphate buffered saline with tween 20; pva-ida: polyvinyl alcohol-iminodiacetate.
competing interest
the authors declare that they have no competing interest.
authors’ contributions
mc conducted the cultivations and magnetic separation experiments, implemented the reactor-cell model and drafted the manuscript.
as contributed to the experiments and provided valuable discussion.
mf provided the mpva-ida- <dig> functionalized magnetic particles and assisted with the hgms setup.
acar supervised the preparation of the triazine particles and provided valuable discussion.
ilb carried out the synthesis of the triazine-functionalized magnetic particles.
acar and ilb helped to design the adsorption and elution experiments.
cp conceived and initiated the project, and assisted with the model.
all authors carried out the revision of the manuscript.
all authors read and approved the final manuscript.

 process integration such as in situ product removal has emerged as a valuable tool to increase the overall process yield and aims at minimizing costs. ispr describes the separation of any target from the bioreaction media, e.g. by adsorption of the target to functionalized surfaces in order to minimize production limitations. these can be proteolytic degradation, inhibition of target functionality and target production. magnetic separation was introduced to selectively adsorb the target product to the surface of functionalized magnetic carrier particles. this technique allows for a high product purity in only one step minimizing overall process costs. potential targets can be proteins, dna or microorganisms. in situ magnetic separation can further increase the overall target protein yield by separating the target protein itself or removing unwanted molecules from the biosuspension during the bioprocess. ligands known from column chromatography can be employed for functionalization of the beads. in this work the overall impact of integrated isms on the production of his-tagged single chain fragment variable lysozyme-specific antibody fragments d from e. coli cultivations is investigated. two types of particles were tested: metal-chelate and triazine-functionalized magnetic beads. immobilized metal affinity ligands such as co2+, zn2+, ni2+ or cu2+ that chelate to covalently-bound iminodiacetic acid are capable of specifically binding histidine residues of his-tagged target proteins. according to the literature these ligands offer important advantages such as chemical stability, high binding capacity, protein recovery, and the possibility of matrix regeneration. the removal of monoclonal antibodies from the biosuspension with magnetic metal chelate particles has been successfully evaluated by morgan et al.. biomimetic affinity ligands based on the triazine scaffold, as the artificial proteins a and l, could also be successfully immobilized on magnetic supports and provide a cost-efficient alternative to isolate igg antibodies. in the current work the triazine beads were tested for the first time to separate scfv d fragments, corroborating evidence already obtained from theoretical studies. as shown by holschuh et al., antibodies were successfully captured from biosuspension with magprep® protein a functionalized magnetic beads after the cultivation process. lysozyme, the antigen of the d, has also been immobilized on magnetic beads to capture fv antibody fragments from clarified e. coli lysate. small affinity ligands such as ida charged with divalent metal ions or triazine functionalization are advantageous over biospecific ligands such as protein a due to lower manufacturing costs, milder elution conditions, higher stability with regards to leakage and disinfection. however, the use of divalent metal ions as ligands bears the risk to intoxicate microorganisms, especially when they are applied during cultivation. to our knowledge, this study is the first in order to test whether isms with metal chelate and triazine beads is compatible with the microbial production system and is suitable to increase the overall d yield. evaluation of isms was performed by comparison of cultivations with and without isms supported by process modeling to gain a deeper understanding of the integrated process. methods microorganism and media extracellular his-tagged lysozyme-specific scfv d antibody fragments were produced by the recombinant e. coli pope strain kindly provided by prof. dr. stefan dübel. production of d was induced by 50 μmol/l isopropyl β-d-1-thiogalactopyranoside while 100 mg/l na-ampicillin served as selection marker in all media. three different media were applied. medium: glucose: 20–30; yeast extract: 10; casamino acids: 5; kh2po4:; k2hpo4:. the yeast extract consisted of % amino acids, % and % cysteine and methionine, respectively. casamino acids consisted of 100% amino acids, % were methionine. medium contained additional  g/l mgso4∙7h2o and tracer according to the literature furthermore referred to as “extra salts”: na2moo4∙4h2o:; cocl2∙6h2o:; mncl2∙4h2o:; cucl2∙2h2o:; h3bo3:; zn-acetate∙2h2o: medium is similar to medium with 16 g/l tryptone instead of casamino acids and was only used in shaking flask cultivations. the reactor feed solution contained 200 g/l glucose, 20 g/l casamino acids,  g/l na-ampicillin and 50 μmol/l iptg provided in one bottle. chemicals were purchased from roth. cultivation conditions in shaking flasks and bioreactor 5 ml cultures were grown as inoculum in either medium, or. inoculation of 195 ml of the corresponding medium followed in 500 ml baffled shaking flasks. each culture was kept at 37°c and 150 rpm for 12 hours while the ph decreased from ph  to during cultivation. one shaking flask culture was either used as inoculum of  l fresh medium in the bioreactor or further used for small scale isms experiments. the  l stirred tank bioreactor was prepared for fed-batch mode equipped with an easyferm ph electrode and the oxyferm amperometric po sensor. cultivations were performed at ph , and the po kept above 20% oxygen saturation at 500 rpm stirrer speed. aeration was realized by a constant flow of  l/min into the bioreactor. co and o off-gas analysis was performed by the exhalyzer system. liquid offline samples could be taken manually or by using the auto sampler 851-as. a two-stage fermentation process was established both in the reactor and shaking flasks: cells were grown in batch mode for -13 h at 37°c until the production of d was induced by addition of iptg and the temperature lowered to 25°c not to overload the secretory pathways of e. coli. after induction, the reactor medium was supplemented by a continuous nutrient feed at 2–4 g/h until the end of cultivation to avoid carbon and nitrogen limitations. k was conducted using the same cultivation parameters and medium as in k. shaking flask cultivations sk and sk were performed in medium containing tryptone instead of casamino acids, but  g ida- beads were applied in sk while triazine beads were used in sk particle treatment and in situ magnetic separation procedures two batches of polyvinyl alcohol -coated iminodiacetate -functionalized beads were used to chelate cu2+-ions and specifically bind his-tagged d: pva-ida- and pva-ida-. cu2+-ions were chosen as ligands because in the case of ida-chelating ligands, the ranking of affinities of different metal ions is cu2+ > ni2+ > zn2+ ≥ co2+. these divalent ions are preferably used for purification of histidine-tagged proteins. triazine-functionalized beads were manufactured according to the literature. disinfection of the beads and the isms system was done by incubation in 20-30% ethanol for 1 hour. ida- and ida- beads were further incubated in -  mol/l cu2+ solution for 60 min. elution of the pva-ida and triazine beads was performed by applying  mol/l na2-edta/pbs buffer at ph  or  mol/l glycine buffer at ph  for 1 hour, respectively. before and after each step, at least 3- subsequent washing steps of the beads occurred in pbs buffer at ph . particle treatment was performed at 22°c and 150 rpm in a 2 l bottle and in 50 ml falcon tubes at 20 rpm. maximum particle concentrations were 70 g/l for disinfection and washing steps as well as 19 g/l and 56 g/l for adsorption and elution steps, respectively. adsorption was conducted in shaking flasks for 10 min, and magnetic separation performed for another 10 min with a ndfeb block permanent magnet according to the procedure described by käppler. adsorption in the closed system was conducted in a 2 l bottle with  l biosuspension for at least 30 min. solid-solid–liquid separation followed for at least 15 min by means of a u-shaped permanent magnet. in general, adsorption was conducted at 25°c and 150 rpm. after separation, the biosuspension was further cultivated as before. particles applied in k were regenerated and reused in k all other beads were only used once. magnetic particle characterization equivalent diameters x of ida- and ida- particle agglomerates were measured in pbs buffer at ph  using the helos analyzer. the hydrodynamic diameter of triazine particle agglomerates was measured in double distilled h2o using the zetasizer nano zs. electron microscopy allowed for detailed examination of the primary particles’ structures. magnetization properties of the beads were measured with the micromag magnetometer. batch adsorption studies were carried out with both particle types to determine the binding characteristics of d. adsorption on triazine and ida beads was conducted for 20 min and 10-60 min at 22°c on a rotation shaker vv, respectively. measurements were approximated by the langmuir adsorption model using a nonlinear least square fitting function to estimate the maximum adsorption load qmax* and the dissociation constant kd. the equilibrium concentration of d in the medium after adsorption is cp*. q∗=qmax∗·cp∗cp∗+kd offline analytical procedures optical densities of the biosuspension were measured at 600 nm and correlated to a bdm standard curve. all buffers were prepared as aqueous solutions. glucose was determined according to using the god-pap test kit glucose liquicolor. measurements of total protein are based on the method by lowry modified by peterson, and bovine serum albumin was used as calibration standard. cu2+ concentrations in culture supernatants were measured by inductively-coupled plasma optical emission spectrometry using the model jy38s, 1200 w equipped with a u5000at + ultrasonic nebulizer. d was determined by means of an antigen-binding, indirect elisa-method using 96-well microtiter-plates. the d calibration standard was received from a shaking flask culture after adsorption and elution, and was stored at -20°c. according to sds-page analysis the elution fraction was considered to contain scfv d only, and the d concentration of the elution sample was then determined with the lowry method. elisa plates were coated overnight with  μg antigen per well at 4°c. soluble antibody fragments were detected with anti-c-myc 9e antibodies and anti-mouse igg antibody conjugated with peroxidase. after treatment with tmb and sulfuric acid, absorbance was measured at 450 nm using a microtiter plate reader, and the blank of a sample without d was subtracted. elisa measurements were performed in the linear range of d concentration. the rate of d degradation was examined by incubation of diluted culture supernatant at and 37°c on a rotation shaker and taking samples hourly. modeling the integrated bioprocess on the reactor and cellular level mathematical models of the stirred tank reactor and the cells were established in matlab-simulink based on differential equations, mass balances, kinetics and stoichiometries. three substrates s,i with i = glucose, total amino acids and cysteine were modeled. the model was established for two reasons. although many assumptions were made, mainly to describe growth and product formation, it shall provide a deeper understanding of the whole bioprocess. however, the main goal was to provide a tool that can be used to optimize the isms steps. this is important because the amount of particles used and the number of required isms steps must be minimized because they are connected to extra costs. a set of simulation parameters is proposed in table  and explanation of their origin is provided in the text. calculation of the reactor volume v and the dilution rate d considered constant substrate and base flow qs,i and qbase as well as volume reduction by the auto sampler qsampler. the flow rate was chosen in that way that glucose was not limiting in the reactor. dvdt=∑iqs,i+qbase−qsampler d=∑iqs,i+qbasev a) estimated by minimizing least squares for eqs. a1) 3- a2) 2- a3) values valid for 37°c; for 25°c calculation according to equation 3- b) estimated from literature. c) estimated manually. d) according to measurements. e) set manually. calculations of the bdm concentration cx took the specific growth rate μ of the microorganisms into consideration which was obtained from equation 3- dcxdt=μ·cx−d·cx substrate concentrations cs,i were derived from equation 3- where rs,i are the specific substrate uptake rates which were calculated from equation 3- the composition of the substrate feed can be taken from microorganism and media section. when i = cysteine is considered as substrate, the assumption was made that cysteine could be synthesized by the cells at a fixed rate rcys when additional sulfur 2so4) was present in the medium and cysteine provided in the medium was exhausted after approx. 40 h of cultivation. otherwise no cysteine was synthesized. dcs,idt=−rs,i·cx−d·cs,i+d·cs,i,f+rcys·cx carbon dioxide production depends on the specific carbon dioxide production rate rco of the microorganisms. hereby, the co flux into the reactor was neglected as it is insignificant in comparison to the co produced. dmco2dt=qco2=rco2·cx·v concentrations of d and other proteins cp,i and cp,j were calculated from equation 3- while rp,i,j describe protein formation rates that were estimated manually by approximation of the measurements, rp,i,deg represents d degradation which was determined experimentally. adsorption and removal of proteins from the bioreactor is considered by the term Γ˙gijm−2h− that takes particle properties such as the bet-surface aspec into account. the total protein content was calculated as cp,tot = cp,i + cp,j dcp,i,jdt=rp,i,j·cx−d·cp,i,j−rp,i,deg·cp,i−Γ˙·mmp·aspecv adsorption kinetics of proteins to a solid matrix is determined by the transport rate ktr of the protein to the surface and the adsorption rate ka. equation 3- is obtained for the assumption ka ≫ ktr with the surface-specific load of protein on the particle Γ. hereby, ktr was estimated by minimizing least squares for equation 3- kd and q*max were estimated from equation 2- dΓdt=Γ˙=1000·ktr·cp,i−kd·Γqmax∗aspec−Γ the substrate uptake was considered as the first step in the metabolic network and was modeled by michaelis-menten kinetics. km was taken from the literature with the assumption km,aa,cys ≈ km,nh4+. rs,i=rs,i,max·cs,ics,i+km maximum substrate uptake rates were calculated by equation 3- where yx,s,i is the integral yield coefficient for bdm on each substrate i that was obtained from measurements or estimated manually. rs,i,max=μmaxyx,s,i μmax was experimentally determined for t = 37°c and estimated by equation 3- for t = 25°c. hereby, aarrh. is a pre-exponential factor that was estimated manually, ea the activation energy, and r =   j mol-1 k- the universal gas constant. μmax=aarrh.·exp−ear·t+ k d production was modeled by a constant specific production rate rp,i,0 >  after induction in medium and after an additional lag phase in medium, 3 mg g-1 h- and 6 mg g-1 h- ). production of proteins beside d was constantly modeled as rp,j,0 > . rp,i,j=rp,i,j,o the total mass balance for glucose in the anabolism and catabolism is modeled as equation 3- rglu=rana+rcata component balances of carbon, amino acids and cysteine in the anabolism are shown in equations 3- - 3- hereby, e is the molar or mass fraction of a chemical compound within another compound as indicated. the following values were taken from the literature: ec,x, ec,p,i,j, eaa,x, ecys,yeast, ecys,x, ecys,p,i ≈ 6∙  g/mol/26000 g/mol. to obtain eaa,p,i,j the assumption was made that proteins only consist of amino acids while ecys,p,j was estimated manually. ec,glu·rana=ec,x·μ+ec,p,i·rp,i+ec,p,j·rp,j raa=eaa,x·μ+eaa,p,i·rp,i+eaa,p,j·rp,j rcys=ecys,x·μ+ecys,p,i·rp,i+ecyc,p,j·rp,j the carbon balance for the respiration is presented in equation 3- ec,co2·rco2=ec,glu·rcata the energy balance couples anabolism and catabolism of the cell, but intracellular atp levels were not quantified. hence, the carbon dioxide production rate rco was modeled as a function of specific growth as well as protein production rates. yield coefficients on atp as well as the maintenance rate rm were taken from the literature and normalized. the maintenance rate mf · rm refers to intracellular energy requirements. estimates for mf were received from. rco2=yco atpyx,atp·μ+yco atpyp,i,atp·rp,i+yco atpyp,j,atp·rp,j+mf·rm yco x=yco atpyx,atp;yco p,i=yco atpyp,i,atp;yco p,j=yco atpyp,j,atp mathematic modeling of d production requires solutions for nine rates r by means of available equations. thus, the system would be over-determined if all equations were used, and rates might possibly contradict each other. however, there can only be one specific growth rate that relies upon the limiting substrate and its uptake rate. thus, the three theoretical substrate uptake rates rglu, raa, rcys were determined separately before the system matrix was calculated. if glucose is assumed to be limiting, the remaining eight rates were calculated by finding a solution for the equation system a∙s = b with the stoichiometric matrix a and the vectors s =  and b. finally, the set of rates corresponding to the minimum solution of μ was chosen for further calculation. two different parameter sets for k5-k and k9-k were applied. the main difference is found in the parameters rp,i, that were obtained due to different d production rates and rcys due to different medium compositions.  magnetic particle characterization equivalent diameters of particle agglomerates x50 =  and  μm were found for ida- and ida- beads in pbs buffer at ph . the hydrodynamic diameter of triazine bead agglomerates was  ±   μm. results obtained from electron microscopy also revealed much smaller primary particles. thus, these particles have a very high surface-to-volume ratio which is desirable to obtain a high protein binding capacity. it is known from the literature that magnetic particles with a diameter ≥  -1 μm can be easily separated by simple magnetic separators, while separation of smaller particles is much more efficient when high gradient magnetic separators are used. this applies because the magnetic force which is experienced by the particles through the external magnetic field is proportional to the particle volume and the gradient of the magnetic field strength inside the separation chamber. especially at higher viscosities of the cultivation medium magnetic particle agglomerates are favorable due to shorter separation times. since particle agglomeration can occur spontaneously in the medium, especially at high particle concentrations, it is important for in situ protein separation that no biomass, i.e. microorganisms, is included or adsorbed in/to those agglomerates. losses of biomass, i.e. escherichia coli cells, after the isms step are discussed in comparison of the total d yield in processes with and without isms section. saturation magnetization ms =  a m2 kg- and  ±  a m2 kg- were measured for ida- and ida- beads, and ms =   ±  a m2 kg- for triazine beads. the remanent magnetization was low at mr =  - a m2 kg- thus particles were considered superparamagnetic. it is important to keep in mind that magnetic particles or agglomerates thereof have superparamagnetic properties only when the size of each individual magnetite core is between 15–50 nm. adsorption of the target protein scfv d to the beads was quantified by measuring isotherms that could be approximated by the langmuir adsorption model. in the experiments maximum loads of qmax* =   g/g and  g/g were found for ida- and ida- beads, respectively, when medium containing extra salts was used. in medium without extra salts, qmax* was significantly decreased to ~  -  g/g. different maximum loads might be based on different particle production processes and are not further discussed. highest loads of ida beads were achieved for 60 min incubation in cu2+-solution and 60 min adsorption. for triazine beads we found qmax* =   g/g in medium with extra salts. according to the low kd-values obtained from the isotherms, all beads can be categorized as affinity adsorbents. the maximum obtained load for ida- beads qmax* =   g/g is very high compared to qmax* =   g/g for green fluorescent protein. possibly, d concentrations were overestimated by the elisa procedure in general due to incomplete elution or loss of d activity in the elution buffer. however, the proposed elisa method is sufficient to compare the results provided in this study among each other. degradation of the scfv d in cultivation media the stability of the d was tested in cell-free cultivation medium up to 50 h of incubation, but no degradation or any loss of d activity could be detected. although the d fragments were found to be stable in cell-free medium, isms was applied to study its impact on the whole bioprocess in presence of the microorganisms with the particle systems characterized in magnetic particle characterization section. production of d and isms in bioreactor cultivations process performance in complex medium with extra salts two reference cultivations k and k were conducted without and two cultivations k and k carried out with isms to investigate the performance of isms. results of k were similar to those in k and are not shown. before induction, cells were grown in medium up to a bio dry mass concentration of 5–  g/l at a maximum specific growth rate of μmax =  -  h- while glucose was rapidly consumed but not limiting. after induction, bdm concentrations increased up to -  g/l at the end of k k and k offline bdm and glucose concentrations were similar in cultivations with and without isms. d was only produced after induction: in k k and k d production was delayed by 30 hours. further investigations are required to clarify whether the delay was due to inhibition of d production by the additional salts provided in the medium. although cultivations k k and k were performed in the same medium under the same conditions, maximum obtained d concentrations differed significantly before the isms step. according to harrison, different leakage rates of the d fragments from the periplasmic space into the medium might be responsible for those differences. furthermore, the obtained extracellular d titers are much higher compared to those reported in literature. although d concentrations might have been overestimated within the elisa procedure, the impact of isms on the process could be studied well. for better comparison of processes with and without isms, d concentrations were normalized to the final d concentration before the isms step. in k and k 92% and 98% of the overall d were separated from the medium by isms, respectively. however, no significant production was observed after separation in k and k instead, d activities decreased towards zero until the end of cultivations. this effect cannot be attributed to significant degradation of the d fragments in the medium since no d degradation could be quantified. thus, d production was supposed to be inhibited as a consequence of the isms step. further explanation is provided in the section response of the microorganisms to the isms steps. during all cultivations, protein other than d was continuously produced and secreted into the medium up to 2 g/l. process performance in complex medium without extra salts two reference cultivations k and k were conducted without isms. bdm and glucose concentrations as well as total protein concentrations are comparable to those obtained from cultivations in medium containing extra salts. however, after induction, bdm concentrations remained constant in k or slightly decreased in k10-k d was immediately produced after isms in k, and maximum d concentrations before the isms step were lower compared to the results obtained from k5-k, and they did not increase above -  g/l at the end of cultivations. investigations of the culture medium suggest that the lower overall bdm and d concentrations are based on potential sulfur limitation in medium after induction. only methionine was supplied by the feed being theoretically sufficient to deliver the sulfur required for d production. sulfur is a compound of cysteine that is required for biomass and protein synthesis. calculations were based on % sulfur and % cysteine content of the bdm and d fragments, respectively. in k 78% of the overall d were separated from the medium by isms. in contrast to k and k d was produced in k after isms reaching 50% of the maximum level before separation. validation of the process simulation by offline data simulation of bdm and glucose concentrations over cultivation time could be validated by offline measurements for all cultivations. as it was observed in all cultivations, biomass growth already declined before induction. the experimentally unknown limitation was modeled as cysteine, i.e. sulfur limitation, and exceptionally as glucose limitation in k after 98 hours. cells were considered capable of producing cysteine from sulfate that was abundant in the medium of k k and k to fulfill the requirements of d production and biomass growth, but that was not abundant in k k and k. this can be an explanation for decreased bdm and d titers in cultivations k9-k degradation of d in the medium was set to zero. significant differences between the d offline data and the simulation occurred after separation in k and k while d was reproduced in the simulation, no d reproduction occurred in the offline data. simulated d and total protein courses could be validated well with experimental data in k9-k including the isms step. d production rates were comparable before and after isms but were not elevated compared to the reference cultivations k and k. similar results were obtained for cultivations with and without isms in shaking flask cultivation ). in the best case similar d production rates before and after isms were expected because no degradation of the d was observed. in the response of the microorganisms to the isms steps an explanation is given why the production of d might have been limited in k and k after the isms steps. response of the microorganisms to the isms steps measurements of co in the off-gas and cu2+ concentrations in the medium were conducted to study the culture viability before and after isms. decreased or no d production after isms correlates well with a decreased respiratory capacity of the cells: in k and k the co fluxes in the off-gas stream dropped from to  g/h and from to  g/h after the initial isms step indicating a decreased metabolic activity. in k qco recovered up to  g/h after 87 h of cultivation. interestingly, after 87 h of cultivation, bdm concentrations almost doubled although no significant d production was observed any more. this implies that cells were either inhibited to produce d after isms or the process suffered from microbial contamination. the second isms step in k did not evoke a drop of the co signal at 116 h. in k no significant drop of the co signal was observed indicating that the metabolic activity was not decreased by isms. overestimation of the co flux by the simulation might be based on the fact that no overflow metabolism was taken into account in the model. measurements of cu2+ concentrations in the medium revealed significant copper contamination of up to 180 mg/l and 300 mg/l after the first and second isms step in k respectively. copper accumulation in the medium due to insufficient washing steps after copper-loading of ida particles seems unlikely because four subsequent washing steps were carried out. partial leakage of copper from the beads most likely occurred during their application in the medium. this suggestion is supported by high availability of up to  mmol free ida groups on the particle surface per gram pva-ida- beads that theoretically chelate equimolar amounts of cu2+ ions. these findings imply that cell metabolism and thus production of d by recombinant e. coli can be strongly inhibited by free cu2+ ions. cu2+ ions might be reduced in the fermentation medium to cu+ ions that are able to structurally damage proteins and decrease their activity due to highly reactive radical intermediates. copper concentrations below 120 mg/l as in k seem to be less inhibiting, and d production was less diminished in k compared to k and k where copper concentrations exceeded 140 mg/l. in sk the copper concentration was not measured before and after isms. however, due to similar concentrations of cu2+-loaded ida- particles in the biosuspension during the isms step, it can be assumed that the copper concentration in the biosuspension after isms in sk did not exceed the value obtained in k. in literature, the minimal inhibitory concentration of cu2+, ni2+, co2+ and zn2+ ions on e. coli cells is  mmol/l. further research can clarify if a potential d production inhibition can be circumvented by using other divalent metal ions: ni2+ and co2+ ions are reported to be less carcinogenic than cu2+ ions but are still potentially harmful if intended for therapeutic purposes. since zn2+ is redox-inactive and less toxic than cu2+, it might be tested as metal ligand to bind his-tagged d. another approach would be to use tetradendate chelating agents such as nitrilotriacetic acid instead of tridendate ligands such as ida to obtain more stable complexes with the metal ion and potentially avoid excessive leakage of me2+ into the medium. cultivation and isms in shaking flasks by means of triazine beads additional isms experiments were conducted in shaking flasks using triazine-functionalized beads. in both complex media and, up to 57-79% of d was separated from the medium, and d was produced after isms. for both media, similar linearized d production rates rp,i,0 =  -  g g-1 h- were found. these results are comparable to those obtained for ida- beads used in sk. however, no copper or other heavy metal ions were required for adsorption using the triazine beads. we have thus shown experimentally that scfv d antibody fragments can be selectively separated from biosuspension by gum arabic coated magnetic carrier particles functionalized with the triazine ligand 22/ a biomimetic of protein a. until now, binding of those beads has only been reported to full igg antibodies although it has been shown recently that ligand 22/ can also bind to the fab portion of igg by molecular modeling studies. comparison of the total d yield in processes with and without isms as can be seen from figure  isms did not yield extra benefit for total d production since the total accumulated d concentrations with and without isms were equal in the best case. this result has been confirmed by simulation including theoretical multi step isms. as shown for extracellular protease that was degraded in biosuspension such a model can be useful to optimize the number of isms steps before the experiment is actually performed. neither an inhibition of d in the medium on d production itself nor losses of d degradation were observed in the extracellular medium. while the total d yield without isms was set yno isms =  the yield with isms was yisms =  - and - for ida and triazine beads, respectively. elution efficiencies between and 100% were received for both pva-ida- and - particle charges. it remains unclear whether low elution efficiencies are to be attributed to incomplete elution or loss of activity in the elution buffer although edta is reported to be a strong eluent which extracts the metal ions from ida and disrupts interactions between proteins and chelating ligands. adsorption and elution efficiency of his-tagged proteins from ida-functionalized magnetic beads can depend on the chelated metal ion: in literature, multi-subunit adsorption of a his-tagged protein onto chelated cu2+ was reported which significantly limited elution efficiency. furthermore, the isolated target protein can potentially be structurally damaged in presence of reduced cu+ ions which leads to reduced activity. this implies to test other divalent metal ions such as ni2+, zn2+ or co2+ which might lead to higher elution efficiencies. elution efficiencies of d from triazine-functionalized beads only reached 6%. in comparison, elution efficiencies of 35% were obtained for igg, and the elution protocol needs further improvement. sds-page should be applied to control the purity of the elution samples. losses of biomass due to the adsorption procedure were 9 ± 2% and 21 ± 4% for ida and triazine beads, respectively. this indicates that biomass is more attracted to adsorb to the triazine beads. coating the beads with an extra layer of biopolymer such as alginate might minimize the risk of bio mass adsorption. the application of a high gradient magnetic separator can further help to minimize biomass losses because formation of one single compact filter cake with inclusion of biomass is avoided.  results have shown that pva-ida particles are well suited to separate and purify his-tagged proteins from complex media. however, their application for isms is limited due to the release of chelated copper ions into the medium which have shown to inhibit d production above ccu > 120 mg/l. other less toxic divalent metal ions should be tested and validated as ligands for the ida-beads. but it must be shown in semi-continuous isms experiments whether this particle type proofs to be compatible with the bioprocess or is better suited for separation of his-tagged proteins after cultivation as it is well known from the literature. in the case of separation of antibodies and their fragments, triazine-functionalized magnetic particles were found to be an excellent low-cost alternative to protein a-functionalized beads. although no negative impact of these beads was observed on the bioprocess, further semi-continuous isms experiments are necessary. in the best case, similar d production rates were observed before and after in situ separation when ida- or triazine-functionalized particle types were applied. no degradation of d was measured in biosuspension and, thus, isms did not increase the total yield of the d -production process as was shown by experiments and simulation. under consideration of the obtained results and proposed improvements on pva-ida particle performance, both pva-ida and triazine-functionalized magnetic beads can serve as isms platform technology to obtain highly purified product in only one separation step. in situ magnetic separation can bring most benefit to those production processes where antibodies or their fragments lack stability in the medium or are subject to degradation. these processes need to be identified. this study provides a route how to proceed with experiments and optimize the process by simulation: small scale experiments should be conducted first and followed by scale up. when the kinetics of degradation or lack of stability of the target are quantified and implemented into the model, the latter can be applied to optimize isms in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required isms steps. abbreviations greek symbols μ: specific growth rate, g g-1 h-1; Γ: surface-specific particle load, g m-2; latin symbols aarrh: arrhenius pre-factor; aspec: bet-surface, m2 g-1; b: magnetic flux density, t; c: concentration, g l-1; d: dilution rate, h-1; e: mass or molar fraction; ea: activation energy, j mol-1; ka: adsorption rate, m h-1; kd: dissociation constant, g l-1; km: michaelis-menten constant, g l-1; ktr: transport rate, m h-1; m: saturation magnetization, a m2 kg-1; m: mass, g; mf: maintenance factor; q: adsorption load, g g-1; q: flow to/from reactor, l/h, g/h; r: specific uptake/production/maintenance rate, g g-1 h-1; t: cultivation time, h; t: temperature,°c; v: reactor volume, l; x50: equivalent diameter, m; yx,s,i: integral yield coefficient, g g-1; y: overall product yield; yx,s,i: differential yield coefficient, g g-; indices *: equilibrium; 0: initial condition; aa: amino acids; ana: anabolism; atp: adenosine triphosphate; c: carbon; cata: catabolism; co2: carbon dioxide; cu2+: divalent copper ions; cys: cysteine; deg: degradation; f: feed; glu: glucose; isms: in situ magnetic separation; m: maintenance; max: maximum; mp: magnetic particles; p,i: scfv d; p,j: other proteins besides d; r: remanent; s: saturation; s,i: substrate; x: biomass.; natural constant r: universal gas constant,  g mol-1 k.; other abbreviations bdm: bio dry mass; bet: brunauer-emmett-teller; isms: in situ magnetic separation; iptg: isopropyl β-d-1-thiogalactopyranoside; scfv: single chain fragment variable; pbs: phosphate buffered saline; mpbst: modified phosphate buffered saline with tween 20; pva-ida: polyvinyl alcohol-iminodiacetate. competing interest the authors declare that they have no competing interest. authors’ contributions mc conducted the cultivations and magnetic separation experiments, implemented the reactor-cell model and drafted the manuscript. as contributed to the experiments and provided valuable discussion. mf provided the mpva-ida- functionalized magnetic particles and assisted with the hgms setup. acar supervised the preparation of the triazine particles and provided valuable discussion. ilb carried out the synthesis of the triazine-functionalized magnetic particles. acar and ilb helped to design the adsorption and elution experiments. cp conceived and initiated the project, and assisted with the model. all authors carried out the revision of the manuscript. all authors read and approved the final manuscript. 
this study investigates the impact of isms on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. after the isms step, elevated copper concentrations above 120 mg/l in the medium negatively influenced d production. the proposed model can be applied to design future isms experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required isms steps. such optimized particle systems can be regarded as isms platform technology, especially for the production of antibodies and their fragments with low stability in the medium. in situ magnetic separation has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. in situ product removalmagnetic separationprotein purificationaffinity ligandstriazine beadsmetal chelate beadsrecombinant scfv antibody fragmentsextracellular proteinescherichia coli fermentationcomplex media in the present work, an integrated isms process was established for the production of his-tagged single chain fragment variable d antibodies produced by e. coli in complex media. both particle systems are well suited for separation of d during cultivation. while the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. we could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb d fragments, and measured maximum loads of  g d per g of beads.
 in situ magnetic separation has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. in the present work, an integrated isms process was established for the production of his-tagged single chain fragment variable d antibodies produced by e. coli in complex media. this study investigates the impact of isms on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.  both particle systems are well suited for separation of d during cultivation. while the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. after the isms step, elevated copper concentrations above 120 mg/l in the medium negatively influenced d production. due to the stable nature of the model protein scfv d in the biosuspension, the application of isms could not increase the overall d yield as was shown by simulation and experiments.  we could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb d fragments, and measured maximum loads of  g d per g of beads. although copper-loaded metal-chelate beads did adsorb his-tagged d well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. such optimized particle systems can be regarded as isms platform technology, especially for the production of antibodies and their fragments with low stability in the medium. the proposed model can be applied to design future isms experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required isms steps. in situ product removalmagnetic separationprotein purificationaffinity ligandstriazine beadsmetal chelate beadsrecombinant scfv antibody fragmentsextracellular proteinescherichia coli fermentationcomplex media 